Positive selection of FLP-mediated unequal sister chromatid exchange products in mammalian cells.
Site-specific recombination provides a powerful tool for studying gene function at predetermined chromosomal sites. Here we describe the use of a blasticidin resistance system to select for recombination in mammalian cells using the yeast enzyme FLP. The vector is designed so that site-specific recombination reconstructs the antibiotic resistance marker within the sequences flanked by the FLP target sites. This approach allows the detection of DNA excised by FLP-mediated recombination and facilitates the recovery of recombination products that would not be detected by available screening strategies. We used this system to show that the molecules excised by intrachromosomal recombination between tandem FLP recombinase target sites do not reintegrate into the host genome at detectable frequencies. We further applied the direct selection approach to recover a rare FLP-mediated recombination event displaying the characteristics of an unequal sister chromatid exchange between FLP target sites. Implications of this approach for the generation of duplications to assess their effect on gene dosage and chromosome stability are discussed.
Aladjem
M I
MI
Gene Expression Laboratory, The Salk Institute, San Diego, California 92037, USA.
Brody
L L
LL
O'Gorman
S
S
Wahl
G M
GM
D000818:Animals; D002460:Cell Line; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D018390:Gene Targeting; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics*; D000882:Haplorhini; D005810:Multigene Family; D009705:Nucleosides / Q000494:pharmacology; D011995:Recombination, Genetic; D012854:Sister Chromatid Exchange
D005819:Genetic Markers; D009705:Nucleosides; C004500:blasticidin S; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase