以生殖细胞为外源基因载体建立异种移植转基因供体动物的研究
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摘要
建立转基因动物是异种移植研究中极为重要的工作基础。我们采用以生殖细胞(卵细胞和精子细胞)为外源基因载体的新型转基因技术建立转基因动物，以简化操作、降低成本、提高外源基因整合和表达的效率。
第一部分卵巢注射法建立hCD59、hCD55、HT单基因转移小鼠的研究
目的探讨卵巢注射法建立异种移植用转基因动物的可行性。方法采用注射法将hC955、HT、hCD59基因重组质粒分别导入雌鼠卵巢内，交配后产下原代(G_0代)小鼠。提取G_0代小鼠基因组DNA，采用PCR方法对目的基因整合进行初筛。对PCR出现特异条带的小鼠基因组DNA进行Southern印迹杂交，进一步证实目的基因整合。抽取目的基因整合阳性小鼠的外周血，用逆转录-聚合酶链反应(RT-PCR)和流式细胞术(FCM)检测目的基因表达。对FCM表达阳性的转基因小鼠肝脏和肾脏进行免疫组织化学染色，观察目的基因在器官中的表达分布情况。对表达目的基因的G_0代小鼠进行交配生产F1代小鼠，同法检测F1代目的基因的整合与表达。分离3种转基因小鼠的脾脏淋巴细胞，进行人血清溶破实验。结果对15只雌性小鼠进行卵巢注射，共产生G_0代鼠130只。其中，注射HT基因重组质粒的小鼠产仔62只，注射hCD59基因重组质粒的小鼠产仔42只，注射hCD55基因重组质粒的小鼠产仔26只。经PCR初筛后，Southern印迹杂交证实染色体中整合HT、hCD55、hCD59基因的小鼠分别有21只、4只、9只，总整合率为26.2％，高于受精卵显微注射法的整合率。经RT-PCR检测共20只G_0代小鼠阳性，包括3只hCD55小鼠、14只HT小鼠、3只hCD59小鼠。FCM检测发现外周血单核细胞表达人H抗原、hCD55、hCD59的小鼠分别有9只、2只、2只，蛋白表达的效率为10.0％，高于受精卵显微注射法的表达率。免疫组化检测显示，在转基因小鼠的肝脏、肾脏等组织中均有相应外源基因的表达。G_0代小鼠交配后产下F1代小鼠37只，PCR检测hCD55、HT、hCD59基因整合的小鼠分别为7只、6只、3只，FCM检测表达的分别为0只、1只、1只。与对照组比较，三种转基因小鼠的脾脏淋巴细胞耐受人血清溶破的能力均有所增强。结论采用卵巢注射法可以使针对异种移植的基因在小鼠体内整合，并可实现RNA水平和蛋白质水平的表达，其整合与表达的效率要高于受精卵显微注射法。三种转基因构件均可遗传给后代(F1代)，并在蛋白质水平表达。人血清溶破实验证实，通过卵巢注射法制备的三种转基因动物可以发挥抗异种排斥的作用。
第二部分卵巢内共注射建立hCD59／HT双基因和hCD55／hCD59／HT三基因转移小鼠的初步研究
目的探讨卵巢内多基因共注射建立多基因转移动物的可行性。方法采用hCD59／HT双基因重组质粒和hCD59／hCD55／HT三基因重组质粒共同注射卵巢的方法，得到原代小鼠。PCR和Southern印迹杂交法检测原代小鼠目的基因整合，FCM检测目的基因表达。结果双基因重组质粒共同注射4只雌鼠，获得G_0代鼠31只。Southern印迹杂交证实仅出现hCD59／HT双基因阳性鼠2只，无单基因整合鼠，仅1只鼠出现hCD59／HT共表达阳性，另1只鼠表达阴性。三基因重组质粒共同注射5只雌鼠，3胎共产生G_0代鼠137只，Southern印迹杂交证实仅出现hCD59／HT双基因阳性鼠2只，无其它整合情况，表达均为阴性。结论多基因共注射卵巢法可以得到相应的转基因动物，该方法确实可靠且周期较短。但多基因共注射后，基因之间会互相影响，整合与表达的效率比单基因注射者明显降低，提示其中有更复杂的机制参与，需要深入研究。
第三部分PAMAM-D对hCD55基因转染猪精子细胞介导作用的研究
目的探讨新型纳米材料PAMAM-D对hCD55基因转染猪精子细胞的介导作用。方法将新型纳米材料PAMAM-D(G_5)和线状hCD55 DNA按照不同氮磷比(电荷比)制备PAMAM-D／hCD55复合物。取部分复合物酶切电泳。将复合物与处理后的1×10~6猪精子细胞共孵育2h，原位杂交法检测hCD55对猪精子细胞的转染效率，经精子质量检测工作站检测孵育后的精子活力和精子畸形率。结果对PAMAM-D／DNA复合物进行酶切消化后，其中的DNA分子不被限制性内切酶降解。经原位杂交法检测，加入PAMAM-D后可以明显提高各组猪精子细胞的转染效率，且基本上随着氮磷比的增加转染效率也随之增高。最高者(400ng，氮磷比20：1)可达对照组的167％(47.5％±3.5％vs 28.5％±1.5％)。经精子质量检测工作站检测PAMAM-D对精子细胞活力和精子细胞畸形率没有明显影响(p＞0.05)。结论PAMAM-D作为载体可以提高目的基因对猪精子细胞的转染效率，其对猪精子细胞无明显毒性，增强了精子载体法的实用性，为生产异种移植用转基因猪提供一种可供参考的方法。
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.
SECTIONⅠReasch on establishing single-transgenic mice carrying humanCD59 or human CD55 or HT gene by ovary injection
Objective 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(G_0) after mating. Genomic DNAwas extracted from all of liveborn G_0 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 G_0 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.
SECTIONⅡPreliminary study on stablishing multi-transgenic mice carryinghCD59 /HT genes & hCD59/hCD55/HT genes by ovary injection
Objective 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 G_0 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 G_0 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.
SECTIONⅢ Research on polyamidoamine dendrimers mediated hCD55 genetransfected pig sperm cells
Objective To investigate effect of the new nanomaterialspolyamidoamine dendrimers(PAMAM-D) mediated hCD55 genetransfectedpig sperm cells. Methods Polyamidoamine dendrimersG_5 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 1×10~6 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.
第一部分卵巢注射法建立hCD59、hCD55、HT单基因转移小鼠的研究
目的探讨卵巢注射法建立异种移植用转基因动物的可行性。方法采用注射法将hC955、HT、hCD59基因重组质粒分别导入雌鼠卵巢内，交配后产下原代(G_0代)小鼠。提取G_0代小鼠基因组DNA，采用PCR方法对目的基因整合进行初筛。对PCR出现特异条带的小鼠基因组DNA进行Southern印迹杂交，进一步证实目的基因整合。抽取目的基因整合阳性小鼠的外周血，用逆转录-聚合酶链反应(RT-PCR)和流式细胞术(FCM)检测目的基因表达。对FCM表达阳性的转基因小鼠肝脏和肾脏进行免疫组织化学染色，观察目的基因在器官中的表达分布情况。对表达目的基因的G_0代小鼠进行交配生产F1代小鼠，同法检测F1代目的基因的整合与表达。分离3种转基因小鼠的脾脏淋巴细胞，进行人血清溶破实验。结果对15只雌性小鼠进行卵巢注射，共产生G_0代鼠130只。其中，注射HT基因重组质粒的小鼠产仔62只，注射hCD59基因重组质粒的小鼠产仔42只，注射hCD55基因重组质粒的小鼠产仔26只。经PCR初筛后，Southern印迹杂交证实染色体中整合HT、hCD55、hCD59基因的小鼠分别有21只、4只、9只，总整合率为26.2％，高于受精卵显微注射法的整合率。经RT-PCR检测共20只G_0代小鼠阳性，包括3只hCD55小鼠、14只HT小鼠、3只hCD59小鼠。FCM检测发现外周血单核细胞表达人H抗原、hCD55、hCD59的小鼠分别有9只、2只、2只，蛋白表达的效率为10.0％，高于受精卵显微注射法的表达率。免疫组化检测显示，在转基因小鼠的肝脏、肾脏等组织中均有相应外源基因的表达。G_0代小鼠交配后产下F1代小鼠37只，PCR检测hCD55、HT、hCD59基因整合的小鼠分别为7只、6只、3只，FCM检测表达的分别为0只、1只、1只。与对照组比较，三种转基因小鼠的脾脏淋巴细胞耐受人血清溶破的能力均有所增强。结论采用卵巢注射法可以使针对异种移植的基因在小鼠体内整合，并可实现RNA水平和蛋白质水平的表达，其整合与表达的效率要高于受精卵显微注射法。三种转基因构件均可遗传给后代(F1代)，并在蛋白质水平表达。人血清溶破实验证实，通过卵巢注射法制备的三种转基因动物可以发挥抗异种排斥的作用。
第二部分卵巢内共注射建立hCD59／HT双基因和hCD55／hCD59／HT三基因转移小鼠的初步研究
目的探讨卵巢内多基因共注射建立多基因转移动物的可行性。方法采用hCD59／HT双基因重组质粒和hCD59／hCD55／HT三基因重组质粒共同注射卵巢的方法，得到原代小鼠。PCR和Southern印迹杂交法检测原代小鼠目的基因整合，FCM检测目的基因表达。结果双基因重组质粒共同注射4只雌鼠，获得G_0代鼠31只。Southern印迹杂交证实仅出现hCD59／HT双基因阳性鼠2只，无单基因整合鼠，仅1只鼠出现hCD59／HT共表达阳性，另1只鼠表达阴性。三基因重组质粒共同注射5只雌鼠，3胎共产生G_0代鼠137只，Southern印迹杂交证实仅出现hCD59／HT双基因阳性鼠2只，无其它整合情况，表达均为阴性。结论多基因共注射卵巢法可以得到相应的转基因动物，该方法确实可靠且周期较短。但多基因共注射后，基因之间会互相影响，整合与表达的效率比单基因注射者明显降低，提示其中有更复杂的机制参与，需要深入研究。
第三部分PAMAM-D对hCD55基因转染猪精子细胞介导作用的研究
目的探讨新型纳米材料PAMAM-D对hCD55基因转染猪精子细胞的介导作用。方法将新型纳米材料PAMAM-D(G_5)和线状hCD55 DNA按照不同氮磷比(电荷比)制备PAMAM-D／hCD55复合物。取部分复合物酶切电泳。将复合物与处理后的1×10~6猪精子细胞共孵育2h，原位杂交法检测hCD55对猪精子细胞的转染效率，经精子质量检测工作站检测孵育后的精子活力和精子畸形率。结果对PAMAM-D／DNA复合物进行酶切消化后，其中的DNA分子不被限制性内切酶降解。经原位杂交法检测，加入PAMAM-D后可以明显提高各组猪精子细胞的转染效率，且基本上随着氮磷比的增加转染效率也随之增高。最高者(400ng，氮磷比20：1)可达对照组的167％(47.5％±3.5％vs 28.5％±1.5％)。经精子质量检测工作站检测PAMAM-D对精子细胞活力和精子细胞畸形率没有明显影响(p＞0.05)。结论PAMAM-D作为载体可以提高目的基因对猪精子细胞的转染效率，其对猪精子细胞无明显毒性，增强了精子载体法的实用性，为生产异种移植用转基因猪提供一种可供参考的方法。
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.
SECTIONⅠReasch on establishing single-transgenic mice carrying humanCD59 or human CD55 or HT gene by ovary injection
Objective 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(G_0) after mating. Genomic DNAwas extracted from all of liveborn G_0 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 G_0 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.
SECTIONⅡPreliminary study on stablishing multi-transgenic mice carryinghCD59 /HT genes & hCD59/hCD55/HT genes by ovary injection
Objective 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 G_0 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 G_0 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.
SECTIONⅢ Research on polyamidoamine dendrimers mediated hCD55 genetransfected pig sperm cells
Objective To investigate effect of the new nanomaterialspolyamidoamine dendrimers(PAMAM-D) mediated hCD55 genetransfectedpig sperm cells. Methods Polyamidoamine dendrimersG_5 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 1×10~6 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.
引文
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