Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters.
The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells. The level of Cre expression is critical to induce loxP site-specific recombination in ES cells. To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (CAG) promoter, human polypeptide chain elongation factor 1alpha (hEF-1alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct. Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression. No selection system was used in the experiments. The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1alpha promoter, the mPGK promoter and the MC1 promoter in order. These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector. Thus, it is important to choose the promoter for effective recombination by Cre.
Araki
K
K
Department of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine.
Imaizumi
T
T
Okuyama
K
K
Oike
Y
Y
Yamamura
K
K
D000199:Actins / Q000235:genetics; D000818:Animals; D017101:Bacteriophage P1 / Q000235:genetics*; D015139:Blotting, Southern; D002460:Cell Line; D002645:Chickens; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000138:chemical synthesis; D019426:Integrases / Q000096:biosynthesis / Q000235:genetics*; D007763:Lac Operon; D051379:Mice; D010957:Plasmids / Q000138:chemical synthesis; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D013234:Stem Cells; D014764:Viral Proteins
D000199:Actins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; C045073:Cre recombinase; D019426:Integrases