基于CRISPR/Cas9技术建立IGF2R基因修饰PK-15细胞

doi:10.13304/j.nykjdb.2022.0363

中国农业科技导报 ›› 2022, Vol. 24 ›› Issue (10): 200-207.DOI: 10.13304/j.nykjdb.2022.0363

• 方法与技术创新 • 上一篇

基于CRISPR/Cas9技术建立IGF2R基因修饰PK-15细胞

谢春嫡¹(), 郭肖蓉¹^,², 张煦栋¹^,³, 周荣¹(), 李奎⁴

^1.中国农业科学院北京畜牧兽医研究所，北京 100193
^2.佛山科学技术学院生命科学与工程学院，广东省动物分子设计与精准育种重点实验室，广东佛山 528225
^3.内蒙古农业大学动物科学学院，呼和浩特 010010
^4.中国农业科学院深圳农业基因组研究所，深圳 518120

收稿日期:2022-04-30 接受日期:2022-08-03 出版日期:2022-10-15 发布日期:2022-10-25
通讯作者: 周荣
作者简介:谢春嫡E-mail：xxiechundi@163.com；
基金资助:
国家自然科学基金项目(31972541);中央级公益性科研院所基本科研业务费专项(Y2021XK20);六安市产学研合作重大专项

Establishment of IGF2R Knockout PK-15 Cells by CRISPR/Cas9 System

Chundi XIE¹(), Xiaorong GUO¹^,², Xudong ZHANG¹^,³, Rong ZHOU¹(), Kui LI⁴

^1.Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
^2.Guangdong Provincial key Laboratory of Animal Molecular Design and Precise Breeding, College of Life Science and Engineering, Foshan University, Guangdong Foshan 528231, China
^3.College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010010, China
^4.Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China

Received:2022-04-30 Accepted:2022-08-03 Online:2022-10-15 Published:2022-10-25
Contact: Rong ZHOU

摘要/Abstract

摘要：

作为我国特色的地方种质资源，藏猪对高海拔、低氧、强紫外线辐射等恶劣环境具有良好的适应性，前期研究发现藏猪的IGF2R（insulin-like growth factor 2 receptor，IGF2R）基因在其内含子上有1段274 bp的缺失，且该缺失为藏猪独有的变异，关于IGF2R是否参与藏猪对低氧等恶劣环境的适应性调节的相关研究尚未报道。为了建立IGF2R基因274 bp修饰的猪细胞系，研究IGF2R基因对藏猪低氧耐受特性的影响，通过CRISPR/Cas9技术建立基因修饰PK-15细胞系，并通过PCR、实时荧光定量PCR（real time quantitative PCR，RT-qPCR）、蛋白免疫印迹（Western-blot）、免疫荧光（immunofluorescence，IF）等方法鉴定其分子特征，随后分别利用CCK-8和RT-qPCR 、Western-blot对基因修饰细胞进行活力和凋亡基因检测。结果显示，已成功构建IGF2R基因274 bp修饰的猪细胞系，IGF2R基因表达量极显著下调（P < 0.01）。基因修饰细胞活力在72 h时极显著上升（P < 0.01），并且含半胱氨酸的天冬氨酸蛋白水解酶9 （cysteinyl aspartate specific proteinase，Caspase9）和BCL-2关联 X （BCL2-associated X，BAX）蛋白的水平显著下调（P < 0.05），并且B淋巴细胞瘤-2（B-cell lymphoma-2，BCL2）基因的mRNA水平极显著上升（P < 0.01）。成功构建了IGF2R基因精确修饰的PK-15细胞系，并初步进行了功能研究，为研究IGF2R基因功能提供细胞模型，并为后续基因编辑猪的制备奠定基础。

关键词: CRISPR/Cas9, IGF2R基因, 猪, 品种改良

Abstract:

As a local quality resource with Chinese characteristics， Tibetan pig has good adaptability to harsh environments such as high altitude， low oxygen and strong ultraviolet radiation. Previous study found that the insulin like growth factor 2 receptor gene of Tibetan pig had a 274 bp deletion in its intron， and this deletion was a unique variation of Tibetan pig. At present， there is no relevant research on whether IGF2R is involved in the adaptive regulation of Tibetan pig to poor environments such as hypoxia. Based on the previous findings， this study aimed to establish a pig cell line modified by 274 bp of IGF2R gene， so as to lay a foundation for the subsequent study of the effect of IGF2R gene on the hypoxia tolerance of Tibetan pigs. The gene knockout PK-15 cell line was established by CRISPR/Cas9 technology， and its molecular characteristics were identified by PCR， real time quantitative PCR （RT-qPCR）， Western-blot and immunofluorescence （IF）. Then the viability and apoptosis genes of gene knockout cells were detected by CCK-8， RT-qPCR and Western-blot. The results showed that a pig cell line with 274 bp deletion of IGF2R gene was successfully constructed， and the expression of IGF2R gene decreased extremely significantly（P < 0.01）. The activity of gene knockout cells increased extremely significantly （P < 0.01）at 72 h， and the protein levels of cysteine containing aspartate specific protein 9 （caspase 9） and Bcl-2 associated X （BAX） decreased significantly （P < 0.05）， The mRNA level of B-cell lymphoma-2 （BCL2） increased extremely significantly （P < 0.01）. In this study， the PK-15 cell line precisely modified of IGF2R gene was successfully constructed， and the function was preliminarily studied， which provided a cell model for the function studying of IGF2R gene and laid a foundation for the preparation of subsequent gene editing pigs.

Key words: CRISPR/Cas9, IGF2R gene, pig, variety improvement

中图分类号:

谢春嫡, 郭肖蓉, 张煦栋, 周荣, 李奎. 基于CRISPR/Cas9技术建立IGF2R基因修饰PK-15细胞[J]. 中国农业科技导报, 2022, 24(10): 200-207.

Chundi XIE, Xiaorong GUO, Xudong ZHANG, Rong ZHOU, Kui LI. Establishment of IGF2R Knockout PK-15 Cells by CRISPR/Cas9 System[J]. Journal of Agricultural Science and Technology, 2022, 24(10): 200-207.

图/表 6

表1 引物序列

Table 1 Primer sequence

基因Gene	序列Sequence（5’-3’）	用途Purpose
sgRNA1	CTGCGTCACTAGGTCTGCAGGGG	载体构建 Vector construction
sgRNA2	TGAACGCACAAGGTTATGTGTGG	载体构建 Vector construction
IGF2R-1	F： ACGGTACTAGCCCTCCTTGT R： TTCTCTTGCCCTCCCTCTGA	PCR
IGF2R-2	F： AGAAAGAGGTGCCGTGCTAC R： CCGGAGCGTGTCTATGTCTC	qPCR
BAX	F： GGCCTCCTCTCCTACTTTGG R： CTCAGCCCATCTTCTTCCAG
BCL-2	F： ATGTGTGTGGAGAGCGTCAAC R： AGAGACAGCCAGGAGAAATCAA
β-actin	F： GGACTTCGAGCAGGAGATGG R： AGGAAGGAGGGCTGGAAGAG

图1 质粒构建及效率检测A：明场图；B：荧光图；C：修饰效率检测。 M为 100 bp DNA相对分子质量标准，1 为空白对照细胞，2为转染细胞

Fig. 1 Plasmid construction and efficiency detectionA： Bright field image； B： Fluorescence image； C： Knockout efficiency detection. M is 100 bp DNA marker，1 is blank control cells，2 is cells after transfection

图2 单克隆细胞基因型分析A：细胞流式分选，方框表示收集的绿色荧光细胞；B：部分细胞基因型PCR鉴定图

Fig. 2 Genotype analysis of monoclonal cellsA： Flow cytometry of cells， the box represent the collected green fluorescent cells； B： Genotype of cells identified by PCR

图3 IGF2R基因修饰细胞分子特征鉴定A：蛋白水平；B：mRNA水平；C：免疫荧光。*和**分别表示P < 0.05和P < 0.01水平差异显著

Fig. 3 Molecular characterization of the IGF2R gene deletion cellA： Protein level；B： mRNA level； C： Immunofluorescence. * and ** indicate significant difference at P < 0.05 and P < 0.01 levels， respectively

图4 IGF2R基因的修饰对细胞活力的影响注：**分别表示P < 0.05和P < 0.01水平差异显著，ns表示差异不显著。

Fig. 4 Effect of IGF2R gene deletion on cell viabilityNote：** indicates significant difference at P < 0.01 level； ns indicates no significant difference.

图5 IGF2R基因的修饰对细胞凋亡的影响A：BCL-2 和BAX mRNA水平；B：凋亡蛋白水平检测。*和**分别表示P < 0.05和P < 0.01水平差异显著，ns表示差异不显著

Fig. 5 Effect of IGF2R gene deletion on apoptosisA： BCL-2 and BAX mRNA level； B： Apoptotic gene protein level. * and ** indicate significant difference at P < 0.05 and P < 0.01 levels， respectively； ns indicates no significant difference

参考文献 27

1	STORZ J F, SCOTT G R, CHEVIRON Z A, Phenotypic plasticity and genetic adaptation to high-altitude hypoxia in vertebrates [J]. J. Exp. Biol.,2010,213(24):4125-4136.
2	何建文,刘秀,李少斌,等.藏绵羊HMOX2基因克隆,表达及其耐低氧适应性进化分析[J]. 农业生物技术学报,2018,26(11): 1890-1899.
	HE J W, LIU X, LI S B, et al.. Cloning, expression and hypoxia adaptation evolution of HMOX2 gene in Tibetan sheep (Ovis aries) [J]. J. Agric. Biotechnol., 2018,26(11) :1890-1899.
3	GARY-BOBO M, NIRDE P, JEANJEAN A, et al.. Mannose 6-phosphate receptor targeting and its applications in human diseases [J]. Curr. Med. Chem.,2007,14(28):2945-2953.
4	REDDY S T, CHAI W, CHILDS R A, et al.. Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor [J]. J. Biol. Chem.,2004,279(37):38658-38667.
5	MACDONALD R, PFEFFER, COUSSENS L, et al.. A single receptor binds both insulin-like growth factor Ⅱ and mannose-6-phosphate [J]. Science,1988,239(4844):1134-1137.
6	HAN J, GOLDSTEIN L A, HOU W, et al.. Involvement of CASP9 (caspase 9) in IGF2R/CI-MPR endosomal transport [J]. Autophagy, 2021,17(6):1393-1409.
7	WANG X, LIN L, LAN B, et al.. IGF2R-initiated proton rechanneling dictates an anti-inflammatory property in macrophages [J]. Sci. Adv.,2020,6(48):7389.
8	CHIANG C, SCOTT A J, DAVIS J R, et al.. The impact of structural variation on human gene expression [J]. Nat. Genet.,2017,49(5):692-699.
9	ZHOU R, LI S T, YAO W Y, et al.. The Meishan pig genome reveals structural variation-mediated gene expression and phenotypic divergence underlying Asian pig domestication [J]. Mol. Ecol. Resour.,2021,21(6):2077-2092.
10	车晶晶,徐奎,张秀玲,等.基于CRISPR/Cas9技术的Wip1基因敲除ST细胞的建立[J]. 畜牧兽医学报,2021,52(10):2814-2821.
	CHE J J, XU K, ZHANG X L, et al.. Establishment of Wip1-knockout ST cells mediated by CRISPR/Cas9 system [J]. Acta Vet. et Zootech. Sin., 2021,52(10):2814-2821.
11	CONG L, RAN F A, COX D, et al.. Multiplex henome rngineering using CRISPR/Cas systems [J]. Science,2013,339(6121):819-823.
12	LEE H, YOON D E, KIM K, Genome editing methods in animal models [J]. Anim. Cells Syst.,2020,24(1):8-16.
13	CANALIS E, YU J, SCHILLING L, et al.. The lateral meningocele syndrome mutation causes marked osteopenia in mice [J]. J. Biol. Chem.,2018,293(36):14165-14177.
14	GAO X, TAO Y, LAMAS V, et al.. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents [J]. Nature,2018,553(7687):217-221.
15	詹群美.利用CRISPR/Cas9基因编辑技术生产MSTN基因敲除巴马香猪[D].佛山:佛山科学技术学院, 2020.
	ZHAN Q M. Producing MSTN gene knockout Bama pig by CRISPR/Cas9 gene editing [D]. Foshan: Foshan University, 2020.
16	姜元.利用CRISPR/Cas9介导的Knock-in技术制备抗PEDV基因编辑猪[D].长春:吉林大学,2021.
	JIANG Y. Generation of PEDV resistant gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy [D].Changchun: Jilin University, 2021.
17	XU K, ZHOU Y, MU Y, et al.. CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance [J]. eLife Sci.,2020,9:e57132.
18	XU K, ZHANG X, LIU Z, et al.. A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection [J]. Sci China Life Sci.,2022,65(8): 1535-1546.
19	WANG Z Q, FUNG M R, BARLOW D P, et al.. Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene [J]. Nature,1994,372(6505):464-467.
20	LAU M M, STEWART C E, LIU Z, et al.. Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality [J]. Genes Dev.,1994,8(24):2953-2963.
21	LUDWIG T, EGGENSCHWILER J, FISHER P, et al.. Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds [J]. Dev. Biol.,1996,177(2):517-535.
22	吕莉,宋陈玲,寇洁,等. IGF1R和IGF2R基因在鸭脂肪组织中的发育表达模式比较[J]. 中国畜牧杂志,2013,49(1):5-10.
23	EL-MAGD M A, ABO-AL-ELA H G, EL-NAHAS A, et al.. Effects of a novel SNP of IGF2R gene on growth traits and expression rate of IGF2R and IGF2 genes in gluteus medius muscle of Egyptian buffalo [J]. Gene,2014,540(2):133-139.
24	VICIKOVA K, PETROVCIKOVA E, MANKA P, et al.. Serum and urinary levels of CD222 in cancer: origin and diagnostic value [J]. Neoplasma,2018,65(5):762-768.
25	VAILLANT O, CHEIKH K E, WARTHER D, et al.. Mannose-6-phosphate receptor: a target for theranostics of prostate cancer [J]. Angew. Chem. Int. Ed.,2015,54(20):5952-5956.
26	刘世博,李刚,谢庆鹏,等.沉默IGF2R促进膀胱癌5637细胞增殖及肿瘤形成的机制探讨[J]. 现代肿瘤医学,2019,27(23):4135-4139.
	LIU S B, LI G, XIE Q P, et al.. Study on the mechanism of sliencing IGF2R promotes cell proliferation and tumorigenesis in bladder cancer 5637 cell line [J]. Modern Oncol., 2019,27(23):4135-4139.
27	CHU C H, TZANG B S, CHEN L M, et al.. Activation of insulin-like growth factor Ⅱ receptor induces mitochondrial-dependent apoptosis through G(alpha)q and downstream calcineurin signaling in myocardial cells [J]. Endocrinology,2009,150(6):2723-2731.

[1]	翟利敏, 李文通, 冯政, 李华, 裴杨莉. 基因编辑猪的研究现状[J]. 中国农业科技导报, 2022, 24(8): 25-34.
[2]	魏艳晨, 陈吉祥, 王永刚, 孟彤彤, 韩亚龙, 李美. 荒漠植物珍珠猪毛菜根际土壤细菌多样性与土壤理化性质相关性分析[J]. 中国农业科技导报, 2022, 24(5): 209-217.
[3]	庄重, 赵龙, 白皓, 毕瑜林, 黄应权, 陈国宏, 常国斌. CRISPR/Cas9技术在家禽育种方面的应用[J]. 中国农业科技导报, 2022, 24(1): 14-23.
[4]	高云, 王瑜, 鲁斯迪, 雷明刚, 罗俊杰, 黎煊, . 猪舍热舒适性评价及夏季湿帘作用下的CFD模拟[J]. 中国农业科技导报, 2021, 23(7): 125-135.
[5]	王巍1,2,罗玉子1,张丽1,马吉飞2,仇华吉1. 消毒技术在非洲猪瘟防控中的应用现状与问题[J]. 中国农业科技导报, 2021, 23(4): 93-102.
[6]	刘广娟,徐泽权,邢世均,陈铮,朱明睿,徐艳丽,张洪瑞,张莉,王子荣*. 磷酸盐对PSE猪肉食用品质和微观结构的影响[J]. 中国农业科技导报, 2021, 23(3): 114-121.
[7]	廖嘉明, §, 李春梅§, 张石虎, 李布野, 欧阳昆唏, 陈晓阳. CRISPR/Cas9基因编辑技术的发展及其在植物中的应用[J]. 中国农业科技导报, 2021, 23(12): 20-28.
[8]	赵云翔1,2§*,高广雄1§,方程2,朱琳2,李晓辉1,杨文俊1,张从林2. 不同品种公猪睾丸、精液品质发育规律及相关性研究[J]. 中国农业科技导报, 2020, 22(7): 61-68.
[9]	王祖力. 新型冠状病毒肺炎疫情对我国生猪产业的影响及对策建议[J]. 中国农业科技导报, 2020, 22(6): 6-11.
[10]	辛红佳,李鹏程,滕守振,李圣彦,汪海,郎志宏*. 拟南芥SWEET1/2/3基因突变体构建及功能鉴定[J]. 中国农业科技导报, 2020, 22(2): 39-49.
[11]	刘杜娟,黄火清,苏小运 . 低纤维素酶背景里氏木霉菌株的构建和应用[J]. 中国农业科技导报, 2020, 22(12): 50-57.
[12]	杨海峰1,2§,李艳艳1,2§,陈晓兰1,姚诗贇1,黄亚奇2,夏良友2,李宇琛2,卜仕金2*. 烯丙孕素内服溶液的临床药效和靶动物安全性研究[J]. 中国农业科技导报, 2020, 22(1): 78-86.
[13]	张丽,罗玉子,王涛,孙元,仇华吉*. 非洲猪瘟诊断技术发展现状与需求分析[J]. 中国农业科技导报, 2019, 21(9): 1-11.
[14]	周丹1,2,高云1,3*,雷明刚3,4,黎煊1,3. 冬季仔猪舍内二氧化碳浓度的数值模拟研究[J]. 中国农业科技导报, 2019, 21(8): 90-98.
[15]	李丹,陈一飞*,李行健,蒲东. 计算机视觉技术在猪行为识别中应用的研究进展[J]. 中国农业科技导报, 2019, 21(7): 59-69.

基于CRISPR/Cas9技术建立IGF2R基因修饰PK-15细胞

Establishment of IGF2R Knockout PK-15 Cells by CRISPR/Cas9 System

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 27

相关文章 15

编辑推荐

Metrics