Conditional
Gene Activation in Cultured Hepatocytes Using
a Ligand-dependent Chimeric Cre Recombinase
ZHU
Huan-Zhang1,2,3, CAO Ying-Ying2, CHENG Guo-Xiang2,
XUE Jing-Lun1*
( 1 State Key Laboratory of Genetic Engineering, Institute of
Genetic, School of Life Sciences, Fudan University, Shanghai 200433, China;
2 Shanghai Transgenic Research Center,
Shanghai 201203, China;
3 Institute of Pathology,
Southwest Hospital, Third Military Medical University, Chongqing 400038, China
)
Abstract 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’ bgeo gene, which is inserted between the promoter and the human alkaline phosphatase (hAP) reporter gene, thereby preventing hAP reporter gene transcription, respectively. After treated with 1 mmol/L 4-hydroxytamoxifen (4-OHT), a proportion of hAP staining positive cells were detected by hAP staining. It was further confirmed that ‘floxed’ βgeo 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.
Key words Cre recombinase; inducible gene expression; hepatocytes; alkaline phosphatase; lacZ
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