Generation of Transgenic Animals by Use of YACs

The use of genomic-type DNA constructs ensures optimal transgene expression, once inserted into the host genome, because their large size includes most if not all the regulatory elements that are needed for correct gene expression. Large heterologous DNA molecules can be easily manipulated in bacterial or yeast cells, through the use of bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs), respectively. YACs are the vectors that allow the manipulation of larger DNA molecules, in excess of 1 Mb (1,000 kb). Some mammalian loci (i.e., the APP locus, ~450 kb) greatly exceed the maximum size for inserts that can be accommodated by BACs. Therefore YACs are currently the only available robust and reliable solution for working with these large genes. In this chapter, we will describe several procedures directed towards the preparation of YAC DNA of a quality suitable for microinjection into mouse embryos, for the successful generation of transgenic mice.