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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