Chapter 15: Molecular and Genomic Surgery

1. Biological sciences developed drastically in last 60 years after the uncovering of DNA structure by Watson and Crick

2. The completion of the human genome sequence in 2003 represents a great milestone in modern science.

3. The technology emerging from molecular and cellular biology has revolutionized the understanding of disease and will radically transform the practice of surgery.

4. The use of genetically modified mouse models and cell lines using gene therapy and RNA interference therapy has greatly contributed to the understanding of the molecular basis for human diseases and targeted therapies.

5. The sequencing of each individual’s genome has the potential to improve the predication, prevention, and targeted treatment of disease, resulting in personalized medicine and surgery.

6. The use of functional genomics and modern molecular analyses will facilitate the discovery of actionable genes to guide choice of care.

The beginning of modern medicine can be traced back to centuries ago when physicians and scientists began studying human anatomy from cadavers in morgues and animal physiology following hunting expeditions. Gradually, from the study of animals and plants in greater detail and the discovery of microbes, scientific principles governing life lead to the emergence of the biological sciences. As biological science developed and expanded, scientists and physicians began to utilize the principles of biological sciences to solve challenges of human diseases while continuing to explore the fundamentals of life in greater detail. With ever-evolving state-of-the-art scientific tools, our understanding of how cells, tissues, organs, and entire organisms function, down to the level of molecular and subatomic structure, has resulted in modern biology with an enormous impact on modern healthcare and the discovery of amazing treatments for disease at an exponential pace. Significant progress has been made in molecular studies of organ development, cell signaling, and gene regulation. The advent of recombinant DNA technology, polymerase chain reaction (PCR) techniques, and next-generation genomic sequencing, which resulted in the sequencing of the human genome, holds the potential to have a transformational influence on healthcare and society this century by not only broadening our understanding of the pathophysiology of disease, but also by bringing about necessary changes in personalized medicine.

Today’s practicing surgeons are becoming increasingly aware that many modern surgical procedures rely on the information gained through molecular research (i.e., personalized surgery). Genomic information, such as deleterious BRCA and RET proto-oncogene mutations, is being used to help direct prophylactic procedures to remove potentially harmful tissues before they do damage to patients. Molecular engineering has led to cancer-specific gene therapy that could serve in the near future as a more effective adjunct to surgical debulking of tumors than radiation or chemotherapy, so surgeons will benefit from a clear introduction to how basic biochemical and biological principles relate to the developing area of molecular biology. This chapter reviews the current information on modern molecular biology for the surgical community.

Basic Concepts of Molecular Research

The modern era of molecular biology, which has been mainly concerned with how genes govern cell activity, began in 1953 when James D. Watson and Francis H. C. Crick made one of the greatest scientific discoveries by deducing the double-helical structure of deoxyribonucleic acid (DNA).^1,2 The year 2003 marked the 50th anniversary of this great discovery. In the same year, the Human Genome Project completed with sequencing approximately 20,000 to 25,000 genes and 3 billion base pairs in human DNA.³ Before 1953, one of the most mysterious aspects of biology was how genetic material was precisely duplicated from one generation to the next. Although DNA had been implicated as genetic material, it was the base-paired structure of DNA that provided a logical interpretation of how a double helix could “unzip” to make copies of itself. This DNA synthesis, termed replication, immediately gave rise to the notion that a template was involved in the transfer of information between generations, and thus confirmed the suspicion that DNA carried an organism’s hereditary information.

Within cells, DNA is packed tightly into chromosomes. One important feature of DNA as genetic material is its ability to encode important information for all of a cell’s functions (Fig. 15-1). Based on the principles of base complementarity, scientists also discovered how information in DNA is accurately transferred into the protein structure. DNA serves as a template for RNA synthesis, termed transcription, including messenger RNA (mRNA, or the protein-encoding RNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). mRNA carries the information from DNA to make proteins, termed translation, with the assistance of rRNA and tRNA. Each of these steps is precisely controlled in such a way that genes are properly expressed in each cell at a specific time and location. In recent years, new classes of noncoding RNAs (ncRNA), for example, microRNA (or miRNA), Piwi-interacting RNA (or piRNA), and long intergenic noncoding RNA (or lincRNA), have been identified. Although the number of ncRNAs encoded in the human genome is unknown and a lot of ncRNAs have not been validated for their functions, ncRNAs have been associated to regulate gene expression through posttranscriptional gene regulation such as mRNA degradation or epigenetic regulation such as chromatin structure modification and DNA methylation induction.⁴ Consequently, the differential gene activity in a cell determines its actions, properties, and functions.

Molecular Approaches to Surgical Research

Rapid advances in molecular and cellular biology over the past half century have revolutionized the understanding of disease and will radically transform the practice of surgery. In the future, molecular techniques will be increasingly applied to surgical disease and will lead to new strategies for the selection and implementation of operative therapy. Surgeons should be familiar with the fundamental principles of molecular and cellular biology so that emerging scientific breakthroughs can be translated into improved care of the surgical patient.

The greatest advances in the field of molecular biology have been in the areas of analysis and manipulation of DNA.¹ Since Watson and Crick’s discovery of DNA structure, an intensive effort has been made to unlock the deepest biologic secrets of DNA. Among the avalanche of technical advances, one discovery in particular has drastically changed the world of molecular biology: the uncovering of the enzymatic and microbiologic techniques that produce recombinant DNA. Recombinant DNA technology involves the enzymatic manipulation of DNA and, subsequently, the cloning of DNA. DNA molecules are cloned for a variety of purposes including safeguarding DNA samples, facilitating sequencing, generating probes, and expressing recombinant proteins in one or more host organisms. DNA can be produced by a number of means, including restricted digestion of an existing vector, PCR, and cDNA synthesis. As DNA cloning techniques have developed over the last quarter century, researchers have moved from studying DNA to studying the functions of proteins, and from cell and animal models to molecular therapies in humans. Expression of recombinant proteins provides a method for analyzing gene regulation, structure, and function. In recent years, the uses for recombinant proteins have expanded to include a variety of new applications, including gene therapy and biopharmaceuticals. The basic molecular approaches for modern surgical research include DNA cloning, cell manipulation, disease modeling in animals, and clinical trials in human patients.

DNA and Heredity

DNA forms a right-handed, double-helical structure that is composed of two antiparallel strands of unbranched polymeric deoxyribonucleotides linked by phosphodiester bonds between the 5′ carbon of one deoxyribose moiety to the 3′ carbon of the next (Fig. 15-2). DNA is composed of four types of deoxyribonucleotides: adenine (A), cytosine (C), guanine (G), and thymine (T). The nucleotides are joined together by phosphodiester bonds. In the double-helical structure deduced by Watson and Crick, the two strands of DNA are complementary to each other. Because of size, shape, and chemical composition, A always pairs with T, and C with G, through the formation of hydrogen bonds between complementary bases that stabilize the double helix.

Recognition of the hereditary transmission of genetic information is attributed to the Austrian monk, Gregor Mendel. His seminal work, ignored upon publication until its rediscovery in 1900, established the laws of segregation and of independent assortment. These two principles established the existence of paired elementary units of heredity and defined the statistical laws that govern them.⁵ DNA was isolated in 1869, and a number of important observations of the inherited basis of certain diseases were made in the early part of the twentieth century. Although today it appears easy to understand how DNA replicates, before the 1950s, the idea of DNA as the primary genetic material was not appreciated. The modern era of molecular biology began in 1944 with the demonstration that DNA was the substance that carried genetic information. The first experimental evidence that DNA was genetic material came from simple transformation experiments conducted in the 1940s using Streptococcus pneumoniae. One strain of the bacteria could be converted into another by incubating it with DNA from the other, just as the treatment of the DNA with deoxyribonuclease would inactivate the transforming activity of the DNA. Similarly, in the early 1950s, before the discovery of the double-helical structure of DNA, the entry of viral DNA and not the protein into the host bacterium was believed to be necessary to initiate infection by the bacterial virus or bacteriophage. Key historical events concerning genetics are outlined in Table 15-1.

For cells to pass on the genetic material (DNA) to each progeny, the amount of DNA must be doubled. Watson and Crick recognized that the complementary base-pair structure of DNA implied the existence of a template-like mechanism for the copying of genetic material.¹ The transfer of DNA material from the mother cell to daughter cells takes place during somatic cell division (also called mitosis). Before a cell divides, DNA must be precisely duplicated. During replication, the two strands of DNA separate, and each strand creates a new complementary strand by precise base-pair matching (Fig. 15-3). The two, new, double-stranded DNAs carry the same genetic information, which can then be passed on to two daughter cells. Proofreading mechanisms ensure that the replication process occurs in a highly accurate manner. The fidelity of DNA replication is absolutely crucial to maintaining the integrity of the genome from generation to generation. However, mistakes can still occur during this process, resulting in mutations, which may lead to a change of the DNA’s encoded protein and, consequently, a change of the cell’s behavior. The reliable dependence of many features of modern organisms on subtle changes in genome is linked to Mendelian inheritance and also contributes to the processes of Darwinian evolution. In addition, massive changes, so-called genetic instability, can occur in the genome of somatic cells such as cancer cells.

Gene Regulation

Living cells have the necessary machinery to enzymatically transcribe DNA into RNA and translate the mRNA into protein. This machinery accomplishes the two major steps required for gene expression in all organisms: transcription and translation (Fig. 15-4). However, gene regulation is far more complex, particularly in eukaryotic organisms. For example, many gene transcripts must be spliced to remove the intervening sequences. The sequences that are spliced off are called introns, which appear to be useless, but in fact may carry some regulatory information. The sequences that are joined together, and are eventually translated into protein, are called exons. Additional regulation of gene expression includes modification of mRNA, control of mRNA stability, and its nuclear export into cytoplasm (where it is assembled into ribosomes for translation). After mRNA is translated into protein, the levels and functions of the proteins can be further regulated posttranslationally. However, the following sections will mainly focus on gene regulation at transcriptional and translational levels.

Transcription

Transcription is the enzymatic process of RNA synthesis from DNA.⁶ In bacteria, a single RNA polymerase carries out all RNA synthesis, including that of mRNA, rRNA, and tRNA. Transcription often is coupled with translation in such a way that an mRNA molecule is completely accessible to ribosomes, and bacterial protein synthesis begins on an mRNA molecule even while it is still being synthesized. Therefore, a discussion of gene regulation with a look at the simpler prokaryotic system precedes that of the more complex transcription and posttranscriptional regulation of eukaryotic genes.

Transcription in Bacteria

Initiation of transcription in prokaryotes begins with the recognition of DNA sequences by RNA polymerase. First, the bacterial RNA polymerase catalyzes RNA synthesis through loose binding to any region in the double-stranded DNA and then through specific binding to the promoter region with the assistance of accessory proteins called σ factors (sigma factors). A promoter region is the DNA region upstream of the transcription initiation site. RNA polymerase binds tightly at the promoter sites and causes the double-stranded DNA structure to unwind. Consequently, few nucleotides can be base-paired with the DNA template to begin transcription. Once transcription begins, the σ factor is released. The growing RNA chain may begin to peel off as the chain elongates. This occurs in such a way that there are always about 10 to 12 nucleotides of the growing RNA chains that are base-paired with the DNA template.

The bacterial promoter contains a region of about 40 bases that include two conserved elements called –35 region and –10 region. The numbering system begins at the initiation site, which is designated +1 position, and counts backward (in negative numbers) on the promoter and forward on the transcribed region. Although both regions on different promoters are not the same sequences, they are fairly conserved and very similar. This conservation provides the accurate and rapid initiation of transcription for most bacterial genes. It is also common in bacteria that one promoter serves to transcribe a series of clustered genes, called an operon. A single transcribed mRNA contains a series of coding regions, each of which is later independently translated. In this way, the protein products are synthesized in a coordinated manner. Most of the time, these proteins are involved in the same metabolic pathway, thus demonstrating that the control by one operon is an efficient system. After initiation of transcription, the polymerase moves along the DNA to elongate the chain of RNA, although at a certain point, it will stop. Each step of RNA synthesis, including initiation, elongation, and termination, will require the integral functions of RNA polymerase as well as the interactions of the polymerase with regulatory proteins.

Transcription in Eukaryotes

Transcription mechanisms in eukaryotes differ from those in prokaryotes. The unique features of eukaryotic transcription are as follows: (a) Three separate RNA polymerases are involved in eukaryotes: RNA polymerase I transcribes the precursor of 5.8S, 18S, and 28S rRNAs; RNA polymerase II synthesizes the precursors of mRNA as well as microRNA; and RNA polymerase III makes tRNAs and 5S rRNAs. (b) In eukaryotes, the initial transcript is often the precursor to final mRNAs, tRNAs, and rRNAs. The precursor is then modified and/or processed into its final functional form. RNA splicing is one type of processing to remove the noncoding introns (the region between coding exons) on an mRNA. (c) In contrast to bacterial DNA, eukaryotic DNA often is packaged with histone and nonhistone proteins into chromatins. Transcription will only occur when the chromatin structure changes in such a way that DNA is accessible to the polymerase. (d) RNA is made in the nucleus and transported into cytoplasm, where translation occurs. Therefore, unlike bacteria, eukaryotes undergo uncoupled transcription and translation.

Eukaryotic gene transcription also involves the recognition and binding of RNA polymerase to the promoter DNA. However, the interaction between the polymerase and DNA is far more complex in eukaryotes than in prokaryotes. Because the majority of studies have been focused on the regulation and functions of proteins, this chapter primarily focuses on how protein-encoding mRNA is made by RNA polymerase II.

Translation

DNA directs the synthesis of RNA; RNA in turn directs the synthesis of proteins. Proteins are variable-length polypeptide polymers composed of various combinations of 20 different amino acids and are the working molecules of the cell. The process of decoding information on mRNA to synthesize proteins is called translation (see Fig. 15-1). Translation takes place in ribosomes composed of rRNA and ribosomal proteins. The numerous discoveries made during the 1950s made it easy to understand how DNA replication and transcription involve base-pairing between DNA and DNA or DNA and RNA. However, at that time, it was still impossible to comprehend how mRNA transfers the information to the protein-synthesizing machinery. The genetic information on mRNA is composed of arranged sequences of four bases that are transferred to the linear arrangement of 20 amino acids on a protein. Amino acids are characterized by a central carbon unit linked to four side chains: an amino group (–NH₂), a carboxy group (–COOH), a hydrogen, and a variable (–R) group. The amino acid chain is assembled via peptide bonds between the amino group of one amino acid and the carboxy group of the next. Because of this decoding, the information carried on mRNA relies on tRNA. Translation involves all three RNAs. The precise transfer of information from mRNA to protein is governed by genetic code, the set of rules by which codons are translated into an amino acid (Table 15-2). A codon, a triplet of three bases, codes for one amino acid. In this case, random combinations of the four bases form 4 × 4 × 4, or 64 codes. Because 64 codes are more than enough for 20 amino acids, most amino acids are coded by more than one codon. The start codon is AUG, which also corresponds to methionine; therefore, almost all proteins begin with this amino acid. The sequence of nucleotide triplets that follows the start codon signal is termed the reading frame. The codons on mRNA are sequentially recognized by tRNA adaptor proteins. Specific enzymes termed aminoacyl-tRNA synthetases link a specific amino acid to a specific tRNA. The translation of mRNA to protein requires the ribosomal complex to move stepwise along the mRNA until the initiator methionine sequence is identified. In concert with various protein initiator factors, the methionyl-tRNA is positioned on the mRNA and protein synthesis begins. Each new amino acid is added sequentially by the appropriate tRNA in conjunction with proteins called elongation factors. Protein synthesis proceeds in the amino-to-carboxy-terminus direction.

The biologic versatility of proteins is astounding. Among many other functions, proteins serve as enzymes that catalyze critical biochemical reactions, carry signals to and from the extracellular environment, and mediate diverse signaling and regulatory functions in the intracellular environment. They also transport ions and various small molecules across plasma membranes. Proteins make up the key structural components of cells and the extracellular matrix and are responsible for cell motility. The unique functional properties of proteins are largely determined by their structure (Fig. 15-5).

Regulation of Gene Expression

The human organism is made up of a myriad of different cell types that, despite their vastly different characteristics, contain the same genetic material. This cellular diversity is controlled by the genome and accomplished by tight regulation of gene expression. This leads to the synthesis and accumulation of different complements of RNA and, ultimately, to the proteins found in different cell types. For example, muscle and bone express different genes or the same genes at different times. Moreover, the choice of which genes are expressed in a given cell at a given time depends on signals received from its environment. There are multiple levels at which gene expression can be controlled along the pathway from DNA to RNA to protein (see Fig. 15-4). Transcriptional control refers to the mechanism for regulating when and how often a gene is transcribed. Splicing of the primary RNA transcript (RNA processing control) and selection of completed mRNAs for nuclear export (RNA transport control) represent additional potential regulatory steps. The mRNAs in the cytoplasm can be selectively translated by ribosomes (translational control) or selectively stabilized or degraded (mRNA degradation control). Finally, the resulting proteins can undergo selective activation, inactivation, or compartmentalization (protein activity control).

Because a large number of genes are regulated at the transcriptional level, regulation of gene transcripts (i.e., mRNA) often is referred to as gene regulation in a narrow definition. Each of the steps during transcription is properly regulated in eukaryotic cells. Because genes are differentially regulated from one another, one gene can be differentially regulated in different cell types or at different developmental stages. Therefore, gene regulation at the level of transcription is largely context dependent. However, there is a common scheme that applies to transcription at the molecular level (Fig. 15-6). Each gene promoter possesses unique sequences called TATA boxes that can be recognized and bound by a large complex containing RNA polymerase II, forming the basal transcription machinery. Usually located upstream of the TATA box (but sometimes longer distances) are a number of regulatory sequences referred to as enhancers that are recognized by regulatory proteins called transcription factors. These transcription factors specifically bind to the enhancers, often in response to environmental or developmental cues, and cooperate with each other and with basal transcription factors to initiate transcription. Regulatory sequences that negatively regulate the initiation of transcription also are present on the promoter DNA. The transcription factors that bind to these sites are called repressors, in contrast to the activators that activate transcription. The molecular interactions between transcription factors and promoter DNA, as well as between the cooperative transcription factors, are highly regulated and context-dependent. Specifically, the recruitment of transcription factors to the promoter DNA occurs in response to physiologic signals. A number of structural motifs in these DNA-binding transcription factors facilitate this recognition and interaction. These include the helix-turn-helix, the homeodomain motif, the zinc finger, the leucine zipper, and the helix-loop-helix motifs.

Human Genome

Genome is a collective term for all genes present in one organism. The human genome contains DNA sequences of 3 billion base pairs, carried by 23 pairs of chromosomes. The human genome has an estimated 25,000 to 30,000 genes, and overall, it is 99.9% identical in all people.^7,8 Approximately 3 million locations where single-base DNA differences exist have been identified and termed single nucleotide polymorphisms. Single nucleotide polymorphisms may be critical determinants of human variation in disease susceptibility and responses to environmental factors.

The completion of the human genome sequence in 2003 represented another great milestone in modern science. The Human Genome Project created the field of genomics, which is the study of genetic material in detail (see Fig. 15-1). The medical field is building on the knowledge, resources, and technologies emanating from the human genome to further the understanding of the relationship of the genes and their mutations to human health and disease. This expansion of genomics into human health applications resulted in the field of genomic medicine.

The emergence of genomics as a science will transform the practice of medicine and surgery in this century. This breakthrough has allowed scientists the opportunity to gain remarkable insights into the lives of humans. Ultimately, the goal is to use this information to develop new ways to treat, cure, or even prevent the thousands of diseases that afflict humankind. In the twenty-first century, work will begin to incorporate the information embedded in the human genome sequence into surgical practices. By doing so, the genomic information can be used for diagnosing and predicting disease and disease susceptibility. Diagnostic tests can be designed to detect errant genes in patients suspected of having particular diseases or of being at risk for developing them. Furthermore, exploration into the function of each human gene is now possible, which will shed light on how faulty genes play a role in disease causation. This knowledge also makes possible the development of a new generation of therapeutics based on genes. Drug design is being revolutionized as researchers create new classes of medicines based on a reasoned approach to the use of information on gene sequence and protein structure function rather than the traditional trial-and-error method. Drugs targeted to specific sites in the body promise to have fewer side effects than many of today’s medicines. Finally, other applications of genomics will involve the transfer of genes to replace defective versions or the use of gene therapy to enhance normal functions such as immunity.

Proteomics refers to the study of the structure and expression of proteins as well as the interactions among proteins encoded by a human genome (see Fig. 15-1).⁹ A number of Internet-based repositories for protein sequences exist, including Swiss-Prot (http://www.expasy.ch). These databases allow comparisons of newly identified proteins with previously characterized sequences to allow prediction of similarities, identification of splice variants, and prediction of membrane topology and posttranslational modifications. Tools for proteomic profiling include two-dimensional gel electrophoresis, time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization, and protein microarrays. Structural proteomics aims to describe the three-dimensional structure of proteins that is critical to understanding function. Functional genomics seeks to assign a biochemical, physiologic, cell biologic, and/or developmental function to each predicted gene. An ever-increasing arsenal of approaches, including transgenic animals, RNA interference (RNAi), and various systematic mutational strategies, will allow dissection of functions associated with newly discovered genes. Although the potential of this field of study is vast, it is in its early stages.

It is anticipated that a genomic and proteomic approach to human disease will lead to a new understanding of pathogenesis that will aid in the development of effective strategies for early diagnosis and treatment.¹⁰ For example, identification of altered protein expression in organs, cells, subcellular structures, or protein complexes may lead to development of new biomarkers for disease detection. Moreover, improved understanding of how protein structure determines function will allow rational identification of therapeutic targets, and thereby not only accelerate drug development, but also lead to new strategies to evaluate therapeutic efficacy and potential toxicity.⁹

Cell Cycle and Apoptosis

Every organism is composed of many different cell types at different developmental stages. Some cell types continue to grow, while some cells stop growing after a developmental stage or resume growth after a break. For example, embryonic stem cells grow continuously, while nerve cells and striated muscle cells stop dividing after maturation. Cell cycle is the process for every cell including DNA replication and protein synthesis, DNA segregation in half, and package DNA and protein in two newly formed cells to enable passage of identical genetic information from one parental cell to two daughter cells. Thus, the cell cycle is the fundamental mechanism to maintain tissue homeostasis. A cell cycle comprises four periods: G₁ (first gap phase before DNA synthesis), S (synthesis phase when DNA replication occurs), G₂ (the gap phase before mitosis), and M (mitosis, the phase when two daughter cells with identical DNA are generated) (Fig. 15-7). After a full cycle, the daughter cells enter G₁ again, and when they receive appropriate signals, undergo another cycle, and so on. The machinery that drives cell cycle progression is made up of a group of enzymes called cyclin-dependent kinases (CDKs). Cyclin expression fluctuates during the cell cycle, and cyclins are essential for CDK activities and form complexes with CDK. The cyclin A/CDK1 and cyclin B/CDK1 drive the progression for the M phase, while cyclin A/CDK2 is the primary S phase complex. Early G₁ cyclin D/CDK4/6 or late G₁ cyclin E/CDK2 controls the G₁-S transition. There also are negative regulators for CDK termed CDK inhibitors, which inhibit the assembly or activity of the cyclin-CDK complex. Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.

The cell cycle is connected with signal transduction pathways as well as gene expression. Although the S and M phases rarely are subjected to changes imposed by extracellular signals, the G₁ and G₂ phases are the primary periods when cells decide whether or not to move on to the next phase. During the G₁ phase, cells receive green- or red-light signals, S phase entry or G₁ arrest, respectively. Growing cells proliferate only when supplied with appropriate mitogenic growth factors. Cells become committed to entry of the cell cycle only toward the end of G₁. Mitogenic signals stimulate the activity of early G₁ CDKs (e.g., cyclin D/CDK4) that inhibit the activity of pRb protein and activate the transcription factor called E2F to induce the expression of batteries of genes essential for G₁-S progression. Meanwhile, cells also receive antiproliferative signals such as those from tumor suppressors. These antiproliferative signals also act in the G₁ phase to stop cells’ progress into the S phase by inducing CKI production. For example, when DNA is damaged, cells will repair the damage before entering the S phase. Therefore, G₁ contains one of the most important checkpoints for cell cycle progression. If the analogy is made that CDK is to a cell as an engine is to a car, then cyclins and CKI are the gas pedal and brake, respectively. Accelerated proliferation or improper cell cycle progression with damaged DNA would be disastrous. Genetic gain-of-function mutations in oncogenes (that often promote expression or activity of the cyclin/CDK complex) or loss-of-function mutations in tumor suppressor (that stimulate production of CKI) are causal factors for malignant transformation.

In addition to cell cycle control, cells use genetically programmed mechanisms to kill cells. This cellular process, called apoptosis or programmed cell death, is essential for the maintenance of tissue homeostasis (Fig. 15-8).

Normal tissues undergo proper apoptosis to remove unwanted cells, those that have completed their jobs or have been damaged or improperly proliferated. Apoptosis can be activated by many physiologic stimuli such as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth factor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing apoptosis: the death receptor and mitochondrial. However, recent advances in apoptosis research suggest an interconnection of the two pathways. What is central to the apoptotic machinery is the activation of a cascade of proteinases called caspases. Similarly to CDK in the cell cycle, activities and expression of caspases are well controlled by positive and negative regulators. The complex machinery of apoptosis must be tightly controlled. Perturbations of this process can cause neoplastic transformation or other diseases.

Signal Transduction Pathways

Gene expression in a genome is controlled in a temporal and spatial manner, at least in part by signaling pathways.¹¹ A signaling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors, ends up in the nucleus (Fig. 15-9). All cells have the ability to sense changes in their external environment. The bioactive substances to which cells can respond are many and include proteins, short peptides, amino acids, nucleotides/nucleosides, steroids, retinoids, fatty acids, and dissolved gases. Some of these substances are lipophilic and thereby can cross the plasma membrane by diffusion to bind to a specific target protein within the cytoplasm (intracellular receptor). Other substances bind directly with a transmembrane protein (cell-surface receptor). Binding of ligand to receptor initiates a series of biochemical reactions (signal transduction) typically involving protein-protein interactions and the transfer of high-energy phosphate groups, leading to various cellular end responses.

Control and specificity through simple protein-protein interactions—referred to as adhesive interactions—is a common feature of signal transduction pathways in cells.¹² Signaling also involves catalytic activities of signaling molecules, such as protein kinases/phosphatases, that modify the structures of key signaling proteins. Upon binding and/or modification by upstream signaling molecules, downstream effectors undergo a conformational (allosteric) change and, consequently, a change in function. The signal that originates at the cell surface and is relayed by the cytoplasmic proteins often ultimately reaches the transcriptional apparatus in the nucleus. It alters the DNA binding and activities of transcription factors that directly turn genes on or off in response to the stimuli. Abnormal alterations in signaling activities and capacities in otherwise normal cells can lead to diseases such as cancer.

Advances in biology in the last two decades have dramatically expanded the view on how cells are wired with signaling pathways. In a given cell, many signaling pathways operate simultaneously and crosstalk with one another. A cell generally may react to a hormonal signal in a variety of ways: (a) by changing its metabolite or protein, (b) by generating an electric current, or (c) by contracting. Cells continually are subject to multiple input signals that simultaneously and sequentially activate multiple receptor- and non–receptor-mediated signal transduction pathways, which form a signaling network. Although the regulators responsible for cell behavior are rapidly identified as a result of genomic and proteomic techniques, the specific functions of the individual proteins, how they assemble, and the networks that control cellular behavior remain to be defined. An increased understanding of cell regulatory pathways—and how they are disrupted in disease—will likely reveal common themes based on protein interaction domains that direct associations of proteins with other polypeptides, phospholipids, nucleic acids, and other regulatory molecules. Advances in the understanding of signaling networks will require methods of investigation that move beyond traditional “linear” approaches into medical informatics and computational biology. The bewildering biocomplexity of such networks mandates multidisciplinary and transdisciplinary research collaboration. The vast amount of information that is rapidly emerging from genomic and proteomic data mining will require the development of new modeling methodologies within the emerging disciplines of medical mathematics and physics.

Signaling pathways often are grouped according to the properties of signaling receptors. Many hydrophobic signaling molecules are able to diffuse across plasma membranes and directly reach specific cytoplasmic targets. Steroid hormones, thyroid hormones, retinoids, and vitamin D are examples that exert their activity upon binding to structurally related receptor proteins that are members of the nuclear hormone receptor superfamily. Ligand binding induces a conformational change that enhances transcriptional activity of these receptors. Most extracellular signaling molecules interact with transmembrane protein receptors that couple ligand binding to intracellular signals, leading to biologic actions.

There are three major classes of cell-surface receptors: transmitter-gated ion channels, seven-transmembrane G-protein–­coupled receptors (GPCRs), and enzyme-linked receptors. The superfamily of GPCRs is one of the largest families of proteins, representing over 800 genes of the human genome. Members of this superfamily share a characteristic seven-transmembrane configuration. The ligands for these receptors are diverse and include hormones, chemokines, neurotransmitters, proteinases, inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs signal through heterotrimeric G proteins, which are guanine-nucleotide regulatory complexes. Thus the receptor serves as the receiver, the G protein serves as the transducer, and the enzyme serves as the effector arm. Enzyme-linked receptors possess an extracellular ligand-recognition domain and a cytosolic domain that either has intrinsic enzymatic activity or directly links with an enzyme. Structurally, these receptors usually have only one transmembrane-spanning domain. Of at least five forms of enzyme-linked receptors classified by the nature of the enzyme activity to which they are coupled, the growth factor receptors such as tyrosine kinase receptor or serine/threonine kinase receptors mediate diverse cellular events including cell growth, differentiation, metabolism, and survival/apoptosis. Dysregulation (particularly mutations) of these receptors is thought to underlie conditions of abnormal cellular proliferation in the context of cancer. The following sections will further review two examples of growth factor signaling pathways and their connection with human diseases.

Insulin Pathway and Diabetes

The discovery of insulin in the early 1920s is one of the most dramatic events in the treatment of human disease.¹³ Insulin is a peptide hormone that is secreted by the β-cell of the pancreas. Insulin is required for the growth and metabolism of most mammalian cells, which contain cell-surface insulin receptors (InsR). Insulin binding to InsR activates the kinase activity of InsR. InsR then adds phosphoryl groups, a process referred to as phosphorylation, and subsequently activates its immediate intracellular effector, called insulin receptor substrate (IRS). IRS plays a central role in coordinating the signaling of insulin by activating distinct signaling pathways, the PI3K-Akt pathway and MAPK pathway, both of which possess multiple protein kinases that can control transcription, protein synthesis, and glycolysis (Fig. 15-10).

The primary physiologic role of insulin is in glucose homeostasis, which is accomplished through the stimulation of glucose uptake into insulin-sensitive tissues such as fat and skeletal muscle. Defects in insulin synthesis/secretion and/or responsiveness are major causal factors in diabetes, one of the leading causes of death and disability in the United States, affecting an estimated 16 million Americans. Type 2 diabetes accounts for about 90% of all cases of diabetes. Clustering of type 2 diabetes in certain families and ethnic populations points to a strong genetic background for the disease. More than 90% of affected individuals have insulin resistance, which develops when the body is no longer able to respond correctly to insulin circulating in the blood. Although relatively little is known about the biochemical basis of this metabolic disorder, it is clear that the insulin-signaling pathways malfunction in this disease. It is also known that genetic mutations in the InsR or IRS cause type 2 diabetes, although which one is not certain. The ­majority of type 2 diabetes cases may result from defects in downstream-signaling components in the insulin-signaling pathway. Type 2 diabetes also is associated with declining β-cell function, resulting in reduced insulin secretion; these pathways are under intense study. A full understanding of the basis of insulin resistance is crucial for the development of new therapies for type 2 diabetes. Furthermore, apart from type 2 diabetes, insulin resistance is a central feature of several other common human disorders, including atherosclerosis and coronary artery disease, hypertension, and obesity.

Transforming Growth Factor-β (TGF-β) Pathway and ­Cancers

Growth factor signaling controls cell growth, differentiation, and apoptosis.¹⁴ Although insulin and many mitogenic growth factors promote cell proliferation, some growth factors and hormones inhibit cell proliferation. TGF-β is one of them. The balance between mitogens and TGF-β plays an important role in controlling the proper pace of cell cycle progression. The growth inhibition function of TGF-β signaling in epithelial cells plays a major role in maintaining tissue homeostasis.

The TGF-β superfamily comprises a large number of structurally related growth and differentiation factors that act through a receptor complex at the cell surface (Fig. 15-11). The complex consists of transmembrane serine/threonine kinases. The receptor signals through activation of heterotrimeric complexes of intracellular effectors called SMADs (which are contracted from homologous Caenorhabditis elegans Sma and Drosophila Mad, two evolutionarily conserved genes for TGF-β signaling). Upon phosphorylation by the receptors, SMAD complexes translocate into the nucleus, where they bind to gene promoters and cooperate with specific transcription factors to regulate the expression of genes that control cell proliferation and differentiation. For example, TGF-β strongly induces the transcription of a gene called p15^INK4B (a type of CKI) and, at the same time, reduces the expression of many oncogenes such as c-Myc. The outcome of the altered gene expression leads to the inhibition of cell cycle progression. Meanwhile, the strength and duration of TGF-β signaling is fine-tuned by a variety of positive or negative modulators, including protein phosphatases. Therefore, controlled activation of TGF-β signaling is an intrinsic mechanism for cells to ensure controlled proliferation.

Resistance to TGF-β’s anticancer action is one hallmark of human cancer cells. TGF-β receptors and SMADs are identified as tumor suppressors. The TGF-β signaling circuit can be disrupted in a variety of ways and in different types of human tumors. Some lose TGF-β responsiveness through downregulation or mutations of their TGF-β receptors. The cytoplasmic SMAD4 protein, which transduces signals from ligand-activated TGF-β receptors to downstream targets, may be eliminated through mutation of its encoding gene. The locus encoding cell cycle inhibitor p15^INK4B may be deleted. Alternatively, the immediate downstream target of its actions, cyclin-dependent kinase 4 (CDK4), may become unresponsive to the inhibitory actions of p15^INK4B because of mutations that block p15^INK4B binding. The resulting cyclin D/CDK4 complexes constitutively inactivate tumor suppressor pRb by hyperphosphorylation. Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. For example, in pancreatic and colorectal cancers, 100% of cells derived from these cancers carry genetic defects in the TGF-β signaling pathway. Therefore, the antiproliferative pathway converging onto pRb and the cell division cycle is, in one way or another, disrupted in a majority of human cancer cells. Besides cancer, dysregulation of TGF-β signaling also has been associated with other human diseases such as Marfan’s syndrome and thoracic aortic aneurysm.

Gene Therapy and Molecular Drugs in Cancer

Modern advances in the use of molecular biology to manipulate genomes have greatly contributed to the understanding of the molecular basis for how cells live, die, or differentiate. Given the fact that human diseases arise from improper changes in the genome, the continuous understanding of how the genome functions will make it possible to tailor medicine on an individual basis. Although significant hurdles remain, the course toward therapeutic application of molecular biology already has been mapped out by many proof-of-principle studies in the literature. In this section, cancer is used as an example to elaborate some therapeutic applications of molecular biology. Modern molecular medicine includes gene therapy and molecular drugs that target genes or gene products that wire human cells.

Cancer is a complex disease, involving uncontrolled growth and spread of tumor cells (Fig. 15-12). Cancer development depends on the acquisition and selection of specific characteristics that set the tumor cell apart from normal somatic cells. Cancer cells have defects in regulatory circuits that govern normal cell proliferation and homeostasis. Many lines of evidence indicate that tumorigenesis in humans is a multistep process and that these steps reflect genetic alterations that drive the progressive transformation of normal human cells into highly malignant derivatives. The genomes of tumor cells are invariably altered at multiple sites, having suffered disruption through lesions as subtle as point mutations and as obvious as changes in chromosome complement. A succession of genetic changes, each conferring one or another type of growth advantage, leads to the progressive conversion of normal human cells into cancer cells.

Cancer research in the past 20 years has generated a rich and complex body of knowledge, revealing cancer to be a disease involving dynamic changes in the genome. The causes of cancer include genetic predisposition, environmental influences, infectious agents, and aging. These transform normal cells into cancerous ones by derailing a wide spectrum of regulatory pathways including signal transduction pathways, cell cycle machinery, or apoptotic pathways.^15,16 The early notion that cancer was caused by mutations in genes critical for the control of cell growth implied that genome stability is important for preventing oncogenesis. There are two classes of cancer genes in which alteration has been identified in human and animal cancer cells: oncogenes, with dominant gain-of-function ­mutations, and tumor suppressor genes, with recessive loss-­of-function mutations. In normal cells, oncogenes promote cell growth by activating cell cycle progression, whereas tumor suppressors counteract oncogenes’ functions. Therefore, the balance between oncogenes and tumor suppressors maintains a well-controlled state of cell growth.

During the development of most types of human cancer, cancer cells can break away from primary tumor masses, invade adjacent tissues, and hence travel to distant sites where they form new colonies. This spreading process of tumor cells, called metastasis, is the cause of 90% of human cancer deaths. Metastatic cancer cells that enter the bloodstream can reach virtually all tissues of the body. Bones are one of the most common places for these cells to settle and start growing again. Bone metastasis is one of the most frequent causes of pain in people with cancer. It also can cause bones to break and create other symptoms and problems for patients.

The progression in the knowledge of cancer biology has been accelerating in recent years. All of the scientific knowledge acquired through hard work and discovery has made it possible for cancer treatment and prevention. As a result of explosive new discoveries, some modern treatments were developed. The success of these therapies, together with traditional treatments such as surgical procedures, is further underscored by the fact that in 2002 the cancer rate was reduced in the United States. Current approaches to the treatment of cancer involve killing cancer cells with toxic chemicals, radiation, or surgery. Alternatively, several new biologic- and gene-based therapies are aimed at enhancing the body’s natural defenses against invading cancers. Understanding the biology of cancer cells has led to the development of designer therapies for cancer prevention and treatment. Gene therapy, immune system modulation, genetically engineered antibodies, and molecularly designed chemical drugs are all promising fronts in the war against cancer.

Immunotherapy

The growth of the body is controlled by many natural signals through complex signaling pathways. Some of these natural agents have been used in cancer treatment and have been proven effective for fighting several cancers through the clinical trial process. These naturally occurring biologic agents, such as interferons, interleukins, and other cytokines, can now be produced in the laboratory. These agents, as well as the synthetic agents that mimic the natural signals, are given to patients to influence the natural immune response agents either by directly altering the cancer cell growth or by acting indirectly to help healthy cells control the cancer. One of the most exciting applications of immunotherapy has come from the identification of certain tumor targets called antigens and the aiming of an antibody at these targets. This was first used as a means of localizing tumors in the body for diagnosis and was more recently used to attack cancer cells. Trastuzumab (Herceptin) is an example of such a drug.¹⁷ Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth factor receptor HER-2, which is overexpressed in approximately 25% of breast cancers. HER-2–overexpressing tumors tend to grow faster and generally are more likely to recur than tumors that do not overproduce HER-2. Trastuzumab is designed to attack cancer cells that overexpress HER-2 by slowing or preventing the growth of these cells, resulting in increased survival of HER-2–positive breast cancer patients. Another significant example is the administration of interleukin-2 (IL-2) to patients with metastatic melanoma or kidney cancer, which has been shown to mediate the durable regression of metastatic cancer. IL-2, a cytokine produced by human helper T lymphocytes, has a wide range of immune regulatory effects, including the expansion of lymphocytes following activation by a specific antigen. Although IL-2 has no direct impact on cancer cells, the impact of IL-2 on cancers in vivo derives from its ability to expand lymphocytes with antitumor activity. The expanded lymphocyte pool enables recognition of the antigen on cancer cells. Thus, the molecular identification of cancer antigens has opened new possibilities for the development of effective immunotherapies for patients with cancer. Clinical studies using immunization with peptides derived from cancer antigens have shown that high levels of lymphocytes with antitumor activity can be produced in cancer-bearing patients. Highly avid antitumor lymphocytes can be isolated from immunized patients and grown in vitro for use in cell-transfer therapies.

Chemotherapy

The primary function of anticancer chemicals is to block different steps involved in cell growth and replication. These chemicals often block a critical chemical reaction in a signal transduction pathway or during DNA replication or gene expression. For example, STI571, also known as Gleevec, is one of the first molecularly targeted drugs based on the changes that cancer causes in cells.¹⁸ STI571 offers promise for the treatment of chronic myeloid leukemia (CML) and may soon surpass interferon-γ as the standard treatment for the disease. In CML, STI571 is targeted at the Bcr-Abl kinase, an activated oncogene product in CML (Fig. 15-13). Bcr-Abl is an overly activated ­protein kinase resulting from a specific genetic abnormality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibition of Bcr-Abl kinase activity not only prevents cell growth of Bcr-Abl–­transformed leukemic cells, but also induces apoptosis. Clinically, the drug quickly corrects the blood cell abnormalities caused by the leukemia in a majority of patients, achieving a complete disappearance of the leukemic blood cells and the return of normal blood cells. ­Additionally, the drug appears to have some effect on other ­cancers including certain brain tumors and gastrointestinal (GI) stromal tumors, a very rare type of stomach cancer.

Gene Therapy

Gene therapy is an experimental treatment that involves genetically altering a patient’s own tumor cells or lymphocytes (cells of the immune system, some of which can attack cancer cells). For years, the concept of gene therapy has held promise as a new, potentially potent weapon to attack cancer. Although a rapid progression in the understanding of the molecular and clinical aspects of gene therapy has been witnessed in the past decade, gene therapy treatment has not yet been shown to be superior to standard treatments in humans.

Several problems must be resolved to transform it into a clinically relevant form of therapy. The major issues that limit its translation to the clinic are improving the selectivity of tumor targeting, improving the delivery to the tumor, and the enhancement of the transduction rate of the cells of interest. In most gene therapy trials for malignant diseases, tumors can be accessed and directly injected (in situ gene therapy). The in situ gene therapy also offers a better distribution of the vector virus throughout the tumor. Finally, a combination of gene therapy strategies will be more effective than the use of a single gene therapy system. An important aspect of effective gene therapy involves the choice of appropriate genes for manipulation. Genes that promote the production of messenger chemicals or other immune-active substances can be transferred into the patient’s cells. These include genes that inhibit cell cycle progression, induce apoptosis, enhance host immunity against cancer cells, block the ability of cancer cells to metastasize, and cause tumor cells to undergo suicide. Recent development of RNAi technology, which uses a loss-of-function approach to block gene functions, ensures a new wave of hopes for gene therapy. Nonetheless, gene therapy is still experimental and is being studied in clinical trials for many different types of cancer. The mapping of genes responsible for human cancer is likely to provide new targets for gene therapy in the future. The preliminary results of gene therapy for cancer are encouraging, and as advancements are made in the understanding of the molecular biology of human cancer, the future of this rapidly developing field holds great potential for treating cancer.

It is noteworthy that the use of multiple therapeutic methods has proven more powerful than a single method. The use of chemotherapy after surgery to destroy the few remaining cancerous cells in the body is called adjuvant therapy. Adjuvant therapy was first tested and found to be effective in breast cancer. It was later adopted for use in other cancers. A major discovery in chemotherapy is the advantage of multiple chemotherapeutic agents (known as combination or cocktail chemotherapy) over single agents. Some types of fast-growing leukemias and lymphomas (tumors involving the cells of the bone marrow and lymph nodes) responded extremely well to combination chemotherapy, and clinical trials led to gradual improvement of the drug combinations used. Many of these tumors can be cured today by combination chemotherapy. As cancer cells carry multiple genetic defects, the use of combination chemotherapy, immunotherapy, and gene therapies may be more effective in treating cancers.

Stem Cell Research

Stem cell biology represents a cutting-edge scientific research field with potential clinical applications.¹⁹ It may have an enormous impact on human health by offering hope for curing human diseases such as diabetes mellitus, Parkinson’s disease, neurologic degeneration, and congenital heart disease. Stem cells are endowed with two remarkable properties (Fig. 15-14). First, stem cells can proliferate in an undifferentiated but pluripotent state and, as a result, can self-renew. Second, they have the ability to differentiate into many specialized cell types. There are two groups of stem cells: embryonic stem (ES) cells and adult stem cells. Human ES cells are derived from early preimplantation embryos called blastocysts (5 days postfertilization) and are capable of generating all differentiated germ layers in the body—ectoderm, mesoderm, and endoderm—and therefore are considered pluripotent. Adult stem cells are present in and can be isolated from adult tissues. They often are tissue specific and only can generate the cell types comprising a particular tissue in the body; therefore, they are considered multipotent. However, in some cases, they can transdifferentiate into cell types found in other tissues, called transdifferentiation. For example, hematopoietic stem cells are adult stem cells. They reside in bone marrow and are capable of generating all cell types of the blood and immune system.

Stem cells can be grown in culture and be induced to differentiate into a particular cell type, either in vitro or in vivo. With the recent and continually increasing improvement in culturing stem cells, scientists are beginning to understand the molecular mechanisms of stem cell self-renewal and differentiation in response to environmental cues. It is believed that discovery of the signals that control self-renewal vs. differentiation will be extremely important for the therapeutic use of stem cells in treating disease. It is possible that success in the study of the changes in signal transduction pathways in stem cells will lead to the development of therapies to replace diseased or damaged cells in the body using stem cell derivatives. Recently, stem cell research has been transformed by the discovery from the Shinya Yamanaka group and the James Thomsen group, who have found that a simple genetic manipulation can reprogram adult differentiated cells back into pluripotent stem cells.^20,21 This exciting discovery not only bypasses the ethical issues of using early embryos to generate ES cells, but also ensures a potentially limitless source of patient-specific stem cells for tissue engineering and regenerative medicine.

The Atomic Theory of Disease

The staggering advances in anatomy, physiology, and molecular biology over the past centuries have led us to our current state in which the atom is now the anatomy of the twenty-first century.²² As 99% of the body is composed of six elements ­(oxygen, carbon, hydrogen, nitrogen, calcium, and phosphorus), the next great advance in medicine will be bridging the subatomic, molecular, and genomic levels by forming an atomic theory of disease, which states that alterations in the composition of ­subatomic particles are the root cause of disease. The atomic theory of disease would include genetic alterations at the atomic/subatomic level that are akin to single nucleotide polymorphisms (SNPs), in which alleles for a gene differ on the exact nucleotide in a single location, which can change the ultimate protein structure. This can lead to subtle changes in function or dramatic results that cause pathology. We hypothesize that on a subatomic level, there could potentially be polymorphisms as well, in which there are subtle changes in the sea of subatomic particles. Isotopes, discovered 100 years ago, would fall into this category of subatomic polymorphism, as they differ in the number of neutrons present in the atom. Differences in other particles may not change the mass of the atom, but may alter some of the characteristics of the atom.

A known example of a change in the subatomic milieu of an element leading to a disease process is that of methemoglobinemia, a disorder characterized by an overabundance of methemoglobin. Methemoglobin contains an oxidized form of iron (carrying an extra electron), as opposed to the reduced form in normal hemoglobin. This results in a shift in the oxygen-hemoglobin dissociation curve to the left, causing hypoxia. Methemoglobinemia can be congenital, due to a defect in an enzyme that normally reduces methemoglobin back to hemoglobin, or acquired, caused by breakdown products of drugs that can oxidize hemoglobin. Although there is less than 1% of methemoglobin normally present in human tissues, affecting local blood flow and inflammation through its effects on nitric oxide and heme, large quantities can lead to respiratory failure and death.

DNA Cloning

Since the advent of recombinant DNA technology three decades ago, hundreds of thousands of genes have been identified. Recombinant DNA technology is the technology that uses advanced enzymatic and microbiologic techniques to manipulate DNA.²³ Pure pieces of any DNA can be inserted into bacteriophage DNA or other carrier DNA such as plasmids to produce recombinant DNA in bacteria. In this way, DNA can be reconstructed, amplified, and used to manipulate the functions of individual cells or even organisms. This technology, often referred to as DNA cloning, is the basis of all other DNA analysis methods. It is only with the awesome power of recombinant DNA technology that the completion of the Human Genome Project was possible. It also has led to the identification of the entire gene complements of organisms such as viruses, bacteria, worms, flies, and plants.

Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is delivered into bacterial or mammalian cells or tissues^24,25 (Fig. 15-15). This represents a very basic technique that is widely used in almost all areas of biomedical research. DNA vectors often are called plasmids, which are extrachromosomal molecules of DNA that vary in size and can replicate and be transmitted from bacterial cell to cell. Plasmids can be propagated either in the cytoplasm or after insertion, as part of the bacterial chromosome in Escherichia coli. The process of molecular cloning involves several steps of manipulation of DNA. First, the vector plasmid DNA is cleaved with a restriction enzyme to create compatible ends with the foreign DNA fragment to be cloned. The vector and the DNA fragment are then joined in vitro by a DNA ligase. Alternatively, DNA cloning can be simply done through the so-called Gateway Technology that allows for the rapid and efficient transfer of DNA fragments between different cloning vectors while maintaining reading frame and orientation, without the use of restriction endonucleases and DNA ligase. The technology, which is based on the site-specific recombination system of bacteriophage l, is simple, fast, robust, and automatable and thus compatible for high-throughput DNA cloning.

Finally, the ligation product or the Gateway reaction product is introduced into competent host bacteria; this procedure is called transformation, which can be done by either calcium/heat shock or electroporation. Precautions must be taken in every step of cloning to generate the desired DNA construct. The vector must be correctly prepared to maximize the creation of recombinants; for example, it must be enzymatically treated to prevent self-ligation. Host bacteria must be made sufficiently competent to permit the entry of recombinant plasmids into cells. The selection of desired recombinant plasmid-bearing E. coli normally is achieved by the property of drug resistance conferred by the plasmid vectors. The plasmids encoding markers provide specific resistance to (i.e., the ability to grow in the presence of) antibiotics such as ampicillin, kanamycin, and tetracycline. The foreign component in the plasmid vector can be a mammalian expression cassette, which can direct expression of foreign genes in mammalian cells. The resulting plasmid vector can be amplified in E. coli to prepare large quantities of DNA for its subsequent applications such as transfection, gene therapy, transgenics, and knockout mice.

Detection of Nucleic Acids and Proteins

Southern Blot Hybridization

Southern blotting refers to the technique of transferring DNA fragments from an electrophoresis gel to a membrane support and the subsequent analysis of the fragments by hybridization with a radioactively or chemiluminescently labeled probe (Fig. 15-16).²⁶ Southern blotting is named after E. M. Southern, who in 1975 first described the technique of DNA analysis. It enables reliable and efficient analysis of size-fractionated DNA fragments in an immobilized membrane support. Southern blotting is composed of several steps. It normally begins with the digestion of the DNA samples with appropriate restriction enzymes, which will discriminate wild-type and mutant DNA by size and the separation of DNA samples in an agarose gel by electrophoresis with appropriate DNA size markers, called the DNA ladder. The DNA gel is stained with a dye, usually ethidium bromide, and photographed with a ruler laid alongside the gel so that band positions can later be identified on the membrane. The DNA gel then is treated so the DNA fragments are denatured (i.e., strand separation). The DNA then is transferred onto a nitrocellulose membrane by capillary diffusion or under electricity. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a radioactively or chemiluminescently labeled probe to be identified.

The development of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the physical organization of single and multicopy sequences in complex genomes. The later application of Southern blotting hybridization to the study of restriction fragment length polymorphisms opened up new possibilities such as genetic fingerprinting and prenatal diagnosis of genetic diseases.

Northern Blot Hybridization

Northern blotting refers to the technique of size fractionation of RNA in a gel and the transferring of an RNA sample to a solid support (membrane) in such a manner that the relative positions of the RNA molecules are maintained. The resulting membrane then is hybridized with a labeled probe complementary to the mRNA of interest. Signals generated from detection of the membrane can be used to determine the size and abundance of the target RNA. In principle, Northern blot hybridization is similar to Southern blot hybridization (and hence its name), with the exception that RNA, not DNA, is on the membrane. Although reverse-transcriptase PCR has been used in many applications (described in the next section, Polymerase Chain Reaction), Northern analysis is the only method that provides information regarding mRNA size and has remained a standard method for detection and quantitation of mRNA. The process of Northern hybridization involves several steps, as does Southern hybridization, including electrophoresis of RNA samples in an agarose-formaldehyde gel, transfer to a membrane support, and hybridization to a radioactively labeled DNA probe. Data from hybridization allow quantification of steady-state mRNA levels and, at the same time, provide information related to the presence, size, and integrity of discrete mRNA species. Thus, Northern blot analysis, also termed RNA gel blot analysis, commonly is used in molecular biology studies relating to gene expression.

Polymerase Chain Reaction

PCR is an in vitro method for the polymerase-directed amplification of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA (Fig. 15-17).²⁷ One cycle of PCR reaction involves template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase. Because the primer extension products synthesized in one cycle can serve as a template in the next, the number of target DNA copies nearly doubles at each cycle. Thus, a repeated series of cycles result in the exponential accumulation of a specific fragment in which the termini are sharply defined by the 5′ ends of the primers. The introduction of the thermostable DNA polymerase (e.g., Taq polymerase) transforms the PCR into a simple and robust reaction. The reaction components (e.g., template, primers, Taq polymerase, 2′-deoxynucleoside 5′-triphosphates, and buffer) could all be assembled and the amplification reaction carried out by simply cycling the temperatures within the reaction tube. The specificity and yield in amplifying a particular DNA fragment by PCR reaction are affected by the proper setting of the reaction parameters (e.g., enzyme, primer, and Mg²⁺ concentration, as well as the temperature cycling profile). Modifying various PCR parameters to optimize the specificity of amplification yields more homogenous products, even in rare template reactions.

The emergence of the PCR technique has dramatically altered the approach to both fundamental and applied biologic problems. The capability of amplifying a specific DNA fragment from a gene or the whole genome greatly advances the study of the gene and its function. It is simple, yet robust, speedy, and most of all, flexible. As a recombinant DNA tool, it underlies almost all of molecular biology. This revolutionary technique enabled the modern methods for the isolation of genes, construction of a DNA vector, introduction of alterations into DNA, and quantitation of gene expression, making it a fundamental cornerstone of genetic and molecular analysis.

Immunoblotting and Immunoprecipitation

Analyses of proteins are primarily carried out by antibody-directed immunologic techniques. For example, Western blotting, also called immunoblotting, is performed to detect protein levels in a population of cells or tissues, whereas immunoprecipitation is used to concentrate proteins from a larger pool. Using specific antibodies, microscopic analysis called immunofluorescence and immunohistochemistry is possible for the subcellular localization and expression of proteins in cells or tissues, respectively.

Immunoblotting refers to the process of identifying a protein from a mixture of proteins (Fig. 15-18). It consists of five steps: (a) sample preparation; (b) electrophoresis (separation of a protein mixture by sodium dodecyl sulfate-polyacrylamide gel electrophoresis); (c) transfer (the electrophoretic transfer of proteins from gel onto membrane support [e.g., nitrocellulose, nylon, or polyvinylidene difluoride]); (d) staining (the subsequent immunodetection of target proteins with specific antibody); and (e) development (colorimetric, chemiluminescent, and recently fluorescent visualization of the antibody-­recognized protein). Thus, immunoblotting combines the resolution of gel electrophoresis with the specificity of immunochemical detection. Immunoblotting is a powerful tool used to determine a number of important characteristics of proteins. For example, immunoblotting analysis will determine the presence and the quantity of a protein in a given cellular condition and its relative molecular weight. Immunoblotting also can be used to determine whether posttranslational modification such as phosphorylation has occurred on a protein. Importantly, through immunoblotting analysis, a comparison of the protein levels and modification states in normal vs. diseased tissues is possible.

Immunoprecipitation, another widely used immunochemical technique, is a method that uses antibody to enrich a protein of interest and any other proteins that are associated with it (Fig. 15-19). The principle of the technique lies in the property of a strong and specific affinity between antibodies and their antigens to locate and pull down target proteins in solution. Once the antibody-antigen (target protein) complexes are formed in the solution, they are collected and purified using small agarose beads with covalently attached protein A or protein G. Both protein A and protein G specifically interact with the antibodies, thus forming a large immobilized complex of antibody-antigen bound to beads. The purified protein can then be analyzed by a number of biochemical methods. When immunoprecipitation is combined with immunoblotting, it can be used for the sensitive detection of proteins in low concentrations, which would otherwise be difficult to detect. Moreover, combined immunoprecipitation and immunoblotting analysis is very efficient in analyzing the protein-protein interactions or determining the posttranslational modifications of proteins. In addition, immunoprecipitated proteins can be used as preparative steps for assays such as intrinsic or associated enzymatic activities. The success of immunoprecipitation is influenced by two major factors: (a) the abundance of the protein in the original preparation and (b) the specificity and affinity of the antibody for this protein.

Recently, immunoprecipitation is even used to enrich modified DNA (for example, 5-methylcytosine) for bisulfite sequencing. Besides proteins of interest, specific antibodies can also be raised against specially modified DNA. Like the protein immunoprecipitation, modified DNA can be pulled down, taking advantage of the specificity and affinity of antibody to antigen.

DNA Microarray

Now that the human genome sequence is completed, the primary focus of biologists is rapidly shifting toward gaining an understanding of how genes function. One of the interesting findings about the human genome is that there are only approximately 25,000 to 30,000 protein-encoding genes. However, it is known that genes and their products function in a complicated and yet orchestrated fashion and that the surprisingly small number of genes from the genome sequence is sufficient to make a human being. Nonetheless, with the tens of thousands of genes present in the genome, traditional methods in molecular biology, which generally work on a one-gene-in-one-experiment basis, cannot generate the whole picture of genome function. In the past several years, a new technology called DNA microarray has attracted tremendous interest among biologists as well as clinicians. This technology promises to monitor the whole genome on a single chip so researchers can have a better picture of the interactions among thousands of genes simultaneously.

DNA microarray, also called gene chip, DNA chip, and gene array, refers to large sets of probes of known sequences orderly arranged on a small chip, enabling many hybridization reactions to be carried out in parallel in a small device (Fig. 15-20).²⁸ Like Southern and Northern hybridization, the underlying principle of this technology is the remarkable ability of nucleic acids to form a duplex between two strands with complementary base sequences. DNA microarray provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns. Microarrays require specialized robotics and imaging equipment that spot the samples on a glass or nylon substrate, carry out the hybridization, and analyze the data generated. DNA microarrays containing different sets of genes from a variety of organisms are now commercially available, allowing biologists to simply purchase the chips and perform hybridization and data collection. The massive scale of microarray experiments requires the aid of computers. They are used during the capturing of the image of the hybridized target, the conversion of the image into usable measures of the extent of hybridization, and the interpretation of the extent of hybridization into a meaningful measure of the amount of the complementary sequence in the target. Some data-analysis packages are available commercially or can be found in the core facility of certain institutions.

DNA microarray technology has produced many significant results in quite different areas of application. There are two major application forms for the technology: identification of sequence (gene/gene mutation) in multiple regions of a genome and determination of expression level (abundance) of large numbers of genes simultaneously. For example, analysis of genomic DNA detects amplifications and deletions found in human tumors. Differential gene expression analysis also has uncovered networks of genes differentially present in cancers that cannot be distinguished by conventional means. Significantly, recent advancements in next-generation sequencing (e.g., Solexa and 454 technology) have demonstrated the precision and speed to analyze gene expression in any genome.

Next-Generation Sequencing

The recombinant DNA technology greatly impacts the completion of the Human Genome Project due to the invention of shotgun sequencing, which includes breaking the genome DNA into small pieces and randomly cloning those pieces into DNA vectors that are easily sequenced.^29,30 Based on the overlapping sequence of each clone, computer analysis can be programmed to map and align the DNA sequence that will ultimately cover the whole human genome.

Based on shotgun sequencing, as the sequencing technologies advance, next-generation sequencing has become one of the most powerful tools to analyze DNA mutation, to identify epigenetic modification, and to profile gene expression or ncRNA expression.³¹ The next-generation sequencing process usually includes library construction, sequencing, and data analysis. Take the Illumina next-generation sequencing as an example: DNA are shared or digested into small pieces and then used to generate a DNA library with adapters on both ends of each DNA piece. Then, the DNA library is diluted and loaded on a chamber of a slide, called a lane, for cluster amplification. Cycled fluorescent deoxyribonucleotide triphosphates (dNTPs) are then added to the chamber to enable DNA polymerization, resulting in different fluorescent emission representing different dNTP reading on different clusters, into a microscope. The fluorescent signal is transformed into sequencing data that will be aligned and mapped to a standard genome database. The advantages of next-generation sequencing include the following: no necessity of DNA cloning; fast and cost-effective; and a huge amount of data to give a good depth and accuracy of the sequence.

Based on the applications, the most common next-­generation sequencing technologies for whole-genome sequencing are whole-genome DNA sequencing, whole-genome bisulfite sequencing (BS-seq), RNA sequencing (RNA-seq), and chromatin immunoprecipitation (ChIP) sequencing ­(ChIP-seq). Whole-genome DNA sequencing is purely to sequence the DNA sequence of a genome without any preprocessing of the DNA, reflecting any deletions, replications, and mutations within the genomic DNA. BS-seq is commonly used to identify DNA methylation on the genome (5-methylcytosine [5mC]). The process always involves a bisulfite treatment of DNA before library construction, during which the unmethylated cytosine will be transformed to a uracil, resulting in reading as a thymine in data output, whereas 5mC is protected and remains as cytosine in data output. Thus, 5mC and cytosine are distinguished by this way. RNA-seq is usually performed to analyze transcription for the same purpose as performing a microarray. However, RNA-seq is more accurate and provides more information such as splicing variants than traditional microarray. Usually, cDNA that is reversely transcribed from extracted RNA is used to generate libraries. Depending on the needs, mRNA and ncRNA can be enriched in different protocols for RNA extraction. ChIP-seq is always used to map the location of a DNA-binding protein in the genome. Prior to library construction, ChIP is performed to enrich DNA bound by the protein of interest (POI). First, POI and DNA are cross-linked before sonication. Then, a specific antibody is used to pull down POI and attached DNA fragments. After the protein and DNA are reverse cross-linked, DNA will be purified to make the ChIP-seq library.

By using next-generation sequencing technology, any potential mutations in a patient will be scrutinized as well as any defects in epigenetic modification, which will greatly facilitate the diagnosis of patients and personalization of medicine in a fast and economic way.

Cell Manipulations

Cell Culture

Cell culture has become one of the most powerful tools in biomedical laboratories, as cultured cells are being used in a diversity of biologic fields ranging from biochemistry to molecular and cellular biology.³² Through their ability to be maintained in vitro, cells can be manipulated by the introduction of genes of interest (cell transfection) and be transferred into in vivo biologic receivers (cell transplantation) to study the biologic effect of the interested genes (Fig. 15-21). In the common laboratory settings, cells are cultured either as a monolayer (in which cells grow as one layer on culture dishes) or in suspension.

It is important to know the wealth of information concerning cell culturing before attempting the procedure. For example, conditions of culture will depend on the cell types to be cultured (e.g., origins of the cells such as epithelial or fibroblasts, or primary vs. immortalized/transformed cells). It is also necessary to use cell type-specific culture medium that varies in combination of growth factors and serum concentrations. If primary cells are derived from human patients or animals, some commercial resources have a variety of culture media available for testing. Generally, cells are manipulated in a sterile hood, and the working surfaces are wiped with 70% to 80% ethyl alcohol solution. Cultured cells are usually maintained in a humidified carbon dioxide incubator at 37°C (98.6°F) and need to be examined daily under an inverted microscope to check for possible contamination and confluency (the area cells occupy on the dish). As a general rule, cells should be fed with fresh medium every 2 to 3 days and split when they reach confluency. Depending on the growth rate of cells, the actual time and number of plates required to split cells in two varies from cell line to cell line. Splitting a monolayer requires the detachment of cells from plates by using a trypsin or collagenase treatment, of which concentration and time period vary depending on cell lines. If cultured cells grow continuously in suspension, they are split or subcultured by dilution.

Because cell lines may change their properties when cultured, it is not possible to maintain cell lines in culture indefinitely. Therefore, it is essential to store cells at various time passages for future use. The common procedure to use is cryopreservation. The solution for cryopreservation is usually fetal calf serum containing 10% dimethyl sulfoxide or glycerol, stored in liquid nitrogen (−196°C [−320.8°F]) for years of preservation. However, the viability and health of cells when thawed will decrease over time even in liquid nitrogen.

Cell Transfection

Cells are cultured for two reasons: to maintain and to manipulate them (see Fig. 15-21). The transfer of foreign macromolecules, such as nucleic acid, into living cells provides an efficient method for studying a variety of cellular processes and functions at the molecular level. DNA transfection has become an important tool for studying the regulation and function of genes. The cDNA to be expressed should be in a plasmid vector, behind an appropriate promoter working in mammalian cells (e.g., the constitutively active cytomegalovirus promoter or inducible promoter). Depending on the cell type, many ways of introducing DNA into mammalian cells have been developed. Commonly used approaches include calcium phosphate, electroporation, liposome-mediated transfection, the nonliposomal formulation, and the use of viral vectors. These methods have shown variable success when attempting to transfect a wide variety of cells. Transfection can be performed in the presence or absence of serum. It is suggested to test the transfection efficiency of cell lines of interest by comparing transfection with several different approaches. For a detailed transfection protocol, it is best to follow the manufacturer’s instructions for the particular reagent. General considerations for a successful transfection depend on several parameters, such as the quality and quantity of DNA and cell culture (type of cell and growth phase). To minimize variations in both of these in transfection experiments, it is best to use cells that are healthy, proliferate well, and are plated at a constant density.

Depending on the transfection method, DNA expression can be transient or stable. Using calcium phosphate and liposome-mediated transfection, after DNA is introduced into the cells, it is normally maintained epitopically in cells and will be diluted while host cells undergo cell division. Therefore, functional assays should be performed 24 to 72 hours after transfection, also termed transient transfection. In many applications, it is important to study the long-term effects of DNA in cells by stable transfection. Thus, electroporation and viral vector are often used in these situations to enable integration of ectopic DNA into the host genome. Stable cell clones can be selected when plasmids carry an antibiotic-resistant marker. In the presence of antibiotics, only those cells that continuously carry the antibiotic-resistant marker (after generations of cell division) can survive. One application of stable transfection is the generation of transgenic or knockout mouse models, in which the transgene has to be integrated in the mouse genome in the ES cells, followed by microinjection of those transgenic ES cells into blastocysts to generate chimera mice. Stable cells also can be transplanted into host organs to test the effect of transgenic cells in vivo.

Genetic Manipulations

Understanding how genes control the growth and differentiation of the mammalian organism has been the most challenging topic of modern research. It is essential for us to understand how genetic mutations and chemicals lead to the pathologic condition of human bodies. The knowledge and ability to change the genetic program will inevitably make a great impact on society and have far-reaching effects on how we think of ourselves.

The mouse has become firmly established as the primary experimental model for studying how genes control mammalian development. Genetically altered mice are powerful tools to study the function and regulation of genes as well as modeling human diseases.³³ The gene function can be studied by creating mutant mice through homologous recombination (gene knockout). A gene of interest (GOI) also can be introduced into the mouse (transgenic mouse) to study its effect on development or diseases. Because mouse models do not precisely represent human biology, genetic manipulations of human somatic or ES cells provide a great means for the understanding of the molecular networks in human cells in addition to mouse models. In all cases, the gene to be manipulated must first be cloned. Gene cloning has been made easy by recombinant DNA technology and the availability of human and mouse genomes (see the Human Genome section). The following section briefly describes the technologies and the principles behind how to combine both mouse genetics and human cell culture in exploring gene function and disease mechanisms.

Transgenic Mice

During the past 20 years, DNA cloning and other techniques have allowed the introduction of new genetic material into the mouse germline. As early as 1980, the first genetic material was successfully introduced into the mouse germline by using pronuclear microinjection of DNA (Fig. 15-22). These animals, called transgenic, contain foreign DNA within their genomes. In simple terms, a transgenic mouse is created by the microinjection of ectopic DNA into the one-celled mouse embryo to induce integration, allowing the efficient introduction of cloned genes into the following developing mouse somatic tissues, as well as into the germline.

Designs of a Transgene

The transgenic technique has proven to be extremely important for basic investigations of gene regulation, creation of animal models of human disease, and genetic engineering of livestock. The design of a transgene construct is a simple task. Like constructs used in cell transfection, a simple transgene construct consists of a protein-encoding gene and a promoter that precedes it. The most common applications for the use of transgenic mice are similar to those in the cell culture system: (a) to study the functions of proteins encoded by the transgene, (b) to analyze the tissue-specific and developmental-stage–specific activity of a gene promoter, and (c) to generate reporter lines to facilitate biomedical studies. Examples of the first application include overexpression of oncogenes, growth factors, hormones, and other key regulatory genes, as well as genes of viral origins. Overexpression of the transgene normally represents gain-of-function mutations. The tissue distribution or expression of a transgene is determined primarily by cis-acting promoter enhancer elements within or in the immediate vicinity of the genes themselves. Thus, controlled expression of the transgene can be made possible by using an inducible or tissue-specific promoter. Furthermore, transgenic mice carrying dominant negative mutations of a regulatory gene also have been generated. For example, a truncated growth factor receptor that can bind to the ligand, but loses its catalytic activity when expressed in mice, can block the growth factor binding to the endogenous protein. In this way, the transgenic mice exhibit a loss of function of phenotype, possibly resembling the knockout of the endogenous gene. The second application of the transgenic expression is to analyze the gene promoter of interest. The gene promoter of interest normally is fused to a reporter gene that encodes β-galactosidase (also called LacZ), luciferase, or green fluorescence protein. Chemical staining of LacZ activity or detection of chemiluminescence/fluorescence can easily visualize the expression of the reporter gene. The third application originates from the second: when the activity of the promoter is known, a fluorescent reporter gene (such as GFP) will be driven by the tissue-specific promoter, therefore labeling a particular type of cells at a particular stage. This application is generally used to isolate a special cell type expressing the GFP reporter by fluorescence-activated cell sorting (FACS), as well as lineage-tracing experiments.

Production of Transgenic Mice

The success of generating transgenic mice is largely dependent on the proper quality and concentration of the DNA supplied for microinjection. For DNA to be microinjected into mouse embryos, it should be linearized by restriction digestion to increase the chance of proper transgene integration. Concentration of DNA should be accurately determined. Mice that develop from injected eggs often are termed founder mice.

Genotyping of Transgenic Mice

The screening of founder mice and the transgenic lines derived from the founders is accomplished by determining the integration of the injected gene into the genome. This normally is achieved by performing PCR or Southern blot analysis with a small amount of DNA extracted from the mouse tail. Once a given founder mouse is identified to be transgenic, it will be mated to begin establishing a transgenic line. Usually, for a given gene, more than one transgenic line is generated to assure that the phenotype is due to transgene but not to the interruption of the gene where the transgene integrates into.

Analysis of Phenotype of Transgenic Mice

Phenotypes of transgenic mice are dictated by both the expression pattern and biologic functions of the transgene. Depending on the promoter and the transgene, phenotypes can be predictable or unpredictable. Elucidation of the functions of the transgene-encoded protein in vitro often offers some clue to what the protein might function to do in vivo. When a constitutively active promoter is used to drive the expression of transgenes, mice should express the gene in every tissue; however, this mouse model may not allow the identification and study of the earliest events in disease pathogenesis. Ideally, the use of tissue-specific or inducible promoter allows one to determine if the pathogenic protein leads to a reversible or irreversible disease process in a cell-autonomous manner. For example, rat insulin promoter can target transgene expression exclusively in the β-cells of pancreatic islets. The phenotype of insulin promoter-mediated transgenic mice is projected to affect the function of human β-cells.

Gene Knockout in Mice

The first recorded knockout mouse was created by Mario R. Capecchi, Sir Martin J. Evans, and Oliver Smithies in 1989. They were awarded the 2007 Nobel Prize in Physiology or Medicine. The isolation and genetic manipulation of mouse ES cells represent one of the most important milestones for modern genetic technologies.³⁴ Several unique properties of ES cells, such as the pluripotency to differentiate into all germ layers in an embryo, including the germline, make them an efficient vehicle to introduce genetic alterations in mice. An important breakthrough from this idea is to generate gene-targeted mutation in mice, first by introducing the targeting vector into the ES cells, allowing selection for successful homologous recombination in a dish, then introducing the selected ES clone into the blastocysts, and finally recovering animals bearing the mutant allele from the germline (Fig. 15-23). This not only makes mouse genetics a powerful approach for addressing important gene functions, but also identifies the mouse as a great system to model human disease.

Targeting Vector

The basic concept in building a target vector to knock out a gene is to use two segments of homologous sequence to a GOI that flank a part of the gene essential for functions (e.g., the coding region). In the targeting vector, a positive selectable marker (e.g., the neo gene) is placed between the homology arms. Upon the homologous recombination between the arms of the vector and the corresponding genomic regions of the GOI in ES cells, the positive selectable marker will replace the essential segment of the target gene, thus creating a null allele. In addition, a negative selectable marker also can be used alone or in combination with the positive selectable marker, but must be placed outside of the homologous arms to enrich for homologous recombination. To create a conditional knockout (i.e., gene knockout in a spatiotemporal fashion), site-specific recombinases such as the popular cre-loxP system are used. If the consensus loxP sequences that are recognized by cre recombinases are properly designed into targeting loci, controlled expression of the recombinase as a transgene can result in the site-specific recombination at the right time and in the right place (i.e., cell type or tissue). This method, often referred as conditional knockout, is markedly useful to prevent developmental compensations and to introduce null mutations in the adult mouse that would otherwise be lethal. Overall, this cre-loxP system allows for spatial and temporal control over transgene expression and takes advantage of inducers with minimal pleiotropic effects.

Introduction of the Targeting Vector into ES Cells

ES cell lines can be obtained from other investigators or commercial sources or established from blastocyst-stage embryos. To maintain ES cells at their full developmental potential, optimal growth conditions should be provided in culture. If culture conditions are inappropriate or inadequate, ES cells may acquire genetic lesions or alter their gene expression patterns, and consequently decrease their pluripotency. Excellent protocols are available in public domains or in mouse facilities in most ­institutions.

To alter the genome of ES cells, the targeting vector DNA then is transfected into ES cells. Electroporation is the most widely used and the most efficient transfection method for ES cells. Similar procedures for stable cell transfection are used for selecting ES cells that carry the targeting vector. High-quality, targeting-vector DNA free of contaminating chemicals is first linearized and then electroporated into ES cells. Stable ES cells are selected in the presence of a positive selectable antibiotic drug. After a certain period of time and depending on the type of antibiotics, all sensitive cells die and the resistant cells grow into individual colonies of the appropriate size for subcloning by picking. It is extremely important to minimize the time during which ES cells are in culture between selection and injection into blastocysts. Before injecting the ES cells, DNA is prepared from ES colonies to screen for positive ES cells that exhibit the correct integration or homologous recombination of the targeting vector. Positive ES colonies are then expanded and used for creation of chimeras.

Creation of the Chimera

A chimeric organism is one in which cells originate from more than one embryo source. Here, chimeric mice are denoted as those that contain some tissues from the ES cells with an altered genome. When these ES cells give rise to the lineage of the germ layer, the germ cells carrying the altered genome can be passed on to the offspring, thus creating the germline transmission from ES cells. There are two methods for introducing ES cells into preimplantation-stage embryos: injection and aggregation. The injection of embryonic cells directly into the cavity of blastocysts is one of the fundamental methods for generating chimeras, but aggregation chimeras also have become an important alternative for transmitting the ES cell genome into mice. Since every tissue type of a chimera should contain cells from different origins, the mixture of recognizable markers (e.g., coat color) that are specific for each the donor mouse and ES cells can be used to identify chimeric mice. However, most experimenters probably use existing mouse core facilities already established in some institutions, or contract a commercial vendor for the creation of a chimera.

Genotyping and Phenotyping of Knockout Animals

The next step is to analyze whether germline transmission of targeted mutation occurs in mice. DNA from a small amount of tissue from offspring of the chimera is extracted and subjected to genomic PCR or Southern blot DNA hybridization. Positive mice (i.e., those with properly integrated targeting vector into the genome) will be used for the propagation of more knockout mice for phenotype analysis. When the knockout genes are crucial for early embryogenesis, mice often die in utero, an occurrence called embryonic lethality. When this happens, only the phenotype of the homozygous (both alleles ablated) knockout mouse embryos and the phenotype of the heterozygous (only one allele ablated) adult mice can be studied. Because most are interested in the phenotype of adult mice, in particular when using mice as disease models, it is recommended to create the conditional knockout using the cre-loxP system so that the GOI can be knocked out at will.

To date, more than 5000 genes have been disrupted by homologous recombination and transmitted through the germline. The phenotypic studies of these mice provide ample information on the functions of these genes in growth and differentiation of organisms and during development of human diseases.

RNA Interference

Although gene ablation in animal models provides an important means to understand the in vivo functions of GOI, animal models may not adequately represent human biology. Alternatively, gene targeting can be used to knock out genes in human cells, including human ES cells. Gene ­targeting in human ES cells by homologous recombination has been extremely low efficiency, although there are more new ­techniques emerging aimed at increasing the targeting efficiency. A number of recent advances have made gene targeting in somatic cells as easy as in murine ES cells.³³ However, gene targeting (knocking out both alleles) in somatic cells is a time-consuming process.

Development of RNAi technology in the past few years has provided a more promising approach to understanding the biologic functions of human genes in human cells.³⁵ RNAi is an ancient natural mechanism by which small, double-stranded RNA (dsRNA) acts as a guide for an enzyme complex that destroys complementary RNA and downregulates gene expression in a sequence-specific manner. Although the mechanism by which dsRNA suppresses gene expression is not entirely understood, experimental data provide important insights. In nonmammalian systems such as Drosophila, it appears that longer dsRNA is processed into 21–23 nt dsRNA (called small interfering RNA or siRNA) by an enzyme called Dicer containing RNase III motifs. The siRNA apparently then acts as a guide sequence within a multicomponent nuclease complex to target complementary mRNA for degradation. Because long dsRNA induces a potent antiviral response pathway in mammalian cells, short siRNAs are used to perform gene silencing experiments in mammalian cells (Fig. 15-24).

For siRNA studies in mammalian cells, researchers have used two 21-mer RNAs with 19 complementary nucleotides and 3′ terminal noncomplementary dimers of thymidine or uridine. The antisense siRNA strand is fully complementary to the mRNA target sequence. Target sequences for an siRNA are identified visually or by software.

The target 19 nucleotides should be compared to an appropriate genome database to eliminate any sequences with significant homology to other genes. Those sequences that appear to be specific to the GOI are the potential siRNA target sites. A few of these target sites are selected for siRNA design. The antisense siRNA strand is the reverse complement of the target sequence. The sense strand of the siRNA is the same sequence as the target mRNA sequence. A deoxythymidine dimer is routinely incorporated at the 3′ end of the sense strand siRNA, although it is unknown whether this noncomplementary dinucleotide is important for the activity of siRNAs.

There are two ways to introduce siRNA to knock down gene expression in human cells:

1. RNA transfection: siRNA can be made chemically or using an in vitro transcription method. Like DNA oligos, chemically synthesized siRNA oligos can be commercially ordered. However, synthetic siRNA is expensive, and several siRNAs may have to be tried before a particular gene is successfully silenced. In vitro transcription provides a more economic approach. Both short and long RNA can be synthesized using bacteriophage RNA polymerase T7, T3, or SP6. In the case of long dsRNAs, RNase such as recombinant Dicers will be used to process the long dsRNA into a mixture of 21–23 nt siRNA. siRNA oligos or mixtures can be transfected into a few characterized cell lines such as HeLa (human cervical carcinoma) and 293T cells (human kidney carcinoma). Transfection of siRNA directly into primary cells may be difficult.

2. DNA transfection: Expression vectors for expressing siRNA have been made using RNA polymerase III promoters such as U6 and H1. These promoters precisely transcribe a hairpin structure of dsRNA, which will be processed into siRNA in the cell (see Fig. 15-24). Therefore, properly designed DNA oligos corresponding to the desired siRNA will be inserted downstream of the U6 or H1 promoter. There are two advantages of the siRNA expression vectors over siRNA oligos. First, it is easier to transfect DNA into cells. Second, stable populations of cells can be generated that maintain the long-term silencing of target genes. Furthermore, the siRNA expression cassette can be incorporated into a retroviral or adenoviral vector to provide a wide spectrum of applications in gene therapy.

There has been a fast and fruitful development of RNAi tools for in vitro and in vivo use in mammals. These novel approaches, together with future developments, will be crucial to put RNAi technology to use for effective disease therapy or to exert the awesome power of mammalian genetics. Therefore, the applications of RNAi to human health are enormous. siRNA can be applied as a new tool for sequence-specific regulation of gene expression in functional genomics and biomedical studies. With the availability of the human genome sequences, RNAi approaches hold tremendous promise for unleashing the dormant potential of sequenced genomes.

Practical applications of RNAi will possibly result in new therapeutic interventions. In 2002, the concept of using siRNA in battling infectious diseases and carcinogenesis was proven effective. These include notable successes in blocking replication of viruses, such as HIV, hepatitis B virus, and hepatitis C virus, in cultured cells using siRNA targeted at the viral genome or the human gene encoding viral receptors. RNAi has been shown to antagonize the effects of hepatitis C virus in mouse models. In cancers, silencing of oncogenes such as c-Myc or Ras can slow down the proliferation rate of cancer cells. Finally, siRNA also has potential applications for some dominant genetic disorders.

The twenty-first century, already heralded as the “century of the gene,” carries great promise for alleviating suffering from disease and improving human health. On the whole, completion of the human genome blueprint, the promise of gene therapy and molecular therapies, and the existence of stem cells have captured the imagination of the public and the biomedical community. Aside from their potential in curing human diseases, these emerging technologies also have provoked many political, economic, religious, and ethical discussions. As more is discerned about the technologic scientific advances, more attention must also be paid to concerns for their inherent risks and social implications. It is important for surgeons to play a leadership role in the emergence of personalized medicine and surgery, as surgeons have access to the diseased tissues. Surgeons should be establishing collaborations with the genomic and molecular scientists to develop genomic biobanks in order to study the genome and molecular signaling of the disease tissues that will help with an understanding of the underlying cause of an individual’s disease and ultimately lead to effective, targeted therapies. Surgeons must take this enormous opportunity to collaborate with basic and clinical scientists to develop the field of personalized genomic medicine and surgery this century.

Bifunctional RNAi Technology

Over the last 20 years, the field has worked to define oncogene and nononcogene addiction, discriminate between driver and passenger genes, and appreciate the complexity of complex, robust, network interactions.³⁶ These insights have led to a preliminary understanding of therapeutically relevant sensitivity and resistance pathway signal patterns requiring multiple target modulation. However, this knowledge has not been effectively or reproducibly clinically translated. Clinical response is usually far greater when a combination of single-target molecular therapy is administered. However, it must also be realized that targeting two or more pathways may also increase the toxicity profile, particularly if target specificity is limited. When attempted, off-target toxicity has been demonstrated with combination small-molecule therapy. In contrast, multitargeting bifunctional short hairpin (bi-shRNA) DNA vectors are designed to limit off-target effect given the high specificity for the genes they are designed to target.

Exogenously applied hairpin constructs can be designed to be incorporated into cleavage-dependent RISC or ­cleavage-independent RISC complexes, or both. The concept of a bifunctional shRNA³⁷ is to increase knockdown efficiency without loss of sequence specificity by engaging both siRNA and miRNA-like (i.e., common biogenic pathway but complementary to target sequence) RISCs, thereby concurrently activating nucleolytic (Ago2-RISC) and nonnucleolytic (Ago1, 3, 4 ± Ago2-RISC) processes.³⁸ Each bi-shRNA contains both a matched stem sequence to promote Ago2-mediated passenger strand cleavage and a second partial mismatched stem sequence for cleavage-independent passenger strand departure. Thus, functionality of the effectors is set by programmed passenger strand guided RISC loading rather than Ago subset distribution in the cancer cell. Both component Ago2 and Ago [1, 2, 4 ± 3] RNAi moieties are fully complementary to the mRNA target sequence. Preliminary data indicate reduced “off-target effects” by shRNA compared with target-identical siRNAs. More than two mismatches in sequences within the target region drastically reduce knockdown effect to undetectable levels (unpublished results). The design process involves in silico scanning of the entire human mRNA RefSeq database to avoid any potential sequence-related “off-target effects.” Published data also indicate persistent susceptibility to shRNA-mediated gene knockdown despite recent evidence of reduced Dicer expression in human cancer cells.³⁹

The first clinical experience with the bi-shRNA platform involved the ex vivo knockdown of furin, a Ca²⁺-dependent, nonredundant proprotein convertase that is essential for proteolytic maturational processing of immunosuppressive TGF-β isoforms (β1 and β2). An autologous whole-cell cancer vaccine, FANG™ (furin-knockdown and GMCSF-augmented),⁴⁰ was produced based on a dual function immunosensitization principle of augmenting tumor antigen expression, presentation, and processing via granulocyte-macrophage colony-­stimulating factor (GMCSF) cytokine transgene expression and attenuating secretory immunosuppressive TGF-β. Harvested, autologous cancer cells are transfected with the GMCSF/bi-shRNA^furin (FANG) expression plasmid via electroporation. A phase I clinical trial (BB-IND 14205) involving 52 cancer patients was recently completed. Results demonstrated better than 90% knockdown of the bi-shRNA target, furin, and better than 90% knockdown of furin-regulated proteins TGF-β1 and TGF-β2, thereby confirming the mechanistic expectation of this novel RNAi platform. Moreover, predicted extensive GMCSF expression verified our ability to successfully construct multi-cassette vectors with good manufacturing practice techniques fulfilling Food and Drug Administration requirements for clinical testing.

Twenty-seven patients received one or more vaccine dose, and 23 patients achieved stable disease as their best response. No toxic effect was identified. Median survival of the FANG™-treated patients from time of procurement was 554 days and has not been reached from time of treatment. Expected survival of similar patients is historically less than 1 year. Sequential enzyme-linked immunosorbent spot (ELISPOT) analysis revealed a dramatic and significant increase in immune response from baseline to month 4 in half of the FANG™-treated patients. Comparison of survival between ELISPOT-positive and ELISPOT-negative patients demonstrated a statistically significant increase in survival from time of procurement (P = .045) and time of treatment (P = .025).

These phase I study results demonstrated mechanism, safety, and effectiveness of the bi-shRNA technology and clinical functionality of a multitargeting (dual) DNA expression vector. Further utilization of bi-shRNAi technology is under way clinically (targeting STMN1, a microtubule modulation critical to cancer program) and preclinically targeting PDX1⁴¹ (an oncogene-like transcription factor for pancreatic embryogenesis using nonviral nanoparticle delivery mechanisms).⁴²

Personalized Genomic Medicine and Surgery

Genes determine our susceptibility to diseases and direct our body’s response to medicine.⁴³ Because an individual’s genes differ from those of another, the determination of each individual’s genome has the potential to improve the predication, prevention, and treatment of disease. Sequencing of individual genomes holds the key to realize this revolution called personalized genomic medicine and surgery. Next-generation sequencing, such as Illumina sequencing and 454 pyrosequencing technology, is promising to reduce the time and cost so that genome sequencing can be affordable within healthcare systems. The goal of personalized genomic medicine and surgery is to identify the gene variations in each individual and to target the specific gene variations causing the disease by choosing personalized treatments that effectively work in association with the individual’s genomic profile. The importance of surgeons in this transformational field of biomedical science is that surgeons have access to the diseased tissues on a daily basis. Surgeons should partner with the genomic scientists to develop genomic biobanks in order to study the genome of the disease tissues. These discovery studies are rapidly leading to the uncovering of mutations and SNPs that are the underlying cause of an individual’s disease and ultimately lead to targeted therapies. Although personalized genomic medicine and surgery holds the potential to revolutionize the practice of modern medicine, there currently exists a gap between our ability to sequence any given individual’s genome and how clinicians can apply this information to guide care. There is a rapidly growing list of single genes that are currently guiding care, and these genes are listed as type 1 personalized genes. Examples of these genes are BRCA1, RET proto-oncogene, and CHD1 mutation, which guide potential use of mastectomy, thyroidectomy, and gastrectomy, respectively; however, the great challenge before the scientific and medical community this century is to learn to use the entire genome to guide personalized care.