Efficient modification of the APRT gene by FLP/FRT site-specific targeting.
The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5 X 10(-5), which were 6-22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT+ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination.
Merrihew
R V
RV
The Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
Sargent
R G
RG
Wilson
J H
JH
D000228:Adenine Phosphoribosyltransferase / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005091:Exons; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping
D017931:DNA Primers; D011994:Recombinant Proteins; D000228:Adenine Phosphoribosyltransferase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase