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.
[1] 谌颜. 2014. fad3b乳腺特异表达盒转基因小鼠的研制[D]. 硕士学位论文, 内蒙古大学, 导师: 李光鹏. pp. 24-31. (Chen Y.2014. The production of transgenic mice by mammary gland specific fad3b transfer body[D]. Thesis for M.S., Inner Mongolia University, Supervisor: Li G P. pp. 24-31.) [2] 胡菡, 王加启, 李发弟, 等. 2010. 游离亚麻酸对奶牛乳腺上皮细胞脂肪酸代谢相关基因mRNA转录的影响[J]. 动物营养学报, 22(5): 1342-1349. (Hu H, Wang J Q, Li F D, et al.2010. Effects of free linolenic acid on transcription of mRNAs of genes related to fatty acid metabolism in mammary epithelial cells of dairy cows[J]. Chinese Journal of Animal Nutrition, 22(5): 1342-1349.) [3] 李光鹏, 左永春, 魏著英, 等. 2013. 亚麻脂肪酸脱氢酶基因及其应用. 中国, 201310344833.5[P]. (Li G P, Zuo Y C, Wei Z Y, et al. 2013. The fatty acid desaturase gene in flax and its application. China, 201310344833.5[P].) [4] 王子东, 苏建国, 段彪, 等. 2012. 牛转FAD3B基因胎儿成纤维细胞中内参基因的稳定性评估[J]. 生物技术通报, 11: 118-126. (Wang Z D, Su J G, Duan B, et al.2012. Evaluations of reference genes in bovine transfected FAD3B gene fetal fibroblasts[J]. Biotechnology Bulletin, 11: 118-126.) [5] 魏著英, 菅璐, 杨磊, 等. 2017. 胡麻脂肪酸脱氢酶基因fad3b过表达小鼠模型的建立及其功能分析[J]. 中国细胞生物学学报, 39(2): 172-181. (Wei Z Y, Jian L, Yang L, et al.2017. The role of fatty acid desaturase in flax fad3b in transgenic mice model[J]. Chinese Journal of Cell Biology, 39(2): 172-181.) [6] 于超然. 2014. 转fat-1基因牛外源基因整合位点鉴定[D]. 硕士学位论文, 内蒙古大学, 导师:李光鹏. pp. 14-17. (Yu C R.2014. Identification of the transgenic integration site in fat-1 transgenic cattle[D]. Thesis for M.S., Inner Mongolia University, Supervisor: Li G P. pp. 14-17.) [7] 张超, 石宁宁, 高景波, 等. 2018. 猪Rosa26位点敲入Loxp修饰的hCD55成纤维细胞系的建立[J]. 农业生物技术学报, 26(6): 1074-1083. (Zhang C, Shi N N, Gao J B, et al.2018. Establishment of knock-in loxp-modified hCD55 gene fibroblast cell line with porcine (Sus scrofa) Rosa26 Locus[J]. Journal of Agricultural Biotechnology, 26(6): 1074-1083.) [8] 周文君, 郭日红, 邓明田, 等. 2017. RS-1提高CRISPR-Cas9系统介导的人乳铁蛋白基因敲入效率[J]. 生物工程学报, 33(8): 1224-1234. (Zhou W J, Guo R H, Deng M T, et al.2017. RS-1 enhanced the efficiency of CRISPR-Cas9 mediated knock-in of human lactoferrin[J]. Chinese Journal of Biotechnology, 33(8): 1224-1234.) [9] Arondel V, Lemieux B, Hwang I, et al.1992. Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis[J]. Science, 258(5086): 1353-1355. [10] Chapman B, Morgan L M, Murphy M C.2000. Maternal and early dietary fatty acid intake: Changes in lipid metabolism and liver enzymes in adult rats[J]. Journal of?Nutrition, 130(2): 146-151. [11] Chu V T, Weber T, Graf R, et al.2016. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes[J]. BMC Biotechnology, 16(1): 1-15. [12] Dobrzyn A, Dobrzyn P, Miyazaki M, et al.2005. Polyunsaturated fatty acids do not activate AMP-activated protein kinase in mouse tissues[J]. Biochemical & Biophysical Research Communications, 332(3): 892-896. [13] Hiller B, Hocquette J F, Cassar-Malek I, et al.2012. Dietary n-3 PUFA affect lipid metabolism and tissue function-related genes in bovine muscle[J]. British Journal of Nutrition, 108: 858-863. [14] Hu X M, Guo J, Bai C L, et al.2016. Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice[J]. Frontiers of Agricultural Science and Engineering, 3(1): 87-96. [15] Indo Y, Tatemizo A, Abe Y, et al.2009. Functional expression of a humanized gene for ane omega-3 fatty acid desaturase from scarlet flax in transfected bovine adipocytes and bovine embryos cloned from the cells[J]. Biochimica et Biophysica Acta, 1791(3): 183-190. [16] Irion S, Luche H, Gadue P, et al.2007. Identification and targeting of the ROSA26 locus in human embryonic stem cells[J]. Nature Biotechnology, 25(12): 1477-1482. [17] Jeong Y H, Kim Y J, Kim E Y, et al.2015. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination[J]. Zygote, 24(3): 442-456. [18] Kobayashi T, Kato-Itoh M, Yamaguchi T, et al.2012. Identification of rat Rosa26 locus enables generation of knock-in rat lines ubiquitously expressing tdTomato[J]. Stem Cells Development, 21(16): 2981-2986. [19] Kraemer F B, Shen W J.2002. Hormone-sensitive lipase: Control of intracellular tri-(di-) acylglycerol and cholesteryl ester hydrolysis[J]. Journal of Lipid Research, 43(10): 1585-1594. [20] Liang P P, Xu Y W, Zhang X Y, et al.2015. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes[J]. Protein Cell, 6(5): 363-372. [21] Liu X F, Bai C L, Ding X B, et al.2015. Microarray analysis of the gene expression profile and lipid metabolism in fat-1 transgenic cattle[J]. PloS One, 10(10): e0138874. [22] Luo J, Rizkalla S W, Vidal H, et al.1998. Moderate intake of n-3 fatty acids for 2 months has no detrimental effect on glucose metabolism and could ameliorate the lipid profile in type 2 diabetic men: Results of a controlled study[J]. Diabetes Care, 21(5): 717-724. [23] Li X P, Yang Y, Bu L, et al.2014. Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing[J]. Cell Research, 24(4): 501-504. [24] Ma Y W, Zhang X, Shen B, et al.2014. Generating rats with conditional alleles using CRISPR/Cas9[J]. Cell Research, 24(1): 122-125. [25] Mali P, Yang L, Esvelt K M, et al.2013. RNA-guided human genome engineering via Cas9[J]. Science, 339(6121): 823-826. [26] Madsen L, Petersen R K, Kristiansen K, et al.2005. Regulation of adipocyte differentiation and function by polyunsaturated fatty acids[J]. Molecular Basis of Disease, 1740(2): 266-286. [27] Nakamura M T, Cho H P, Clarke S D.2000. Regulation of hepatic delta-6 desaturase expression and its role in the polyunsaturated fatty acid inhibition of fatty acid synthase gene expression in mice[J]. Journal of Nutrition, 130(6):1561-1565. [28] Ruan J X, Li H G, Xu K, et al.2015. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs[J]. Scientific Reports, 5: 142-153. [29] Shen Wei, Lan Goucheng, Yang Xueyi,et al.2007. Targeting the exogenous htPAm gene on goat somatic cell beta-casein locus for transgenic goat production[J]. Molecular reproduction and development, 74(4): 428-434. [30] Schmitz G, Ecker J.2008. The opposing effects of n-3 and n-6 fatty acids[J]. Progress in Lipid Research, 47(2): 147-155. [31] Tasic B, Hippenmeyer S, Wang C, et al.2011. Site-specific integrase-mediated transgenesis in mice via pronuclear injection[J]. Proceedings of the National Academy of?Science, 108(19): 7902-7907. [32] Vrinten P, Hu Z Y, Munchinsky M A, et al.2005. Two FAD3 desaturase genes control the level of linolenic acid in flax seed[J]. Plant Physiol, 139(1): 79-87. [33] Wang X J, Wang J, Wang Y Y M, et al.2016. Sus scrofa matrix attachment region enhances expression of the PiggyBac system transfected into HEK293T cells[J]. Biotechnology Letters, 38(6): 949-958. [34] Wang L Z, You J H, Zhong B S, et al.2014. Scd 1 mammary-specific vector constructed and overexptession in goat fibroblast cells resulting in an increase of palmitoleic acid and oleic acid[J]. Biochemical and Biophysical Research Communications, 443(2): 389-394. [35] Yuan J X, Li H G, Xu K, et al.2015. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs[J]. Scientific Reports, 5: 142-153. [36] Yang H, Wang H, Shivalila C S, et al.2013. One step generation of mice carrying reporter and conditional alleles by CRISPR/Cas9-mediated genome engineering[J]. Cell, 154(6): 1370-1379. [37] Zhu S Y, Rong Z L, Lu X F, et al.2015. Gene targeting through homologous recombination in monkey embryonic stem cells using CRISPR/Cas9 system[J]. Stem Cells Development, 24(10): 1147-1149. [38] Zambrowicz B P, Imamoto A, Fiering S, et al.1997. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells[J]. Proceedings of the National Academy of?Science, 94(8): 3789-3794. [39] Zhu F, Gamboa M, Farruggio A P, et al.2014. DICE, an efficient system for iterative genomic editing in human pluripotent stem cells[J]. Nucleic Acids Research, 42(5): e34.
