Pronuclear Microinjection of DNA

Gain of function (overexpression) of a gene is achieved by microinjecting a DNA construct containing a promoter or other regulatory sequences linked to a gene of interest. The minimal construct might also contain additional gene regulatory elements such as enhancers, insulators, or locus control regions. Transgene size ranges from small cDNA constructs to large genomic inserts from bacterial artificial chromosomes (BACs). There are many laboratory manuals describing the preparations of transgene constructs (22,23) for successful microinjections. Founder mice (those carrying the original transgene insertion) are bred with nontransgenic mice of the same strain to establish a line of transgenic mice for which the expression of the transgene can be studied. Analysis of the transgenic phenotype can be done in embryos (transient transgenics) or adult mice (see Fig. 1).

Analysis of transgenic mice can be confounded by several factors: Possibilities include a construct that inserts randomly into the genome but causes lethality or masking of a trans-gene's phenotype because the insertion site disrupts an essential gene's function. Transgene integration at a random genomic site also might repress expression of the transgene. Mosaic mice contain more than one transgene integration site because of late integration of the transgene into the genome at the two-cell stage or beyond. This situation confounds analysis of the transgene's expression pattern. In practice, several transgenic founders are identified and evaluated for a consistent phenotype.

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