Construction And In Vitro Expression Of Doxycycline Inducible Cre Recombinase Vector

Conditional gene knockout mediated by Cre/loxP system is one of the most important technologies for the study of gene function.It circumvented some limitations of conventional knockout technology.On its basis , the inducible conditional gene knockout technology which combinates Tet gene expression system with Cre / loxP system can be used to study the function of genes more precisely.This technology requires two types of genetically engineered mice,one type is transgenic mice which can express Cre recombinase in the inducible manner,the other is gene targeting mice with loxP sites on both sides of targeting gene fragments.By the cross breeding of these two mouse lines, the inducible time-specific gene knockout can be achieved.In this study two eukaryotic expression vectors were constructed and identified.They can inducibly express Cre recombinase. And the transcription activity of the vectors was also studied. This study lays the foundation for the generation of the mice inducibly expressing Cre and the conditional ADAM10 gene knockout mice, which will be helpful to understand the function of ADAM 10 gene.By using the molecular biology technology, pTRE vectors of the Tet-On Gene Expression System were linearized and ligated to a 2.7kb fragments from the plasmid pCre-IRES-EGFP, as a result, the recombinant plasmid pTRE-Cre-EGFP (P1)was constructed.Then P1 and the pTet-On plasmid which was also from the Tet-On Gene Expression system were cotransfected into 293T cells with or without Dox, we observed whether or not there would be the green fluorescence and the expression of Cre recombinase. Phcmv-Cre-EGFP DNA fragments from the P1 vectors were ligated to Pcmv-rtTA fragments from the pTet-On vectors to form the vector P2. Then P2 vectors was transfected into 293T cells with or without Dox, we also observed green fluorescence and the expression of Cre recombinase, respectively.Plasmid pCre-IRES-EGFP was used as a positive control,either in the cotransfection of P1 and pTet-On or in the transfection of P2. And the inducible expression of Cre recombinase was identified by RT-PCR.Result: Restriction endonuclease digestion and sequencing showed that vector P1 and P2 were successfully constructed.After P1 and pTet-On plasmid were cotransfected into 293T cells mediated by LP2000, without the existence of Dox,there was little or no expression of green fluorescence,while the positive control group expressed green fluorescence. after dox was added, the cotransfection of P1 and the pTet-On plasmid could induce the expression of green fluorescence. 48 hours after the transfection, RNAs were extracted for RT-PCR detection, respectively. The expression of Cre gene can be detected at mRNA levels, so long as the green fluorescence was induced.Otherwise, there was no expression of Cre gene.After P2 was transfected into 293T cells mediated by LP2000, without the Dox,there was little or no expression of green fluorescence.Only the positive control group expressed green fluorescence. But when dox was added, the transfection of P2 could induce the expression of green fluorescence. 48 hours after the transfection, RNAs were extracted for RT-PCR detection. When green fluorescence was induced, the expression of Cre gene can be detected at the mRNA level.Or there was no expression of Cre gene.In conclusion, we successfully constructed and identified vectors that can inducibly express Cre recombinase (P1 and P2). The cotransfection of P1 and the pTet-On plasmid can induce the expression of green fluorescence in 293T cells.And the transfection of P2 can also induce the expression of green fluorescence in 293T cells.And the expression of Cre gene can be detected at the mRNA level. The results prove that the vector P1 and P2 we constructed induce the expression of Cre recombinase gene in time-specific manner, confirming that the gene elements in the vector P1 and P2 play a normal role in the cells. Both vector P1 and P2 can be used to prepare the Cre transgenic mouse. Moreover, we only prepare a kind of transgenic mice with the vector P2, which will satisfy the requirements for our research.