Transgenic And Genetargeting Techniques

Over the past two decades, progress in the development of molecular genetic methods has enabled the manipulation of genes in intact organisms, such as mice. The technologies have provided a powerful and useful tool that allows the study of gene function and promotes understanding of the molecular mechanisms of disorders of the brain and mind. The mouse genome is by far the most accessible mammalian genome for manipulation. Many successful procedures for introducing new genes, expressing elevated levels of genes, and eliminating or altering the function of identified target genes have been reported. Many mouse models produced by manipulating genes may be used in a variety of fields relevant to neuroscience. It is noteworthy, however, that behavioral studies in mice lag far behind those in rats. For this reason, genetically induced behavioral alterations in mice must be interpreted with caution (Lucki et al. 2001).

Generally, transgenic mice are those expressing exogenous DNA because of the insertion of a gene into the mouse genome. Usually that gene is randomly located within the mouse genome, often as several copies. The use of transgenic mice has represented a major strategy for the investigation of genetic questions since the feasibility and reproducibility of stably introducing DNA by microinjection into individual male mouse embryos were established (Markert 1983). In DNA constructs used for the generation of transgenic mice, the gene of interest is located 3' to promoter sequences to produce a desired distribution of gene expression. The selection of the promoter is the most important consideration in generating transgenic mice. Some promoters, such as platelet-derived growth factor (PDGF), thy1 (a cell surface glycosylphosphatidylinositol-linked glycoprotein), prin (PrP), neuron-specific enolase (NSE), and glial fibrillary acidic protein (GFAP), have demonstrated the ability to direct high-level expression of exogenous genes in brain and/or in the neurons of mice (Hsiao et al. 1996; Kan et al. 2004; Nolte et al. 2001; Sturchler-Pierrat et al. 1997). This level of expression can be modified by incorporation of a "tet-on" or "tet-off" vector into the desired inserted gene. Depending on the nature of the switch (on or off), the mouse will express the gene of interest when ingesting (or taken off) doxycycline.

"Gene targeting" refers to the homologous recombination that occurs between a specifically designed targeting construct and the chromosomal target of interest, in which recombination at the target locus leads to replacement of the native target sequence with construct sequence. The method enables the precise introduction of a mutant into one of many murine genes and has proven invaluable in examining the roles of gene functions in complex biological processes. Most of the target constructs are used to disrupt a target and to eliminate gene function (conventional "knockout"). Generally, a gene-targeting construct that contains positive-negative selection markers is prepared such that the target gene is interrupted by the gene for neomycin resistance, which also serves as a positive selection marker, and a thymidine kinase (TK; Gusella et al. 1983) gene is adjacent to either one or both ends of the homologous genomic sequence for negative selection. The positive-negative deletion scheme is employed to enrich for homologous recombination.

The targeting construct is often introduced into mouse embryonic stem (ES) cells by electroporation. Cells that fail to integrate the targeting construct into the genome are killed by application of neomycin in the culture medium (positive selection). The majority of the remaining cells, in which the entire construct (including the TK gene) inserts randomly, will die as a result of the incorporation of ganciclovir or fialuridine (inactive thymidine analogs), which block DNA synthesis. Homologous recombinant clones that do not contain the TK gene are used to prepare chimeric mice. Cells from these clones are microinjected into the fluid-filled cavity of 3.5-day-old embryos at the blastocyst stage. The injected embryos are then surgically transferred into the uterus of pseudopregnant females. These animals will give birth to chimeric mice. Breeding can be used to generate mice that are heterozygous and homozygous for the mutation. Homozygous mutants may express the gene of interest in any cell of the body (Figure 2-7).

FIGURE 2-7. Conventional gene disruption ("knockout").

FIGURE 2-7. Conventional gene disruption ("knockout").

(A) Producing chimeric mice. First, a mutant allele is produced by replacing the coding exons of the desired gene with a neomycin (neo) cassette and transferring it into embryonic stem (ES) cells. Second, genetically altered ES cells are reintroduced into a developing blastocyst, where they contribute to the developing embryo. (B) Breeding chimeric mice. When the germ cells of the resulting chimeric mouse (chimera) are ES cell-derived (germ-line mutation), the heterozygotes

(+/-) can be produced by breeding. One-half of the offspring will be heterozygous. The heterozygous animals may be bred to produce homozygous mice (-/-). TK = thymidine kinase; WT = wild type.

Pregnancy And Childbirth

Pregnancy And Childbirth

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