Aging Aging

362 Andersenmouse embryo at the one-cell stage (1). The transgenic DNA thus introducedwill randomly integrate into the genomic DNA of the embryo, where it can betranscribed and translated into functional protein. Following injection of theforeign DNA, the embryos are implanted into pseudopregnant recipientfemales. Because the transgenic DNA integrates into the mouse’s genomicDNA at the one-cell stage, the integrated DNA will be contained in the DNA ofall cells of the mouse’s body including its germ line cells and will thereforebecome a heritable component of its genetic make-up. Following their birth,founder animals are tested for presence of the transgene via the polymerasechain reaction (PCR) or Southern blot analysis, and positive founders bred outto create transgenic lines in which patterns and levels of expression of the genecan be observed as well as its physiological consequences. The extent of expressionof the transgene appears to be more related to its site of genomic integrationas opposed to copy number. Many independent lines must be examined toseparate out the effects that the integration site vs the transgene itself have onthe observed phenotype.3. Further Considerations in Transgene DesignNormally, the injected foreign DNA used for transgenic production isdesigned to contain the gene of interest expressed under the control of a regulatorypromoter element that designates at what time and in what cell types theexpression of the transgene will occur. The injected DNA can be either theentire gene itself including its native promoter element and all of its intronsand exons and 5' and 3' regulatory DNA sequences, or it may consist of a heterologouspromoter driving expression of the cDNA that is missing the intronsand regulatory sequences. It has been demonstrated in previous studies thatgenomic constructs are expressed more efficiently than cDNAs. However,owing to the large size of some genes, it is not always possible to use the entiregene although there have now been several reports of successful integration oftransgenic yeast artificial chromosome (YAC) DNAs of several hundredkilobases in size into the mouse genome (2–7). It has been suggested that thepresence of the first intron is necessary for high levels of expression of a cDNAeven if the intron is a heterologous one (8). The choice of the promoter (nativevs heterologous) is normally dependent on when, where, and to what extentexpression of the gene product is desired.4. Making a “Knockout” Mouse: The BasicsAlthough expression of an antisense mRNA in a transgenic construct hasbeen used in a few instances to reduce levels of a desired gene product (9–14),the success of antisense RNA expression is dependent on several factors includingthe level of expression of the endogenous RNA and its turnover rate as well