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

Generation of a flexed Presenilin-1 (PS1) allele involved two recombination events in the embryonic stem (ES) cells. First, a targeting vector contain ing a loxP site in intron 1 and a flexed CMV-HYG/TK double selection casset te in intron 3 was integrated into the PS1 locus by homologous recombinatio n. The use of a negative selection cassette, PGK-DTA, dramatically increase d the recombination efficiency within the targeted locus (75-fold), Second, an expression vector encoding ore recombinase was introduced to excise the flexed CMV-HYG/TK cassette via site-specific recombination, However, all f ive ES cell clones testing positive for the proper removal of the CMV-HYG/T K cassette also contained a proportion of ES cells in which recombination h ad occurred between the distal loxP sites in introns 1 and 3, resulting in excision of the entire flexed region, It is therefore critical to screen fo r possible recombination events involving all 3 loxP sites, in order to ide ntify ES cells clones bearing high proportions of the desired ES cells, (C) 2000 Wiley-Liss, Inc.