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| Site-specific Integration of hFAD3 Gene in Bovine (Bos taurus) NCAPG-LCORL Locus Mediated by CRISPR/Cas9 |
| GUANG Lu, ZHANG Ying, GUO Jing, BAI Chun-Ling*, WEI Zhu-Ying, YU Chao-Ran, HU Ting-Mao, LI Guang-Peng |
| State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock, Inner Mongolia University, Hohhot 010070, China |
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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-3 PUFAs 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) were successfully 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 knocked-in 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 up-regulated 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 hFAD3 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.
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Received: 26 July 2018
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Corresponding Authors:
* , chunling1980_0@163.com
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