Study On Establishing Transgenic Animals By Germ Cells As Exogenous Gene Vector For Xenotransplantation

The establishment of transgenic animals is an extremelyimportant foundation for future work in Xenotransplantation. Weused new methods of gene transfer by germ cells (eggs and spermcells) as exogenous gene vector to establish transgenie animals. Wehope the methods can simplify operations, reduce costs and improvethe efficiency of integration and expression securely.SECTIONâ… Reasch on establishing single-transgenic mice carrying humanCD59 or human CD55 or HT gene by ovary injectionObjective To investigate the feasibility of establishingtransgenic animals by ovary injection for xenotransplantation.Methods The hCD55, HT, hCD59 recombinant plasmid was injected intothe ovary of female mice respectively. Then the female mice gavebirth to the original generation mice(G₀) after mating. Genomic DNAwas extracted from all of liveborn G₀ mice. Initially screening ofgene integration was used by polymerase chain reaction, subsequ-ently Southern blot analysis of mice genomic DNA which specificPCR bands appeared, further confirmed integration of those targetgenes. Expression of the target genes on peripheral bloodmononuclear cells (PBMCs) of transgenic mice were detected bytranscriptase-polymerase chain reaction (RT-PCR) and flowcytometry (FCM). For those FCM positive expression mice,immunohistochemieal staining was performed to examine thedistribution of target genes in the liver and kidney tissues fromthe transgenic mice. The gene expression mice were bred with commonmice to generate F1 mice. Screening was performed as described above. After Separation of three transgenic mouse spleenlymphocytes, the human serum-mediated cytolysis experiment wasconducted. Results There were 15 female mice by injectionovary, and then the mice produced 130 G₀ generation. Among them,these mice included 62 pregnant mice by injected HT recombinantplasmid, 42 pregnant mice by injected human CD59 recombinantplasmid and only 26 pregnant mice by injected human CD55recombinant plasmid. After screening by PCR, Southern blotconfirmed chromosome integration of HT, hCD55, hCD59 gene in micewere 21,4,9. The total integration rate was 26.2%, higher than thegene integration rate through microinjection. There were 20positive mice by RT-PCR detection, including three hCD55 mice, 14HT mice, three hCD59 mice. FCM detected in peripheral bloodmononuclear cells expressing human H antigen, hCD55, hCD59 micewere 9, 2, 2. The efficiency of protein expression was 10.0%,higher than the expression rate of micro-injection. Immunohist-ochemical detection showed that the transgenic mice liver andkidney tissues were corresponding exogenousgeneexpression. Among37 F1 generation mice, the hCD55, HT, hCD59 gene integrated micewere seven, six, three by PCR, and FCM detection of the three geneexpression was 0, 1, 1. Compared with the control group, the threetransgenic mouse spleen lymphocytes in human serum have higersurvival rate. Conclusion The method of ovary injection canintegrate target genes for xenotransplantation in mice genome, andlead to the expression of RNA and protein levels. The efficiencyof gene integration and expression were higher than that ofmicroinjection. Three transgenic components can be transmitted tosubsequent generations (F1), and caused protein expression. Humanserum-mediated cytolysis experiment confirmed that the threetransgenic animals by ovary injection can play the role inanti-xenograft rejection. SECTIONâ…¡Preliminary study on stablishing multi-transgenic mice carryinghCD59 /HT genes & hCD59/hCD55/HT genes by ovary injectionObjective To investigate the feasibility of establishingmulti-gene transfer animals by ovary injection. Methods The HTtransgene construct mixed with the human CD59 transgene contructwere coinjected into mouse ovaries. And so were the above threetransgene contructs. PCR and Southern blot analysis were operatedto confirm chromosome integration of G₀ generation mice.Expression of the target genes was detected by FCM. ResultsThere were 4 female mice by ovary co-injected hCD59/HT genes, andthen these female mice produced 31 G₀ generation mice. Southernblot analysis confirmed that there were only two hCD59/HT doublegenes positive mice, not any single gene integration mouse. Boththem one expressed hCD59 and HT double genes, and the otherexpressed neither hCD59 gene nor HT gene. The three gene constructswere coinjeoted into five female mouse ovaries, and they produced137 offsprings in three broods. Southern blot analysis confirmedthat there were only two hCD59/HT double genes positive mice, noother integration mouse. Both expressed negative. ConclusionTransgenic mice have been cultured successfully by multi-genecoinjected ovary, which was authoritative and shorter. But aftermore genes injection, gene between them will affect each other.Efficiency of integration and expression of multi-gene coinjec-tion were less than that of the single gene injection, whichsuggested that we need study in depth because of a more complexsystem in participation.SECTIONâ…¢ Research on polyamidoamine dendrimers mediated hCD55 genetransfected pig sperm cellsObjective To investigate effect of the new nanomaterialspolyamidoamine dendrimers(PAMAM-D) mediated hCD55 genetransfectedpig sperm cells. Methods Polyamidoamine dendrimersG₅ and linear hCD55 DNA were incubated in different N/P ratio, andthey formed the PAMAM-D/hCD55 compounds. The compounds weredigested by restriction enzymes. At the same time, the compoundswere incubated with 1×10⁶ washed pig sperm cells for 2 hours. Thentransfection efficiency was detected by in situ hybridization andthe effects on sperm motility and deformity were evaluated by spermtesting workstation. Results After digested the compounds byrestriction enzymes, those DNAs in the compounds can not bedegraded. Detected by in situ hybridization, PAMAM-D can improvetransfection efficiency of pig sperm cells markedly. With theincrease of the N/P ratio transfection efficiency basically havealso been increased. Among them the maximum (400ng, N/P=20:1) wasup to 167% compared with control group (47.5％±3.5% vs 28.5%±1.5%). PAMAM-D had no impact on sperm motility and deformity(p＞0.05). Conclusion Polyamidoamine dendrimers as a vector canimprove the gene transfection efficiency and have no cytotoxi-city, so it increase the. practicality of SMGT. The approach mayprovid a available method for the production of transgenic pigsfor xenotransplantation.