Establishment Of Transgenic Mice Of EB Virus Early Gene BHRF1

EBV early gene BHRF1 was confirmed to be a new EBV oncogene in recent years.It can cause cell transformation in vitro,and probably play an important role in occurrence and development in EBV-associated tumor.However,EBV can only infect human being and a few primates,and have no suitable replication system in vitro. Transgenic mice could correctly mimic the physiological and pathological environment in vivo.Therefore,establishment of BHRF1 transgenic mice clearly contribute to elucidate the exact anti-apoptotic mechanisms of BHRF 1 coding protein, as well as the similarities and differences in function compared with the bcl-2.It will become ideal animal model for further study on the biological characteristics of BHRF 1 and exploring the mechanism of EBV oncogenicity.Objective To construct recombinant vector that stably express BHRF1 protein, and to establish EBV early gene BHRF1 transgenic mouse model by microinjection.Method①B95-8 cell RNA was extracted by TRIzol.BHRF1 gene specific primers were designed by Primer premier 5.0 software,and the restriction sites and the protective bases of EcoR I and EcoR V respectively were inserted into the 5' end of upstream and downstream primers.BHRF1 gene was amplificated by RT-PCR.②The PCR products of BHRF1 gene was ligated with pMD18-T vector,making up pMD18-T-BHRFI.③EcoR I and EcoRV were used to digest pMD18-T-BHRF1 and pcDNA(TM)3.1(+) vector,and then BHRF1 gene was ligated with pcDNA3.1 (+),establishing a recombinant vector pcDNA3.1-BHRF1.④pcDNA3.1-BHRF1 was digested by DraⅢand NruI,obtaining about 2000bp DNA fragment containing CMV promoter,the inserted fragment BHRF1 and BGH polyadenylation.This DNAfragment was purified with QIAquick Gel extraction Kit purification from 1% agarose gel,then dissolved in microinjection buffer(pH7.4),the concentration of 3ng/μL.⑤First,ligation of male mice is prepared to make pseudopregnancy mouse and make donor mouse at the same times,then donor mouse were killed for getting the fertilized eggs.The DNA fragment containing target gene was injected into the fertilized eggs by microinjection,and then transplante the fertilized eggs into pseudopregnant mouse uterine tube,development and giving birth offspring mice.⑥The offspring mice tails DNA was extracted and tested by PCR to identify whether the integration of target genes.Result①The 699bp target gene BHRF1 was ligated with pcDNA 3.1, establishing recombinant vecotr pcDNA3.1-BHRF1.The recombinant vecotr was idetified by PCR,restriction endonuclease analysis and sequencing.②About 2000bp of DNA fragment containing the CMV promoter,the inserted fragment BHRF1 and BGH polyadenylation and the 4100bp vector fragment could be observed when digested products was analyzed by electrophoresis.Target DNA fragment was purified,then dissolved in microinjection buffer for microinjection.③20 superovulation mouse were elected,18 mice were seen vaginal suppository,acquired 480 healthy fertilized eggs which were injected purified CMV+BHRF1+polyA DNA fragment by microinjection,390 fertilized eggs were still healthy after short-term culturing,and then were transplanted into 14 pseudopregnant mice uterine tube,11 were pregnant,giving birth 74 offspring mice.④Offspring mice tails DNA was extracted and tested by PCR,sixteen were positive,the positive rate is 21.6%(16/74), 16 founder mouse were acquired.Conclusion BHRF1 transgenic founder mice were successfully established,it provides an ideal animal model for further research on biological characteristics and the role of BHRF 1 in EBV-related tumor oncogenesis.