Heterogeneous populations of ES cells in the generation of a floxedPresenilin-1 allele

Abstract

Generation of a floxed Presenilin-1 (PS1) allele involved two recombination events in the embryonic stem (ES) cells. First, a targeting vector containing a loxP site in intron 1 and a floxed CMV-HYG/TK double selection cassette in intron 3 was integrated into the PS1 locus by homologous recombination. The use of a negative selection cassette, PGK-DTA, dramatically increased the recombination efficiency within the targeted locus (75-fold). Second, an expression vector encoding Cre recombinase was introduced to excise the floxed CMV-HYG/TK cassette via site-specific recombination. However, all five ES cell clones testing positive for the proper removal of the CMV-HYG/TK cassette also contained a proportion of ES cells in which recombination had occurred between the distal loxP sites in introns 1 and 3, resulting in excision of the entire floxed region. It is therefore critical to screen for possible recombination events involving all 3 loxP sites, in order to identify ES cells clones bearing high proportions of the desired ES cells. genesis 26:5-8, 2000.