The Study On Establishing Transgenic Tupaias And Mouse With HBx, Kras Gene By Spermatozoa-mediated Gene Transfer

Objective The recombinant plasmids pcDNA3.1-HBx and pEGFP-N1-Kras coated with liposome were directly injected into the male tree threw's testis. To evaluate the feasibility of establishing transgenic tree threw with HBx and Kras gene by means of seminiferous tubule microinjection and sperm-mediated gene transfer, and to provide experiment base for investigating the role of HBx and Kras genes in pathogenesis of liver cancer with HBV.Methods Kras gene with Nheâ… and Kpnâ… endoenzyme sites was obtained by using PCR ,and subcloned into pEGFP-N1 vector. The reconstructed plasmid was identified by restrictive enzymes digestion and sequencing. The male and female adult tupaias were matched for breeding pairs. Files were made for each animal. The numbers of all pups, the annually average birthrate and the average days of pregnancy interval were calculated. The pup's diet and body weight daily were recorded. Three methods (Formulated milk, passively and actively maternal milk) were used to feed the pups. Sixteen male tree shrews were divided into pcDNA3.1-HBx group, pEGFP-N1-Kras group,pcDNA3.1-HBx + pEGFP-N1-Kras group and 0.9% NaCl group at random. The recombinant vectors of pcDNA3.1-HBx and/or pEGFP-N1-Kras coated with liposome were injected into the testicle tissue of these male tupaias after being diluted by DMEM. The treated animals were fertilized with females four weeks later. Liver biopsies were performed on the baby tupaias after one month of their birth. Polymerase chain reaction (PCR) was performed to detect integration of the target gene into the genomic DNA of the offspring. All cases were detected by immunohistochemical technique using a monoclonal anti-HBx anti-body and Rabbit polyclonal to GFP.Results The target gene Kras fragment about 567 bp was obtained. We performed the group experiment thrice during 22 months. All the tupaias injected with recombinant plasmids had reproductive ability. 128 first generation tupaias were born, and 58 survived. 4 of them were positive for HBx gene(positive rate: 3.57%).But all of them were negative for EGFP gene. 3 of the first generation mouse were positive for HBx. One of them were positive for EGFP. All cases were detected by immunohistochemical technique. The antibody-positive responses were detected in tupaia's hepatocelluar tissue while the antibody-negative responses were in comparison tissue.Conclusion Eukaryotic expression vector pEGFP-N1-Kras was constructed successfully. It is practical for using the spermatogonial cells as the vector to produce the transgenic tree shrews of HBx gene. But this method needs to be further tested for its potential utility in EGFP-Kras transgenic tree shrews generation.