An in vivo assay for measuring the recombination potential between DNA sequences in mammalian cells.
Mammalian intermolecular recombination vectors that place the recombination junction within the intron of a selectable marker gene are presented. Many of the previously reported recombination assays require that recombination occur homologously and that they occur within the coding region of the selectable marker. This vector system involves the use of a human thymidine kinase (tk) minigene and measures the recombination frequency between any chosen DNA sequences, in mammalian thymidine kinase negative cells. The tk minigene is divided into a 5' vector and a 3' vector. In the 5' vector, the DNA sequence of interest is inserted in the proximal portion of tk intron 2. In the 3' vector, the DNA sequence of interest is inserted in the intron sequence between the proteolipid protein exon 2 and tk exons 3-7. Recombination through the DNA sequences of interest, either homologous or illegitimate, will reconstruct a functional tk minigene. The recombination junction is spliced out of the transcribed mRNA and thymidine kinase positive cells can be selected in hypoxanthine-aminopterin-thymidine medium. We have tested these vectors to measure the recombination potential of two Alu repetitive sequences (BLUR 8 and BLUR 11) against a control DNA sequence. BLUR 8 and BLUR 11 do not seem to recombine at a significantly higher frequency over that of the control DNA sequence. These recombination vectors display similar sensitivity to previous recombination systems, but allow tremendous flexibility in the choice of potentially recombinogenic sequences.
Shen
M R
MR
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Deininger
P L
PL
D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D051379:Mice; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D013937:Thymidine Kinase / Q000235:genetics
D012333:RNA, Messenger; D004247:DNA; D013937:Thymidine Kinase