Establishment And Application Of TTA Conditional Transgenic Mice Based On Cre/LoxP System

Tumor is a serious disease that causes serious damage to human life and health, andtransgenic mice have become the powerful research technique for the mechanisms oftumorigenesis.The combined application of Cre/LoxP system and Tetracycline inducible systemis the recent development of the inducible gene expression regulation system which is efficientand non-toxic。Its application in the transgenic mice has realized the regulation of temporal andspatial exogenous gene expression.This makes the mouse tumors closer to the actual situationand then the mouse tumor model can effectively reveal the mechanisms of tumortumorigenesis.Ras gene mutations in human tumour are the most frequent oncogeneabnormalities.K-ras gene mutations have significant influences on human.For further research on carcinogenic mechanism of K-ras gene mutations, proper tumormodels and regulation of temporal and spatial of K-ras mutant gene expression are needed inorder to achieve the goal of treatments of tumors.On the premise that reaction component TRE-Kras-G12D mice of Tetracycline inducible system has been established in the laboratory, theregulatory element mice are needed to be produced.The two kinds of mice mate and then doublepositive mice that can induce the expression of Kras G12D are born.The main work of thisarticle is based on the principles of Cre/LoxP and Tetracycline inducible system.The CL-tTAtransgenic mice which have the potential to express tTA protein highly.They can be used ascommon laboratory transgenic mice and lay the foundation for establishing different oncogenetumor models.In this article pClkhl-tTA-IRES-GFP recombinant plasmid was established throughcommon molecular biology technology，ES cells were treated through electroporation andscreened by use of Hygromycin B and tamoxifen.Then after the copy number and function atthe cellular level were identified and ES cells were treated through blastocyst injection，the chimeric mice were gained.After Chimeric mice mate with FVB mice, the filial mice wereanalysed through PCR, CL-tTA positive mice can be obtained.The following identification of the copy number of positive mice and regulation function at the cellular level were conducted。CL-tTA positive mice mate with TRE-Kras-G12D mice and then double positive mice weregained.Finally, respectively some double positive mice were treated with Adeno-cre virusthrough the nasal cavity injection and others were treated with Adeno-cre virus through theintradermal injection.Then the mice were dissected after three months and HE staining wereconducted to observe whether the organizations form tumors.The experimental results show that the CL-tTA transgenic mice which were single copynumber and can express tTA protein highly were successful to be established.Mating with TRE-Kras-G12D mice, double positive mice were gained.After the double positive mice were treatedwith Adeno-cre virus through the nasal cavity and intradermal injection, skin tumors wasinduced and lung tissues were abnormal three months later.In this article the CL-tTA transgenicmice have the potential to express tTA protein highly and the double positive mice that weretreated with Adeno-cre virus at the right time and different parts form some kinds of tumors.Allof these can provide premise and foundation for the further research on the role of Kras-G12Din the development of these tumors.