Preparation of enzymatically active human Myc-tagged-NCre recombinase exhibiting immunoreactivity with anti-Myc antibody.
The Cre-loxP system has been recognized as a tool for conditional gene targeting in mice. However, most anti-Cre antibodies fail to react with Cre expressed in vivo. In an attempt to directly detect Cre by antibodies in vivo, we constructed the tagged-NCre (NCreMH) gene by connecting the human Myc and His tag sequences to the 3' end of the NCre gene carrying a nuclear localizing signal (NLS) sequence. The production of NCre protein and the recombinase activity were detected after co-transfection with pCMV-NCreMH and pCETZ-17 carrying the loxP-flanked lacZ gene into NIH3T3 cells. This activity was also confirmed in vivo after gene transfer of pCMV-NCreMH and pCRTEIL-6 carrying loxP-flanked HcRed1 and EGFP cDNAs, into oviductal epithelium by electroporation. Immunohistochemical staining using anti-Myc antibody demonstrated that the area positive for enhanced green fluorescent protein (EGFP) fluorescence was immunostained with the antibody. These findings indicate that NCreMH is useful as an alternative to NCre for gene targeting.
Watanabe
Satoshi
S
Department of Developmental Biology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. kettle@affrc.go.jp
Honma
Daisuke
D
Furusawa
Tadashi
T
Sakurai
Takayuki
T
Sato
Masahiro
M
D000818:Animals; D000906:Antibodies / Q000276:immunology; D018390:Gene Targeting; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000276:immunology; D014162:Transfection
D000906:Antibodies; C489427:MYC protein, human; D019913:Nuclear Localization Signals; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C045073:Cre recombinase; D019426:Integrases