Abstract
Here we report a new transgenic expression system by combination of liver-specific expression, mifepristone induction and Cre-loxP recombination to conditionally control the expression of oncogenic krasV12. This transgenic system allowed expression of krasV12 specifically in the liver by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system. We found that liver tumors were generally induced from multiple foci due to incomplete Cre-loxP recombination, thus mimicking naturally occurring human tumors resulting from one or a few mutated cells and clonal proliferation to form nodules. Similar to our earlier studies by both constitutive and inducible expression of the krasV12 oncogene, hepatocellular carcinoma (HCC) is the main type of liver tumor induced by krasV12 expression. Moreover, mixed tumors with hepatocellular adenoma and hepatoblastoma (HB) were also frequently observed. Molecular analyses also indicated similar increase of phosphorylated ERK1/2 in all types of liver tumors, but nuclear localization of β–catenin, a sign of malignant transformation, was found only in HCC and HB. Taken together, our new transgenic system reported in this study allows transgenic krasV12 expression specifically in the zebrafish liver only by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system.
Highlights
Continuously present throughout the course of experiments in order to maintain the transgenic oncogene expression, as withdrawal of the inducing chemical would cause repression of the transgenic oncogene and regression of the induced liver tumors
We have developed a new transgenic system by combining liver-specific transgenic expression, mifepristone inducibility and Cre-loxP recombination for conditional activation of an oncogene to develop inducible liver tumors in zebrafish
Unlike our previous inducible liver tumor models where the inducing chemical is required to be present throughout the duration of experiment as withdrawal of the inducing chemical causes tumor regression[9,11], the current conditional inducible system abolishes the dependence of the inducing chemical for maintenance of the tumor state, as it requires only a brief chemical induction to cause permanent genetic recombination to activate the krasV12 oncogene by transient induction of the Cre recombinase
Summary
Continuously present throughout the course of experiments in order to maintain the transgenic oncogene expression, as withdrawal of the inducing chemical would cause repression of the transgenic oncogene and regression of the induced liver tumors. To overcome this hassle, in the present study, we made an improvement to our transgenic strategy by introducing the Cre/LoxP system to induce a permanent expression. Together with our previously generated liver-driver line to express LexPR activator[9], we generated triple transgenic zebrafish by selective breeding to activate the oncogenic krasV12 gene through a brief treatment using mifepristone and demonstrated the successful generation of liver tumors of various grades. The current liver tumor model might provide a new valuable tool to analyze the development of individual liver tumors and potential chemical intervention
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