CRISPRCas9介导hFAD3基因在牛CSN2位点定点整合研究.docx

优秀毕业论文 精品参考文献资料 CRISPR／ CRISPR／Cas9介导hFAD3基因在奶牛CSN2位点定点整合研究 摘要 脂肪酸去饱和酶一3(fatty acid desaturase 3，FAD3)基因编码n．3多不 饱和脂肪酸脱氢酶，能够将n-6多不饱和脂肪酸(polyunsaturated fatty aci ds，PUFAs)转化成n．3 PUFAs。本试验成功构建基因打靶载体pCSN2．hF AD3和具有切割活性的pCas．Guide一1载体。利用CRISPR／Cas9技术将hFA D3基因定点整合到CSN2位点，研究hFAD3基因在乳腺细胞中特异表达， 及其对脂肪酸相关基因表达的影响。结果表明， (1)外源基因己定点整合 到了CSN2靶位点。 (2)实时定量PCR检测hFAD3基因在转染后乳腺细 胞和成纤维细胞中的表达结果显示，乳腺细胞中hFAD3基因的表达量为成 纤维细胞中的2．5倍(PO．01)，说明hFAD3基因的表达具有一定的乳腺特 异性。 (3)脂肪酸含量分析结果显示，转染后细胞n一6／n．3比值较对照细胞 显著下降(P0．01)。 (4)脂肪酸相关基因的表达检测结果显示，两种细 胞中脂肪分解相关基因(LPL、LIPE和PPARg)表达量大于脂肪合成相关 基因(FASN、ACC和SCD)。 (5)通过流式分选法及无限稀释法筛选单 克隆细胞，经PCR鉴定和测序分析，共得到7株定点整合阳性细胞株。并 检测单克隆细胞中脂肪酸含量变化和脂肪酸相关基因表达，结果与转染后细 胞中的结果一致。 上述结果表明，利用内源性p一酪蛋白启动子可以实现hFAD3基因在细胞 水平良好表达，为生产乳腺特异表达hFAD3基因牛提供实验数据。 关键词：CRISPR／Cas9；hFAD3；CSN2；定点整合；乳腺特异性；n一3 PUFAs； 脂肪酸相关基因 万方数据 CRISPR／ CRISPR／Cas9介导hFAD3基因在奶牛CSN2位点定点整合研究 张英 INTEGRATIoN oF HFAD3 GENE AT THE CSN2 LoCUS VIA CRISPR／CAS9 IN HoLSTEIN COW ABSTRACT Fatty acid desaturase 3(FAD3)gene encoding n一3 polyunsaturated fatty acid desaturases，which convert n-6 polyunsaturated fatty acids(PUFAs)to n-3 PUFAs．In the present study，we constructed a cassette gene targeting vector pC SN2一hFAD3 and a activated pCas—Guide一1 vector，which integrated hFAD3 gene into the CSN2 locus via CRISPR／Cas9．The vectors were transfected respectively to bovine fetal fibroblast cells and mammary gland cells．The integration and expression of the transferred hFAD3 genes were studied，and the compositions of PUFAs and the expressions of the fatty aids associated genes were also investigated．The results showed that exogenous gene has been integrated into CSN2 site．The expression level of hFAD3 gene in mammary gland cells is 2．5 times higher than that in the fibroblast cells(P0．0 1)verified by quantitative polymerase chain reaction，which indicated the more specific expression of exogenous genes in gland cells than the fibroblasts．Fatty acid analysis showed that the n一6 PUFAs／n-3 PUFAs ratio significantly decreased in the transfected cell