Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 12
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After all, there are now close to200 animal models of human cardiovascular disease, which have been producedthrough the use of genetic manipulation using either transgenesis or gene ablation. The use of these mouse-based genetic models where the primary etiology isknown and the disease course can be closely monitored should, at first glance,easily provide the basic reagent – a relatively well defined disease in an organismwith a well-defined genetic background, in which the disease’s progression can bemonitored closely.
Thus, using large groups of animals, the longitudinal progression of disease development becomes a timeline along which points can be takenfor global transcriptional analyses and indeed, large data sets are beginning to begathered across a number of these disease models (http://www.nhlbi.nih.gov/resources/pga/resource.htm). However, considering the dependence on these animal models, it is worthwhile to take a closer look at how they are made and whatthe phenotypes might mean in terms of genetic output.Our ability to precisely manipulate the mammalian genome of the mouse hasgiven us access to exciting new approaches for studying gene function in a physiological context.
Selective changes in a protein or the protein profile of the heartcan be accomplished in order to study both gain-of-function disorders (where anactive protein is overexpressed or a mutant protein with altered function is expressed) and loss-of-function disorders (an endogenous gene is inactivated or amutant protein can serve as a dominant-negative mutation). Even the most abundant cardiomyocyte proteins, the components of the contractile apparatus, cannow be altered or replaced in a defined manner [15–17].29302 Cardiac Disease and The TranscriptomeFor models of cardiovascular disease it is highly desirable that expression be restricted to the heart and preferably, to a specific cell type, e. g.
the cardiomyocyte,within the heart. Systemic or ectopic expression would certainly raise the “biological noise” of the transcriptome analyses as the heart responded to ectopic stimuli.For transgene expression the ideal promoter should drive high levels of gene activity in a cardiac-specific manner at appropriate times during development andshould also display a minimum of position-dependent effects.
The most successful and widely used elements are derived from contractile genes themselves [18]with the a-myosin heavy chain promoter most often chosen due to its high levelsof expression in both the adult atria and ventricles, its cardiac specificity, and thepresence of sequences necessary for copy number-dependent and position-independent expression [19, 20].Creation of a transgenic animal is not genomically benign.
Using pronuclearmicroinjection techniques, the construct is introduced into fertilized eggs, whichare then transferred to pseudopregnant females. Inside the pronucleus, the transgene will either randomly integrate into the mouse genome or be degraded byexonuclease activity. Usually several copies of the transgene are found in a tandem head-to-tail array. As transgene insertion occurs early during development,most or all of the cells of the resulting mouse will carry the genetic modification.Occasionally, the transgene is integrated at more than one site in the genome andoffspring of such founders may inherit one or the other copy of the transgene, resulting in multiple lines with different copy numbers and potentially distinct phenotypes from a single founder [21].
This could, if undetected, have a significantdetrimental effect on any subsequent transcriptome-based analysis, as it is morethan likely that the different transgene numbers would result in phenotypes ofvarying severity.In cardiovascular disease models, the expression level of the transgene is oftenpositively correlated with the copy number and therefore the severity of the phenotype, but the relationship is not a simple one. The site of integration within thegenome, the proximity of the transgene to transcriptional activators and silencerscan both alter the level and tissue selectivity of protein expression. Moreover,transgene insertion can also influence the expression of neighboring genes, sinceit might disrupt a protein coding sequence, a promoter element, or other regulatory regions [22–24]. Fortunately, the consequences of such insertional mutageniceffects are recessive in many cases and therefore do not alter the phenotype unless the transgene is bred to homozygosity.In contrast to the random insertion of the construct into the genome with thetransgenic approach, gene targeting replaces a DNA sequence with the exogenoussequence at a specific site within the genome using homologous recombination[25].
Deletions, point mutations, or replacements can be used to achieve gene inactivation or mutation. The construct is introduced into embryonic stem cells viaelectroporation: the rare homologous recombination can be selected for and thosecells are then microinjected into blastocysts, which are then implanted into pseudopregnant females. Mice heterozygous for the targeted allele can then be intercrossed to generate homozygous mice. If the introduced genetic alteration is, for2.2 The Starting Line: Garbage In – Garbage Out?example, a deletion of a critical region of the protein of interest and results in anull allele, the gene will be completely ablated, producing a “gene knockout” inthe homozygous mice.Gene targeting can also be used to introduce subtle mutations into a gene of interest [26], or as part of a strategy to generate a tissue-specific or inducible knockout [27].
Since this strategy directs integration of the construct to a particular site,the site-specific effects and copy-number dependency that can confound the transgenic approach can be avoided [28]. Moreover, the targeted protein will be expressed under the control of the endogenous promoter, resulting in endogenousexpression levels that reflect the correct temporal and spatial distribution of thewild-type protein. Although more precise, gene targeting is often not theapproach chosen since it is much more time-consuming than transgenesis. Moreover, if the gene of interest plays an important role in early development, eitherhigh prenatal mortality or distortion of the phenotype due to developmental defects can occur.For transcriptome-based analyses, more precise manipulation of altered gene expression would be a significant advantage, as one could lower the biological noisegenerated either by systemic expression of the altered gene (as is the case in mostgene targeting experiments, with rare exceptions [29]), or by transgene expressionthroughout development.
Tissue-specificity of a gene ablation can be achievedusing the E. coli bacteriophage P1 enzyme Cre recombinase. Cre recombinasebinds to a 34 bp DNA sequence called a loxP site, which contains two 13 bp repeated sequences in opposite orientation flanking an 8 bp spacer. Removal of theDNA fragment flanked by loxP (a ‘floxed’ allele) occurs when Cre is expressed.Mice homozygous for a floxed gene are bred to a mouse line expressing Cre recombinase driven by a promoter specific for the tissue of interest: the offspringwill carry a deletion of the floxed gene only in that tissue.
The Cre/lox system hasbeen successfully used by a number of groups to create cardiac-specific knockouts[30] and can also be combined with an inducible system to add temporal control[27, 31, 32].In order to accomplish temporal control (including expression of a transgene ina reversible manner), a variety of inducible systems have been developed and potentially offer the ability to create a well-defined start point for transgene expression of gene ablation in the adult, freeing the transcriptome of the noise resultingfrom the consequences of transgene expression during other developmentaltimes. All systems share a common theme, exemplified by the tetracycline regulatory system. Mice with tissue-specific expression of the tetracycline transactivator(tTA) are generated.
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