Heterogeneous populations of ES cells in the generation of a floxed Presenilin-1 allele.
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.
Yu
H
H
Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Kessler
J
J
Shen
J
J
D000483:Alleles; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005813:Genes, Synthetic; D006921:Hygromycin B / Q000494:pharmacology; D019426:Integrases / Q000378:metabolism; D007438:Introns / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053764:Presenilin-1; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013234:Stem Cells / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins
D008565:Membrane Proteins; D053764:Presenilin-1; D014764:Viral Proteins; D006921:Hygromycin B; D013937:Thymidine Kinase; C045073:Cre recombinase; D019426:Integrases