摘要
转基因家畜是生物医学和农业研究领域的重要材料。而转基因动物生产过程中一般都需要用到抗生素筛选标记基因。获得转基因动物后，其体内残留的抗生素筛选标记存在生物安全方面的隐患，对转基因动物安全及环境安全造成了潜在的威胁。本研究旨在建立一种安全高效的方法，为体细胞核移植（SCNT）提供无抗生素筛选标记的具有良好发育潜能的转基因供体细胞，为培育无抗生素筛选标记的转基因动物奠定基础。研究内容如下：
1.基于假attP位点整合的通用型表达载体构建及功能验证
利用重叠延伸PCR（SOE-PCR）合成attB序列及其他载体元件，构建基于假attP位点整合的通用型表达载体pARNG，评估载体pARNG的主要功能元件。结果显示：通过SOE-PCR合成的attB片段能够在phiC31整合酶的介导下发生位点特异性重组，载体中的双荧光报告系统可以正确指示转染阳性细胞和Cre介导的重组反应。
2.利用phiC31整合酶mRNA生产转基因胎牛成纤维细胞
通过体外转录制备phiC31整合酶（Int）mRNA和突变失活型整合酶（mtInt）mRNA。构建验证phiC31整合酶功能的载体pPBstop-eGFP并与phiC31mRNA共电转胎牛成纤维细胞，通过流式细胞术（FACS）分析和γ-H2AX免疫荧光染色确定转染试验中phiC31IntmRNA最优使用剂量为1μg。构建含有attB位点的HBD3乳腺特异性表达载体pARNG-HBD3，在phiC31mRNA的介导下转染胎牛成纤维细胞，在稳定转染的细胞克隆中检测到7个假attP位点，其中BFF2为整合热点，28%稳定转染克隆中存在该位点的整合。利用生物信息学软件分析这些位点序列特征，并按照“基因组安全港”标准重新评估牛基因组中已公布的所有假attP位点。经过进一步鉴定获得HBD3基因单拷贝整合在BFF2“基因组安全港”的细胞克隆。
3.利用Cre穿膜肽切除转基因细胞中的抗性筛选标记
原核表达、纯化His-NLS-TAT-Cre穿膜蛋白，同时构建验证Cre重组酶功能的胎牛成纤维细胞株BFF2-L2stop优化Cre穿膜肽转导条件。结果显示使用终浓度为100μg/mLHis-NLS-TAT-Cre穿膜蛋白转导胎牛成纤维细胞时，重组效率可达70%，并且对细胞无明显毒性。通过细胞免疫荧光染色证实His-NLS-TAT-Cre重组蛋白在细胞中主要定位于细胞核。利用His-NLS-TAT-Cre重组蛋白转导单拷贝整合pARNG-HBD3的转基因胎牛成纤维细胞并进行FACS分选，Southern blot证实分选得到的只表达绿色荧光的细胞即是切除了抗生素筛选标记的转基因细胞。此外，本研究还证实了phiC31Int mRNA介导的BFF2位点的整合及后续His-NLS-TAT-Cre重组蛋白介导的抗生素筛选标记的切除不会对整合位点旁侧基因的表达造成影响，且获得的转基因细胞染色体数目正常（2n=58+XX）。
4.通过SCNT生产无抗生素筛选标记的转HBD3基因克隆牛
以去除抗性筛选标记的转HBD3基因胎牛成纤维细胞作为供体细胞进行体细胞核移植，获得转基因囊胚后经过胚胎移植，最终获得12头健康的无抗生素筛选标记的转基因奶牛，对其中5头泌乳期的转基因奶牛进行分析，在乳汁中均能检测到HBD3表达，含量为3.9~10.4μg/mL。利用体外琼脂糖扩散试验和乳腺内攻菌试验检验转基因牛抗S.aureus和E.coli感染能力。在分别灌注S. aureus和E.coli细菌培养物的15个转基因奶牛乳房中，检测到33.3%和6.7%的乳腺发生感染，而灌注非转基因奶牛的15个乳房分别检测到93.3%和86.7%的乳腺发生感染。结果表明，转HBD3基因克隆牛可以显著抵抗S. aureus和E. coli的感染。值得注意的是，HBD3基因整合在BFF2位点的转基因奶牛乳汁中HBD3含量较高,在攻菌试验中从未感染。
研究表明通过联合使用phiC31整合酶mRNA、His-NLS-TAT-Cre穿膜蛋白、双荧光报告系统和FACS分选技术可以安全高效地生产无抗生素筛选标记的转基因供体细胞，为SCNT准备具有良好发育潜能的转基因供体细胞，并培育出对乳腺炎致病菌具有抵抗能力的转基因克隆牛。
Transgenic farm animals are important materials for biomedical and agricultural research.Antibiotic selectable marker genes have been widely used to generate transgenic animals.Once transgenic animals have been obtained, the selectable marker is no longer necessary butraises public concerns regarding biological safety. The aim of this study was to establish asafe and efficient method to prepare competent antibiotic selectable marker free transgeniccells for somatic cell nuclear transfer (SCNT), which may lay the foundation for breeding ofantibiotic selectable marker free transgenic animals. The main contents of this research are asfollows:
1. Construction and function evaluation of general expression carrier based onpseudo attP site integration
Full-length attB and other vector components were generated using splicing by overlapextension (SOE) PCR, and then the general expression carrier pARNG was constructed.Functional evaluation was performed on attB site and fluorescent double reporter of pARNG,and the results showed that the synthetic attBwas competentin site-specific integrationmediated by phiC31integrase, and the fluorescent double reporter could be used to monitortransfected cells and Cre-mediated excision.
2. Generation of transgenic bovine fetal fibroblasts using phiC31integrase mRNA
PhiC31integrase (Int) mRNA and inactive mutant phiC31integrase (mtInt) mRNA weregenerated by in vitro transcription. In order to optimize phiC31mRNA mediated transfection,bovine fetal fibroblasts were electroporated with the phiC31integrase activity reporter vectorpBP-stop-eGFP in the presence of phiC31mRNA. The optimized dosage was determined as1μg by using flow cytometry analysis (FACS) and immunofluorescence assay against γ-H2AX.An attB-containing human β-defensin-3(HBD3) mammary gland expression vectorpARNG-HBD3was constructed and electroporated into bovine fetal fibroblasts with phiC31mRNA. A total of seven pseudo attP site were detected and analyzed in stably-transfected cellcolonies,28%of which were detected to integrate at BFF2hot spot. In addition, all published pseudo attP sites in bovine genome were re-analyzed according to the criteria of “genomicsafe harbor”. Finally, transgenic cell colonies with single-copy integration ofHBD3gene intoBFF2genomic safe harbor were obtained.
3. Excision of the antibiotic selectable marker from transgenic cells usingcell-permeant Cre recombinase
Prokaryotic expression and purification of His-NLS-TAT-Cre recombinant protein wasperformed. The Cre recombinase activity reporter fetal fibroblast cell line BFF2-L2GFP wasgenerated to optimize conditions of His-NLS-TAT-Cre mediated protein transduction.Immunofluorescence results showed that His-NLS-TAT-Cre was mainly localized in nucleolus.Excision of antibiotic selectable marker was verified by Southern blot in the EGFP positivecells sorted by FACS after His-NLS-TAT-Cre protein transduction on pARNG-HBD3stably-transfected cell colonies. RT-PCR results showed that phiC31mRNA mediatedinsertion and His-NLS-TAT-Cre protein mediated excision did not alter the expression offlanking genes. In addition, transgenic cells generated by this procedure had the normalchromosome count (2n=58+XX).
4. Generation of cloned transgenic cattle that express human β-defensin-3
Antibiotic selectable marker free cells from different clones were used as nuclei donorsfor production of transgenic cattle. Finally, twelve transgenic cattle were obtained. Byevaluation of transgenic milk samples from five transgenic cows, which were lactatingnormally in the observation period, the expression of HBD3was detected, ranged from3.9to10.4μg/mL. The transgenic cows’ ability to resist infection by S. aureus and E. coli wasconfirmed by intramammary infusion of viable bacterial cultures. Notably, the higherexpressing transgenic cows, which derived from the evaluated BFF2safe harbor integratedtransgenic cells, never became infected after S. aureus or E. coli infusion.
All of above results indicated that combination of phiC31integrase mRNA,cell-permeant Cre protein and fluorescent double reporterprovided a safe and efficientalternative to prepare competent antibiotic selectable marker free transgenic cells for SCNTand sequentially produced antibiotic selectable marker free mastitis-resistant transgenic cattle.
引文
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