Mus musculus

Although worms, flies, and now fish accommodate efficient functional genomic and disease-relevant target identification studies in the context of a multicellular entity, their resemblance to humans and thus their relevance to human disease biology may be limited, especially as pertains to neuronal function, immunological status, and other highly evolved physiological systems. And although human cells are components of higher-order systems, such as organs, and integrate environmental signals from numerous other cell types, it is still argued that cellular assays, which normally interrogate a single cell type, may not accurately recapitulate function in the context of an organism (in vivo). To perform genetic analysis in whole animal systems, biologists frequently utilize Mus musculus, the common house mouse, as a useful experimental model for understanding human gene function because it is anatomically and physiologically similar to Homo sapiens. The similarity of human and mouse genomes is approximately 85%, and more than 100 mouse genetic models mirror human diseases attributed to mutations in the same gene.136 Experimental advantages to using mice include short generation (breeding) time, relative ease of genetic manipulation, and the availability of comprehensive sequence data and genetically defined inbred strains. Three primary approaches are utilized to study gene function in rodents: targeted manipulation of the genome, random mutagenesis, and examination of naturally occurring genetic variations which result in phenotypic disparities.

3.01.2.6.1 Gain-of-function analysis: the transgenic mouse model

As in cellular systems, alterations in gene dosage levels can provide important insights into the function of a gene in the context of a whole organism. The approach is particularly useful in studying diseases such as cancer, where mutations result in the activation of genes that contribute to disease pathology. Transgenic models are established by injecting a DNA construct (usually a cDNA) into the pronuclei of fertilized mouse eggs at the single cell stage (Figure 3a). The introduced DNA randomly integrates into the mouse genome, and the eggs are transferred into a pseudopregnant female mouse for the gestational development. Approximately 10-20% of the pups born to this female will have the foreign DNA inserted into their genome, and each can be used to establish an independent transgenic strain.

Each cell of these transgenic animals will contain the introduced DNA; however, the expression of the encoded protein is dependent upon the promoter used to express the gene of interest. Constitutive ectopic expression, or expression in every cell of the organism, is achieved mainly though use of promoters derived from viruses such as simian virus 40 (SV40), herpes simplex virus (HSV), and mouse mammary tumor virus (MMTV). Direct expression of transgenes to specific tissues has been performed by incorporating promoter/enhancer elements derived from genes whose expression is known to be restricted to those compartments. For example, the CD19 protein is expressed almost exclusively in B cells, and regulatory elements from the CD19 promoter have been utilized to restrict transgenic expression of several heterologous genes strictly to B cells.138 Alternatively, transgenic constructs have been placed under the control of 'inducible' promoters, i.e., promoters that are activated in response to exogenous substances. These include the metallothionein MMTV-LTR, and OAS promoters, which respond to heavy metals, glucocorticoids, and interferon stimulation, respectively.139-141 Since the DNA construct used for transgenesis integrates randomly into the mouse genome, it is subject to regulation by neighboring chromosomal elements. This issue can make it difficult to achieve complete temporally and/or spatially restricted expression. In general, using these techniques, mouse geneticists can direct expression of a gene in a whole animal, or in a selected tissue, or at select times. Transgenic models are useful tools for biological discovery and recapitulating human disease in an experimental in vivo system. For example, the BCR-abl gene of the Philadelphia chromosome was first definitively shown to cause chronic myelogenous leukemia using a transgenic model.142 However, since this approach entails expressing genes in nonphysiological contexts and with synthetic promoters, a biologist must examine critically whether each model faithfully represents the biological function of the transgene.

3.01.2.6.2 Loss-of-function analysis: the knockout/reverse genetics

In the late 1980s, scientists developed techniques to selectively remove a gene from the genome of an embryonic stem cell (ES), a process known as gene targeting. This modified stem cell can then be inserted into a blastocyst, which develops into a mouse that is haploid for the inactivated gene. Inbreeding of these offspring results in a fraction of the pups containing a homozygous deletion in the locus of interest, allowing the scientist to study the effects of the disruption of this gene in a whole animal. In contrast to transgenesis, this procedure utilizes genomic fragments homologous to the gene of interest which is modified to contain a selectable marker and translational termination sequences. This DNA fragment is electroporated into ES cells, and through a process known as homologous recombination, the modified DNA replaces the endogenous sequence (Figure 3b). Thus, the chromosomal and promoter context of the gene is maintained, but no protein product can be produced. Variations to this approach include the incorporation of subtle mutations, as opposed to the complete ablation of the gene product. This approach can be used to assess the contribution of particular amino acids or domains toward the in vivo function of a protein. For example, to model Huntington's disease, which is associated with the extended repeats of glutamines in the relevant gene, Detloff and colleagues inserted a stretch of CAG repeats into the mouse Huntington's disease gene using this technique.143 Furthermore, a 'knock-in' approach, which employs similar strategies, can be used to place a cDNA under ho

Microinjection of DNA

Implantation into pseudopregnant female

Screen offspring for transgenic construct

Transgenic founder mouse

Targeting construct introduced into ES cells

ES cells expanded under selection

ES cells screened for homologous recombination

Positive cells injected into blastocyst and implanted into pseudopregnant female

Offspring are chimeric for deletion

Breeding to isolate germline deletion of genetic loci

Intercross

ENU mutation

Mutant

Figure 3 Mouse genetic systems. (a) Transgenic: typically, the DNA construct of interest is microinjected into the pronuclei of fertilized eggs. In a percentage of cases, the foreign DNA is randomly integrated in the genome of the egg, which is transplanted into a pseudopregnant female. The offspring determined to have received the transgene through genetic analysis (e.g., PCR or Southern blot), and positive animals are utilized to establish founder colonies for the transgenic lines. (b) Knockout: to generate a genetically null mutant, first a targeting vector containing regions homologous to the gene of interest, an incorporated mutation (e.g., stop codon) and a positive selectable marker must be constructed. This is then introduced into embryonic stem (ES) cells, which are subsequently propagated in media containing the corresponding chemical selection. ES cells in which the targeting vector has replaced the endogenous genetic loci through homologous recombination are identified through either Southern blot or PCR analysis, and subsequently injected into a mouse blastocyst. These are then transplanted into a pseudopregnant female, and chimeric offspring of the targeting vector are bred to isolate germline deletions of a genetic loci. (c) ENU mutagenesis. Top panel: a homozygous (ENU) mutant is crossed to a wild-type mouse from another genetic background (e.g., C57/BL6/J mutant crossed to BALB/CByJ wild-type), producing mice that are heterozygous for the mutation in a mixed background (F1 progeny). These offspring are inbred and the mutation is mapped through segregation analysis of single nucleotide polymorphisms (SNPs) and the mutant phenotype. Bottom panel: a depiction of the same process tracking a possible chromosomal mutation.

the control of an endogenous promoter. This strategy, although more time-consuming and labor-intensive than transgenesis, can enable robust temporal and spatial expression of a transgene.

In some cases, the disruption of a gene results in embryonic lethality, making it impossible to study the function of this gene in the adult organism. To circumvent this event, geneticists employ a technique known as conditional knockouts. In this approach, two recombination sites are incorporated into a homologous targeting construct, specifically engineered not to effect the expression of the encoded protein until the expression of a recombinase protein. The mouse that is homozygous for this insert construct is crossed to a mouse that transgenically expresses a recombinase protein, such as Cre or Flp, specifically in the adult or in particular tissues.144'145 Thus, the gene of interest remains intact during embryogenesis; however, the temporally or spatially restricted expression of a recombinase enzyme directs the excision of the sequences between the engineered recombination sites. This results in a loss of function of the gene in specified cells or at appropriate times, enabling the bypass of embryonic lethality.

These knockout techniques have led to assignment of function to hundreds of previously unannotated genes, and in some cases have lead to supporting evidence for target validation and subsequent drug finding activities. Zambrowicz et al. performed a retrospective study to determine how accurately the biology of established drug target knockout strains predicts the phenotype of the small-molecule drug.3 This study revealed that of the 100 best-selling drugs only 43 distinct targets are represented, and of those 43 only 34 have been knocked out by homologous recombination. Surprisingly, 29 of the 34 have been informative in terms of describing the putative function and therapeutic potential of the target, of which many showed a direct correlation. Specifically, knockout of the hydrogen/potassium ATPase, the target for drugs such as Prilosec and Prevacid for the treatment of gastroesophageal reflux disease (GERD), exhibited the expected increase in gastric pH levels (pH = 6.9 in the knockout versus 3.19 in the wild-type). Similarly, knockout of estrogen receptors alpha and beta, targets of the highly marketed menopause and osteoporosis agonist drugs Evista and Premarin, have the expected sterility and absence of breast tissue development. In contrast, peroxisome proliferator-activated receptor gamma (PPAR-g) agonists used to increase insulin sensitivity in the treatment of type II diabetes have the opposite effect of the observation that PPAR-g heterozygous mice have an insulin-sensitive phenotype. And although finding a correlative phenotype between a mouse harboring a gene deletion and a small molecule that antagonizes the respective gene product is sometimes akin to finding a needle in a haystack (the correlation is not always obvious), companies such as Lexicon Genetics are taking it upon themselves to knock out the druggable genome one at a time to harvest more validated targets.

Although gene knockout and its derivative technologies present biologists with powerful tools to study gene function and model disease, these approaches also have certain limitations. These include phenomena referred to as redundancy and compensation. In certain instances, when a gene is disrupted, the organism will either already possess or will activate another gene to fulfill the function of the lost protein, thus making it complicated to decipher the true function of the protein of interest. Also, engineering these models requires significant investment in time, infrastructure, and other costs, thus limiting the ability to conduct these types of studies on a genome scale. Other technologies, including the use of siRNA transgenesis,146 which mimics the effects of a gene knockout at tremendous time and cost savings, gene trap mutagenesis,147 and proposed large-scale efforts,148 may enable a genome-saturating analysis of gene inactivation.

3.01.2.6.3 Forward genetics: chemical mutagenesis in the mouse

Identification of the genetic basis of heritable mutant phenotypes in model organisms is a powerful methodology used to determine the function of genes involved in the specification of biological characteristics. For example, Mendelian inheritance of coat color in mice indicates that this trait is controlled by one or more genes. In most mouse strains, black or brown pigmentation predominates throughout most of the animal's coat with the exception of a thatch of yellow banded hairs found on the belly. Certain mouse mutant strains, known as 'lethal yellow' and 'viable yellow,' maintain an exclusively yellow fur. The genetic locus responsible for this phenotype was identified as Agouti, and its encoded protein was subsequently shown to cause hair follicles to synthesize a yellow pigment.149

In order to take advantage of this forward genetic approach, methods have been established for efficient mutagenesis and screening of mutated mice strains for unique phenotypes correlative with disease or suppressors of a studied disease state (Figure 3c). For instance, to accelerate the process of creating heritable mutations, geneticists typically use chemical agents such as ethylmethane sulfonate (EMS) or N-ethyl-N-nitrosourea (ENU) to induce genetic lesions in progenitor cells, such as those of the male germline. EMS and ENU induce point mutations in DNA, which can lead to several alterations of a gene's function. These include gain-of-function (hypermorphic), loss-of-function, reduction of function (hypomorphic), or alteration of function (neomorphic) effects. Male mice treated with these mutagenic reagents are used to inseminate female to produce colonies of pups carrying heritable mutations in their genomes. These animals are then screened for phenotypes of interest (e.g., high cholesterol, high blood pressure, or obesity). Once a mutant has been selected, a series of interbreedings are done to identify the chromosomal locus of the mutated gene.150 For example, Wen and co-workers employed a genome-wide recessive screening strategy to identify the inositol (1,4,5) trisphosphate 3 kinase B (Itpkb) gene as an important player in T-cell activation,151 which can be potentially exploited as a therapeutic target for immunosuppression. While a number of new technologies are facilitating the identification of genomics sequences mutated in these screens, identification of the causative mutation of the observed phenotype remains a challenging endeavor.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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