Conditional gene activation in cultured hepatocytes using a ligand-dependent chimeric Cre recombinase.
By combining liver-specific promoter and a chimeric Cre recombinase, conditional gene activation could be finely achieved in hepatocytes at selected time points. To this end, the expression vector of Cre-ERt under the control of the mouse albumin gene promoter/enhancer, alb-Cre-ERt, was constructed, and transfected into engineering BRL (Rat hepatocytes) and BRK (Rat kidney) reporter cells which carries a chromosomally integrated 'floxed' beta geo gene, which is inserted between the promoter and the human alkaline phosphatase( hAP) reporter gene, thereby preventing hAP reporter gene transcription, respectively. After treatment with 1 micromol/L 4-hydroxytamoxifen(4-OHT), a proportion of hAP staining positive cells were detected by hAP staining. It was further confirmed that 'floxed' beta geo cassette was removed by Cre excision by using PCR analysis of cellular DNA. No background recombinase activity could be detected in the absence of 4-OHT. Moreover, no hAP-positive cells could be detected in BHK cells untreated or treated with 4-OHT. These data suggested that alb-Cre-ERt expression vector was constructed successfully, and 4-OHT could induce Cre-mediated recombination only in hepatocytes expressing Cre-ERt, thereby activating a stably integrated hAP reporter gene. This provides a further foundation for producing transgenic mice expressing such an 4-OHT inducible Cre recombinase specifically in mouse liver.
Zhu
Huan-Zhang
HZ
State Key Laboratory of Genetic Engineering, Institute of Genetic, School of Life Sciences, Fudan University, Shanghai 200433, China. jlxue@fudan.ac.cn
Cao
Ying-Ying
YY
Cheng
Guo-Xiang
GX
Xue
Jing-Lun
JL
D000469:Alkaline Phosphatase / Q000235:genetics*; D000818:Animals; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D019426:Integrases / Q000235:genetics*; D007763:Lac Operon / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D051381:Rats; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; D013629:Tamoxifen; C016601:afimoxifene; C045073:Cre recombinase; D019426:Integrases; D000469:Alkaline Phosphatase