A Cre-lox recombination system for the targeted integration of circular yeast artificial chromosomes into embryonic stem cells

The ability to produce embryonic stem (ES) cell lines containing different yeast artificial chromosomes (YACs) integrated into the same location in th e genome provides a system for comparing the biological effects of YAC tran sgenes without the confounding influences of integration site and copy numb er, A targeting system was developed for the directed integration of circul ar YACs into mouse ES cells. The system combines Cre-lox recombination tech nology, specifically a positive-selection integration system, with circular YAC lipofection technology to achieve single copy targeted integration of a transgene, Three independent germline competent ES cell lines [lox-contai ning ES lines (designated LES)] were created that contain a '1/2-neo-lox' c assette integrated at different sites within the ES genome, A plasmid conta ining YAC vector sequences and a complementary '1/2-neo-lox' cassette was u sed to circularize two linear YACs containing genomic DNA from human chromo some 21, The circularized YACs were then targeted to the lox sites of the L ES cell lines. Polymerase chain reaction and Southern analysis demonstrated that 21% (5 of 24) of lex-recombinants contain a full-length intact YAC, T his system will make the study of YAC transgenic mice more reliable and rep roducible, allowing the potential for direct comparison of different transg enes expressed from the same site within the genome.