January 14, 2007

Whereas transgenesis involves ‘positive’ or ‘additive’ gene transfer, Gene-targeting procedures are used to disable or ‘knock out’ a candidate gene (23). A construct is designed that has two regions of homology to the target gene, flanking a selectable marker. These markers are bacterial genes that offer resistance to neomycin, hygromycin, or puromycin, which will disrupt the segments of DNA that are either essential for transcription and/or translation. Recombination between the flanking sequences of homology in the targeting construct and the genomic locus replaces the host gene sequence with the selectable marker. [Again the mouse is used as an example animal] The cells that have the selectable marker integrated into their genome are chosen by means of molecular screening techniques prior to being microinjected into blastocysts and implanted into pseudopregnant mice. The heterozygous mice that are produced are mated together to generate homozygous knockout mice, which lack a functional copy of the candidate gene. Gene-targeting is sometimes used to generate mice with a mutation in, rather than inactivation of, the candidate gene. In these cases, a construct identical to the endogenous DNA (other than the desired mutation) is made.

Compared to transgenesis, gene-targeting is an elegant system because it can control both the number of copies as well as the point of insertion. More accurate phenotype modeling is achieved via the absence of insertional mutagenic effects, the complete elimination of the endogenous candidate gene, and its expression in specific cells and tissues. Furthermore, in cases where gene-targeting is used for expressing mutated genes, the mutated gene will be expressed under the same transcriptional patterns as the gene of interest. The experimental advantages of gene-targeting are offset to some degree by its relative labor-intensiveness; transgenesis can yield a stable mouse-line in a matter of months, whereas a gene-targeting line can take years to come to fruition.

Like transgenesis, significant refinement of techniques are necessary before gene-targeting procedures can be applied to therapeutic use in humans.


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