Production of Yeast Artificial Chromosome Transgenic Mice by Pronuclear Injection of One-Cell Embryos

Abstract

The generation of transgenic animals has become an important tool in helping to understand the roles of genes in maintaining health and the roles of human gene variants in contributing to disease susceptibility. In order to model the subtleties of gene expression, which include tissue-specific transcriptional regulation and alternative gene splicing, it is useful to include entire genes that comprise all of the exons, introns, and regulatory sequences required for proper gene expression. A significant proportion of the genes contained in the human genome have been found to span areas of chromosomes that frequently exceed 200 kb. This limits the use of many large cloning vectors, such as P1 artificial chromosomes or bacterial artificial chromosomes, whose upper capacity is around 200 kb of genomic DNA, to be used as transgenes that efficiently model the mechanisms that determine the role of complete genes and their variants in the development of disease. The only cloning vector that can be used to model the mechanisms controlling the expression and functioning of these genes are yeast artificial chromosomes (YACs). In this chapter, a detailed account is given of how YAC DNA can be prepared and purified using pulse-field gel electrophoresis, how this DNA can be introduced into the mouse genome by pronuclear microinjection into one-cell mouse embryos, and how the transgene can be rapidly detected in the resulting transgenic animal by polymerase chain reaction. A brief account of a method of assessing YAC transgene copy number is also outlined.