The Study On Transferring Of Human Tumor Suppressor Gene P53 In Chicken

Objective:The recombinant plasmid pEGFP-N1-P53 with human tumor suppressor gene P53 and enhanced green fluorescent protein (EGFP) gene was constructed, transfection of the chicken blastoderm X,cultivate gene transfer of human tumor suppressor gene chicken.Methods:Human tumor suppressor gene P53 was cloned by RT-PCR from early lung cancer patient peripheral blood,amplification Plasmid pMD18-T-P53 was constructed by the method of digestion ligation and transformation.Then constructed eukaryotic expression plasmid pEGFP-N1-P53 with enhanced green fluorescent protein (EGFP) gene.Chicken embryonic fibroblast cells transfected by the method of Liposome mediated pEGFP-N1-P53,continued incubation,EGFP gene expression was observed under fluorescence microscope.Design experiments,to find out the right way to seal chicken eggs fenestration.liposome-mediated eukaryotic expression plasmid pEGFP-N1-P53 transfected into chicken X blastoderm by microinjection,continue incubation After sealing,detection of exogenous gene expression and integration.Results: The main results of this study are as follows:(1)Human tumor suppressor gene P53 was cloned from Human peripheral blood, the recombinant plasmid pEGFP-N1-P53 was constructed With pEGFP-N1 eukaryotic expression vector,recombinant plasmid was identified by PCR amplification, restriction enzyme digestion and sequencing analysis,transfected chicken blastoderm fibroblasts, Confirmed that Successfully constructed of the recombinant plasmid pEGFP-N1-P53.(2)To study different sealing methods on Hatchability,six different sealing ways was established,they were the eggshell membrane+sealed plastic,eggshell membrane +plastic wrap,eggshell membrane+paraffin wax,eggshell membrane+sealed membrane, eggshell membrane+eggshell and eggshell membrane+egg white,the control group was established.results suggested that embryo hatching rate of the six experimental group were10%,3.3%, 3.3%, 6.7%,13.3% and16.7%,hatching rate of the control group 86.7%,The hatching rate was the highest relatively by Sealing method of eggshell membrane+egg white.Experiment was designed by combination blastoderm microinjection with eggshell membrane+egg white sealing method,test group included 150 eggs,121 embryos dead,the cumulative rate of dead embryos was 80.6%;hatched 29 chicks, hatching rate was 19.3%,21 dead,the mortality rate was 72.4%.In the 121 dead chicken embryo, the expression of green fluorescent protein was detected from the tissue of 20 eggs, the detection rates was 16.5%. in the 21chicken tissues,the expression of green fluorescent protein was detected from the tissue of 1 chicken,the detection rates was 4.7%.PCR method detected the presence of the target gene,found that the five positive chick embryo for the purpose of gene integration, integration rate was 5.33% (8 / 150).Conclusion: The human tumor suppressor gene P53 was cloned in wild type and its eukaryotic expression vector pEGFP-N1-P53 was constructed successfully, Confirmed that exogenous gene mediated by liposome,with blastoderm microinjection could make transgenic chicken.Blastoderm microinjection was an effective method to produce transgenic chickens.