Generation of transgenic mice with yeast artificial chromosomes.

Abstract

Since the original descriptions of methods on how to generate transgenic mice, scientists have been confronted with the limited cloning capacity of the standard plasmid vectors available. Constructs transferred to the germline often lack important cis-regulatory elements and, therefore, fail to precisely reproduce the expression pattern of the endogenous counterpart. When integrated into the genome, the constructs might be influenced by nearby regulatory elements, leading to silenced, enhanced, or altered expression of the transgene. Only rarely have transgenes been found to express in a way which is not dependent on their integration site. In order to overcome the limitations mentioned above, yeast artificial chromosomes (YACs) have been introduced as a new vector system. YACs are linear molecules with a cloning capacity of over one megabase (Mb). Cloned DNA is flanked by two vector arms containing all the necessary elements for stable maintenance of the artificial chromosome in yeast cells. Telomeric sequences guarantee chromosomal stability. The long vector arm harbors a centromer and an autonomously replicating sequence required for chromosomal segregation and replication, respectively. Making use of yeast metabolic marker genes in both arms, one can select for the presence of the YAC. The highly active homologous recombination system in yeast offers the possibility to introduce any desired mutations into the YAC.