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 the genome provides a system for comparing the bio-logical effects of YAC transgenes without the confounding influences of integration site and copy number. A targeting system was developed for the directed integration of circular YACs into mouse ES cells. The system combines Cre-lox recombination technology, 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-containing ES lines (designated LES)] were created that contain a '-neo-lox' cassette integrated at different sites within the ES genome. A plasmid containing YAC vector sequences and a complementary '-neo-lox' cassette was used to circularize two linear YACs containing genomic DNA from human chromosome 21. The circularized YACs were then targeted to the lox sites of the LES cell lines. Polymerase chain reaction and Southern analysis demonstrated that 21% (5 of 24) of lox-recombinants contain a full-length intact YAC. This system will make the study of YAC transgenic mice more reliable and reproducible, allowing the potential for direct comparison of different transgenes expressed from the same site within the genome.
Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. lcall@rics.bwh.harvard.edu
L M
LM
Call
C S
CS
Moore
G
G
Stetten
J D
JD
Gearhart
D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D004270:DNA, Circular; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D011995:Recombination, Genetic; D013234:Stem Cells; D014170:Transformation, Genetic; D014764:Viral Proteins
D004270:DNA, Circular; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases