Trapping of mammalian promoters by Cre-lox site-specific recombination.
One of the challenges in human genome research is to identify the promoter sequences which play a key role in the regulation of gene expression. We report here a new promoter trapping system for use with mammalian cells comprised of the following three steps: 1) Cloning of DNA fragments into a promotertrapping vector, 2) integration of the trapping vector into a designated target in the mammalian genome using the Cre site-specific recombinase, and 3) screening of integrants for trapped promoter sequences by activation of the luciferase gene. To assess the efficiency of this system, lox trapping vectors containing sense tk promoter, antisense tk promoter, or a non-promoter sequence of the neo gene were employed. The resulting levels of luciferase activity of the site-specific integrants were measured directly. Luciferase activity of the integrants can be assayed under conventional culture conditions by simply replacing the culture medium with potassium phosphate buffer containing luciferin. Only those G418r colonies carrying the tk promoter in the normal orientation exhibited a 21-to 35-fold increase in luciferase activity over that of the other integrants. These results indicate that this system is an effective means of trapping promoter sequences from random mammalian genomic DNA fragments.
Fukushige
S
S
Ikeda GenoSPHERE Project, Tokai University School of Medicine, Kanagawa, Japan.
Ikeda
J E
JE
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005821:Genetic Techniques; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics*; D019868:Kanamycin Kinase; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008322:Mammals / Q000235:genetics*; D051379:Mice; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins
D014764:Viral Proteins; D008156:Luciferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D013937:Thymidine Kinase; D019868:Kanamycin Kinase; C045073:Cre recombinase; D019426:Integrases