Targeted rearrangement of a chromosomal repeat sequence by transfection of a homologous DNA sequence using purified integrase.
Using a liposomal transfection with purified bovine leukemia virus (BLV) integrase, we observed an efficient DNA rearrangement of a chromosomal repeat sequence and targeted integration of a part of the transfected plasmid. The BLV integrase recognition sequence (IRS) including the 3' end of the BLV LTR U5, one of the sites cleaved by the integrase, was essential for the DNA rearrangement, and a sequence homologous to the chromosomal DNA neighboring the repeat target site had to be placed downstream of the IRS on the transfected plasmid. The pSV2neo DNA, including the pBR322 sequence preintegrated into L929 cells (primary transfectants), was rearranged by a secondary transfection of a pBR322-based hygromycin-resistance plasmid carrying the IRS. We present a model to explain the chromosomal DNA rearrangement of the primary clones through a homologous recombination-like reaction and amplification of the neighboring sequences.
Tanaka
A S
AS
Department of Safety Research on Biologics and Blood Products, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
Komuro
K
K
D000818:Animals; D015139:Blotting, Southern / Q000379:methods; D002478:Cells, Cultured; D002934:Cinnamates / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D015321:Gene Rearrangement; D018390:Gene Targeting / Q000379:methods*; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D019426:Integrases / Q000235:genetics* / Q000302:isolation & purification; D001909:Leukemia Virus, Bovine / Q000201:enzymology; D051379:Mice; D008957:Models, Genetic; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods*
D002934:Cinnamates; D006921:Hygromycin B; C026273:hygromycin A; D019426:Integrases