利用CRISPR/Cas9技术构建PPARγ基因敲除的IPEC-J2细胞系

doi:10.16431/j.cnki.1671-7236.2023.07.008

摘要/Abstract

摘要： 【目的】利用CRISPR/Cas9技术敲除猪小肠上皮细胞（IPEC-J2）的过氧化物酶体增殖激活受体γ（PPARγ）基因，进而在细胞水平上研究PPARγ基因与肠道炎症反应相关的生物学机制。【方法】根据猪PPARγ基因序列（GenBank登录号：NM_214379），在第2外显子区域设计2对sgRNA序列，经退火形成DNA双链后连接至真核表达载体gRNA_GFP-T1；将构建成功的PPARγ正向打靶载体、反向打靶载体、Cas9-nickase质粒共转染至IPEC-J2细胞，经G418药物筛选获得单克隆细胞株，提取单克隆细胞株DNA后进行PCR扩增，经凝胶电泳鉴定后送测序；挑选基因敲除效果最佳的细胞株，利用免疫荧光法检测PPARγ蛋白表达情况。【结果】打靶载体测序结果显示，打靶序列正确连接；经转染、筛选后共获得48株单克隆细胞；测序结果显示，6株单克隆细胞出现双峰，分别为KO-15、KO-17、KO-21、KO-25、KO-32和KO-35；选择底峰较高的KO-25进行免疫荧光检测，结果显示PPARγ蛋白表达降低。【结论】本研究利用CRISPR/Cas9技术成功获得了PPARγ基因敲除的IPEC-J2细胞，为后续进一步研究PPARγ基因在炎症反应中的功能奠定了基础。

关键词: CRISPR/Cas9; 过氧化物酶体增殖激活受体γ(PPARγ); IPEC-J2细胞; 基因敲除

Abstract: 【Objective】 The purpose of this study was to use CRISPR/Cas9 technique to knock out the peroxisome proliferation-activated receptor γ (PPARγ) gene in porcine small intestinal epithelial cells (IPEC-J2), and to investigate the biological mechanism of PPARγ gene associated with intestinal inflammation at the cellular level.【Method】 According to porcine PPARγ gene sequence (GenBank accession No.:NM_214379), two pairs of sgRNA sequences were designed in exon 2 region.After annealing, DNA double strand was formed and connected to the eukaryotic expression vector gRNA_GFP-T1.The constructed PPARγ vector (forward target vector, reverse target vector) and Cas9-nickase plasmid were co-transfected into IPEC-J2 cells, after G418 drug screening, the monoclonal cell lines were sampled, DNA was extracted, PCR amplified, identified by gel electrophoresis and sequenced to select the cell lines with the best gene knockout effect, the expression of PPARγ protein was detected by immunofluorescence.【Result】 The target vector sequencing results showed that the target sequences were correctly connected.A total of 48 monoclonal cells were obtained after transfection and screening.The sequencing results showed that 6 monoclonal cells showed double peaks, namely KO-15, KO-17, KO-21, KO-25, KO-32 and KO-35, respectively.The KO-25 with higher basal peak was selected for immunofluorescence detection, and the results showed that the expression of PPARγ protein decreased.【Conclusion】 In this study, IPEC-J2 cells with PPARγ gene knockout were successfully obtained using CRISPR/Cas9 technology, which laid the foundation for further research on the function of PPARγ gene in inflammatory response.

Key words: CRISPR/Cas9; peroxisome proliferative activated recepter gamma (PPARγ); IPEC-J2 cells; gene knockout

中图分类号:

张家翔, 韩佃刚, 师亚玲, 毛晓月, 赵凯薇, 杜萱, 信吉阁. 利用CRISPR/Cas9技术构建PPARγ基因敲除的IPEC-J2细胞系[J]. 中国畜牧兽医, 2023, 50(7): 2670-2677.

ZHANG Jiaxiang, HAN Diangang, SHI Yaling, MAO Xiaoyue, ZHAO Kaiwei, DU Xuan, XIN Jige. Construction of IPEC-J2 Cell Lines with PPARγ Gene Knockout Mediated by CRISPR/Cas9 Technology[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2670-2677.

参考文献

[1] 姜懿轩, 王亚玲, 王婷婷, 等.家兔PPARγ基因的克隆表达及肌内脂肪含量的相关性分析[J].黑龙江畜牧兽医, 2022, 7:119-125. JIANG Y X, WANG Y L, WANG T T, et al.Rabbit PPARγ gene cloning and expression and correlation analysis of intramuscular fat content[J].Heilongjiang Animal Science and Veterinary Medicine, 2022, 7:119-125.(in Chinese)
[2] 李琳, 郑卓, 贺红专.PPARγ基因调控猪脂肪沉积研究进展[J].中国猪业, 2016, 11(8):57-60. LI L, ZHENG Z, HE H Z.Research progress on regulation of porcine fat deposition by PPARγ gene[J].China Swine Industry, 2016, 11(8):57-60.(in Chinese)
[3] 蒋晓梅, 刘翀, 朱延焱.黄连总生物碱对溃疡性结肠炎模型大鼠肠黏膜损伤及p38-PPARγ/NF-κB通路的影响[J].中国药师, 2019, 22(12):2188-2193. JIANG X M, LIU C, ZHU Y Y.Effects of the total alkaloids of coptis chinensis on intestinal mucosal injury and p38-PPARγ/NF-κB pathway in rats with ulcerative colitis[J].China Pharmacist, 2019, 22(12):2188-2193.(in Chinese)
[4] 袁伟, 刘辉, 陈美雪, 等.罗格列酮预处理能减轻小肠缺血再灌注损伤[J].胃肠病学, 2014, 19(8):472-475. YUAN W, LIU H, CHEN M X, et al.Rosiglitazone pretreatment ameliorates intestinal ischemia-reperfusion injury[J].Chinese Journal of Gastroenterology, 2014, 19(8):472-475.(in Chinese)
[5] 马晶鑫, 郭金洲, 陈海宁, 等.淡豆豉异黄酮通过PPARγ/LXRα/ABCA1信号通路改善动脉粥样硬化小鼠脂质代谢的作用[J].中国实验方剂学杂志, 2022, 28(11):110-118. MA J X, GUO J Z, CHEN H N, et al.Effect of isoflavones from semen sojae preparatum on lipid metabolism in atherosclerotic mice through PPARγ/LXRα/ABCA1 signaling pathway[J].Chinese Journal of Experimental Traditional Medical Formulae, 2022, 28(11):110-118.(in Chinese)
[6] 朱奕骋.CRISPR/Cas9技术的研究进展及其在肺癌研究中的应用[J].科技传播, 2020, 12(14):172-174. ZHU Y C.Research progress of CRISPR/Cas9 technology and its application in lung cancer research[J].Public Communication of Science & Technology, 2020, 12(14):172-174.(in Chinese)
[7] TANG Y D, GUO J C, WANG T Y, et al.CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates Pseudo-rabies virus editing[J].FASEB Journal, 2018, 32(8):4293-4301
[8] RYCZEK N, HRYHOROWICZ M, ZEYLAND J, et al. CRISPR/Cas technology in pig-to-human xenotransplantation research[J].International Journal of Molecular Sciences, 2021, 22(6):3196.
[9] ZHANG Y, SHOWALTER A M.CRISPR/Cas9 genome editing technology:A valuable tool for understanding plant cell wall biosynthesis and function[J].Frontiers in Plant Science, 2020, 20(11):589517.
[10] 张婷婷, 杨漫漫, 魏强, 等.CRISPR/Cas9介导人HBB基因突变体在猪成纤维细胞的靶向敲入[J].中国实验动物学报, 2020, 28(6):779-787. ZHANG T T, YANG M M, WEI Q, et al.Generation of human HBB mutant knock-in porcine fibroblasts using CRISPR/Cas9-mediated homologous recombination[J].Acta Laboratorium Animalis Scientia Sinica, 2020, 28(6):779-787.(in Chinese)
[11] 王紫君, 任红艳, 肖红卫, 等.CRISPR/Cas9基因编辑技术介导SST基因敲除细胞的制备[J].江西农业学报, 2020, 32(8):70-75. WANG Z J, REN H Y, XIAO H W, et al.Preparation of SST knockout cells mediated by CRISPR/Cas9 technology[J].Acta Agriculturae Jiangxi, 2020, 32(8):70-75.(in Chinese)
[12] 和林洁.CRISPR/Cas9介导鸡细胞PPARγ基因的敲除[D].杨凌:西北农林科技大学, 2016. HE L J.Knock out PPARγ gene of chicken cells by CRISPR/Cas9[D].Yangling:Northwest A&F University, 2016.(in Chinese)
[13] TONTONOZ P, SPIEGELMAN B M.Fat and beyond:The diverse biology of PPARγ[J].Annual Review of Biochemistry, 2008, 77:289-312.
[14] UNGARO P, MIRRA P, ORIENTE F, et al.Peroxisome proliferator-activated receptor-γ activation enhances insulin-stimulated glucose disposal by reducing ped/pea-15 gene expression in skeletal muscle cells:Evidence for involvement of activator protein-1[J] Journal of Biological Chemistry, 2012, 287(51):42951-42961.
[15] 孔路军, 王爱国, 傅金恋, 等.猪PPARs基因组织表达特性的研究[J].中国畜牧杂志, 2006, 19:1-4. KONG L J, WANG A G, FU J L, et al.Study on tissue expression characteristics of porcine PPARs gene[J].Chinese Journal of Animal Science, 2006, 19:1-4.(in Chinese)
[16] 刘寒, 李苏, 赵玉华, 等.PPARα和PPARγ的克隆表达研究进展[J].中国农学通报, 2013, 29(14):31-36. LIU H, LI S, ZHAO Y H, et al.Advance in the clone and expression of PPARα and PPARγ[J].Chinese Agricultural Science Bulletin, 2013, 29(14):31-36.(in Chinese)
[17] 余希婧, 黄辉, 胡秀武, 等.长蛇灸激活PPARγ改善初发强直性脊柱炎大鼠的机制研究[J].中国中医急症, 2022, 31(9):1372-1376. YU X J, HUANG H, HU X W, et al.Mechanism of long snake moxibustion activation of PPARγ to improve the initial onset of ankylosing spondylitis in rats[J].Journal of Emergency in Traditional Chinese Medicine, 2022, 31(9):1372-1376.(in Chinese)
[18] DA S S, KEITA, MOHLIN S, et al.A novel topical PPARγ agonist induces PPARγ activity in ulcerative colitis mucosa and prevents and reverses inflammation in induced colitis models[J]. Inflammatory Bowel Diseases, 2018, 24(4):792-805.
[19] 张永刚, DAVIN R.猪小肠上皮细胞作用的新发现[J].中国畜牧杂志, 2015, 51(8):57-60. ZHANG Y G, DAVIN R.Importance of intestinal cells forgut health-IPEC studies[J].Chinese Journal of Animal Science, 2015, 51(8):57-60.(in Chinese)
[20] 杨宏清.植物提取物生物碱对IPEC-J2细胞RegⅢγ基因表达及信号转导机制研究[D].昆明:云南农业大学, 2021. YANG H Q.Study on the mechanisms of plant extract alkaloids on RegⅢγ gene expression and signal transduction in IPEC-J2 cells[D].Kunming:Yunnan Agricultural University, 2021.(in Chinese)
[21] 白祥慧.CRISPR/Cas9基因敲除研究进展.[J].现代畜牧兽医, 2021, 1:90-92. BAI X H.Research progress of CRISPR/Cas9 gene knockout[J].Modern Journal of Animal Husbandry and Veterinary Medicine, 2021, 1:90-92.(in Chinese)
[22] 史梦然, 沈宗毅, 张楠, 等.CRISPR/Cas9系统在疾病研究和治疗中的应用.[J].生物工程学报, 2021, 37(4):1205-1228. SHI M R, SHEN Z Y, ZHANG N, et al.CRISPR/Cas9 technology in disease research and therapy:A review[J].Chinese Journal of Biotechnology, 2021, 37(4):1205-1228.(in Chinese)
[23] 周金伟, 徐绮嫔, 姚婧, 等.CRISPR/Cas9基因组编辑技术及其在动物基因组定点修饰中的应用[J].遗传, 2015, 37(10):1011-1020. ZHOU J W, XU Q P, YAO J.CRISPR/Cas9 genome editing technique and its application in site-directed genome modification of animals[J].Hereditas, 2015, 37(10):1011-1020.(in Chinese)
[24] 田甜, 周小明.基于CRISPR/Cas9的基因分析方法研究进展[J].激光生物学报, 2020, 29(1):18-25. TIAN T, ZHOU X M.Research progress of genetic analysis methods based on CRISPR/Cas9[J].Acta Laser Biology Sinica, 2020, 29(1):18-25.(in Chinese)
[25] YANG D S, YANG H Q, LI W, et al.Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning[J].Cell Research, 2011, 21(6):979-982.
[26] 李凤丽, 石素华, 李爱芹, 等.CRISPR/Cas9基因编辑技术研究进展[J].山东农业科学, 2020, 52(4):164-172. LI F L, SHI S H, LI A Q, et al.Research progress of CRISPR/Cas9 gene editing technology[J].Shandong Agricultural Sciences, 2020, 52(4):164-172.(in Chinese)