Modification and amplification of yeast artificial chromosomes

The development of yeast artificial chromosome (YAC) technology permits cloning of DNA segments that can be thousands of kilobases in size. This facilitates techniques such as the generation of transgenic animals as a YAC clone can, in most cases, carry an entire gene with all its regulatory elements. Similar to the endogenous yeast chromosomes, YAC DNA is replicated in yeast at one copy per cell. This presents problems when attempting to isolate sufficiently concentrated YAC DNA for pronuclear microinjection. To overcome this problem, we have constructed a YAC amplification vector (pYAM4) that concentrates YAC DNA up to three to eight copies per cell, thus ensuring that more concentrated YAC DNA can be purified. To simplify the analyses of transgene expression in transgenic animals, we have also developed a series of YAC modification vectors (pYIV1, 2, 3, and 4) that can be used to introduce a lacZ reporter gene into YAC DNA by homologous recombination. The protocols described in this chapter with the use of these vectors have led to generation of up to 10% of YAC transgenic mice born after microinjection.