【摘要】 亚麻(Linum usitatissimum)脂肪酸去饱和酶基因3(fatty acid desaturase 3, FAD3)是一种脂肪酸脱氢酶基因,编码ω-3多不饱和脂肪酸(polyunsaturated fatty acids, PUFAs)脱氢酶,能够将ω-6 PUFAs转化成ω-3PUFAs。本研究利用CRISPR/Cas9介导,将无抗性、无标记的人(Homo sapiens)源化h FAD3基因表达载体C2(5’同源臂-CAG-hFAD3-PolyA-3’同源臂)和M2(5’同源臂-MAR-CAG-hFAD3-PolyA-MAR-3’同源臂)定点敲入牛(Bos taurus)胎儿成纤维细胞NCAPG-LCORL位点。运用气质联用仪对脂肪酸含量进行分析,结果表明hFAD3转基因细胞中ω-6/ω-3 PUFAs比值(0.565)较非转基因细胞(1.549)显著下降(P<0.01)。qRT-PCR结果表明h FAD3基因整合并表达后,脂肪分解相关的过氧化物酶体增殖物激活受体基因(peroxisome proliferator activated receptor1, PPARg)、激素敏感脂肪酶基因(hormone-sensitive lipase, LIPE)和脂蛋白脂酶(lipoprotein lipase, LPL)表达总量上调倍数大于脂肪合成相关的乙酰辅酶A羧化酶基因(acetyl CoA carboxylase, ACC)、硬脂酰辅酶A去饱和酶基因(stearoyl CoA desaturase, SCD)和脂肪酸合酶基因(fatty acid synthase, FASN)表达总量,说明hFAD3基因的表达使得细胞内的脂肪趋于分解。通过流式分选获得59株C2打靶载体单克隆细胞株,PCR鉴定及测序结果表明,其中定点整合阳性单克隆细胞3株,定点整合效率为5.1%。同时检测定点整合阳性单克隆细胞株中脂肪酸的含量以及脂肪相关基因的表达量,结果与转染后细胞一致。比较有无核基质结合区(matrix attachment region, MAR)序列介导两种转基因细胞中hFAD3基因的表达量,结果显示,MAR介导组是无MAR组的5.91倍(P<0.01)。本研究利用CRISPR/Cas9介导可实现hFAD3基因在牛NCAPG-LCORL位点的定点整合以及在细胞水平正常行使其生物学功能,同时MAR的介导可显著提高外源基因的表达量,为安全高效生产转基因动物提供科学依据。更多还原

【Abstract】 Linum usitatissimum ω-3 polyunsaturated fatty acids(PUFAs) exert multiple physiology functions,which has to be provided from foods in mammals. The fatty acid desaturase-3 gene(FAD3) encodes a ω-3PUFAs desaturase and converts ω-6 PUFAs to ω-3 PUFAs. In the present study, the hFAD3 gene expression cassette C2(5’arm-CAG-hFAD3-PolyA-3’arm) and M2(5’arm-MAR-CAG-hFAD3-PolyA-MAR-3’arm) weresuccessfully knocked-in NCAPG-LCORL locus of bovine(Bos taurus) fetal fibroblast cells using CRISPR/Cas9 gene editing system. In the obtained transgenic cells, the ratio of ω-6 PUFAs and ω-3 PUFAs was significantly decreased from 1.549 to 0.565 investigated by gas chromatograph attached to mass spectrometer for fatty acid determinator(P<0.01). The expressions of lipolysis associated genes including peroxisome proliferator activated receptor 1 gene(PPARg), hormone-sensitive lipase gene(LIPE) and lipoprotein lipase gene(LPL)were more up-regulated than fatty acid synthesis associated genes including acetyl CoA carboxylase gene(ACC), stearoyl CoA desaturase gene(SCD) and fatty acid synthase gene(FASN) in the obtained transgenic cells detected by qRT-PCR. 59 monoclones were obtained by flow cytometry, in which 3 strains were knockedin cells. The knock-in efficiency was 5.1%. In the number 89 knocked-in monoclonal cells, the ratio of ω-6 and ω-3 PUFAs was significantly decreased from 1.589 to 0.575 compared with the negative monoclonal cells number 21(P<0.01). The expressions of lipolysis associated genes(PPARg, LIPE, LPL) were more upregulated than fatty acid synthesis associated genes(ACC, SCD, FASN) in the knocked-in monoclonal cells.The expressions of fatty acids and fatty acids metabolism related genes in the monoclonal cells were in consistent with the routine transgenic cells. In addition, the h FAD3 expression of transgenic cells mediated with matrix attachment region(MAR) was 5.91 times as much as without MAR(P<0.01), thus the MAR significantly increased hFAD3 expression than that of without MAR in transgenic cells. In conclusion, the hFAD3 gene could successfully knock-in at NCAPG-LCORL locus of bovine fetal fibroblast cells through CRISPR/Cas9 system and expressed in transgenic cells. The involvement of MAR element significantly increased the hFAD3 expression. This study may provide an alternative protocol to construct transgenic vector and probably be useful to produce transgenic animals.更多还原