基于CRISPR-Cas9技术构建TET2基因敲除的鸡胚成纤维细胞系

doi:10.13304/j.nykjdb.2022.0497

摘要/Abstract

摘要：

为研究甲基胞嘧啶双加氧酶2 （tet methylcytosine dioxygenase 2，TET2）及其介导的羟甲基化修饰在调控天然免疫反应中的作用，利用CRISPR-Cas9基因编辑技术构建TET2基因敲除的鸡胚成纤维细胞系。针对鸡TET2 mRNA的2种剪接变体的共有CDS序列设计4组sgRNA，构建sgRNA表达质粒，连同Cas9-T2A-GFP质粒共转染DF-1细胞，筛选高切割效率的sgRNA；将转染该sgRNA的DF-1细胞，进行流式细胞分选，获取表达绿色荧光蛋白（green fluorescent protein，GFP）的细胞，再通过有限稀释法筛选单克隆细胞，并用DNA测序进行鉴定。单克隆敲除细胞扩大培养后，Western blot和Dot blot检测TET2蛋白的表达水平和羟甲基化（5hmC）水平。DNA测序结果表明，TET2-gRNA-2靶位点附近分别发生2和28 bp的缺失突变，引起TET2蛋白移码突变，将该细胞株命名为DT-6。DT-6细胞传代15次以上，细胞形态稳定。Western blot结果显示，与DF-1细胞相比，DT-6细胞几乎不表达TET2蛋白。Dot blot结果显示，与DF-1细胞相比，DT-6细胞全基因组羟甲基化水平显著降低。利用CRISPR-Cas9技术成功构建了敲除TET2基因的鸡胚成纤维细胞系，为研究鸡TET2及其介导羟甲基化修饰参与调控天然免疫反应的途径和分子机制奠定基础。

关键词: 鸡, TET2, CRISPR-Cas9, TET2 基因敲除, 鸡胚成纤维细胞

Abstract:

In order to studying the role of tet methylcytosine dioxygenase 2 （TET2） and its mediated hydroxymethylation in regulating innate immune responses， CRISPR-Cas9 mediated gene editing systems were used to construct a TET2 knockout chicken embryonic fibroblast cell line. 4 pairs of single guide RNAs （sgRNAs） were designed for targeting the conserved CDS sequences of the two RNA spliced isoforms of chicken TET2 gene. The plasmids expressing sgRNA was constructed， and were co-transfected with Cas9-T2A-GFP into DF-1 cells. DF-1 cells were transfected with a higher cleavage activity sgRNA. Then， the cells expressing GFP fluorescent were sorted by flow cytometry， and cultured and screened by limiting dilution method. The positive monoclonal cells were identified by DNA sequencing. When large numbers of the monoclonal knockout cells were genearated， the TET2 protein expression was detected by western blot and the level of hydroxymethylation （5hmC） was detected by dot blot. The results of DNA sequence showed that 2 and 28 bp deletion were detected in TET2 gene at position of the target site of TET2-gRNA-2. Both 2 deletions caused a reading frame shift and a premature stop codon. The clonal knockout cells were named DT-6. DT-6 cells were passaged for more than 15 times， the cell morphology was stable and the TET2 protein was not expressed. Compared with the DF-1 cells， the hydroxymethylation level of the cells was significantly reduced in DT-6 cells. In this study，the TET2 gene knockout chicken embryo fibroblast cell line was successfully constructed by applying CRISPR-Cas9 technology， which would provide a foundation for the further study of TET2 and its hydroxymethylation-mediated pathways and molecular mechanism in regulating innate immune response.

Key words: chicken, TET2, CRISPR-Cas9, TET2 gene knockout, chicken embryo fibroblast cell

中图分类号:

S831.2

王强州, 潘诗雨, 方梦雅, 李微, 王佳兴, 刘茵茵, 陈世豪. 基于CRISPR-Cas9技术构建TET2基因敲除的鸡胚成纤维细胞系[J]. 中国农业科技导报, 2023, 25(11): 227-233.

Qiangzhou WANG, Shiyu PAN, Mengya FANG, Wei LI, Jiaxing WANG, Yinyin LIU, Shihao CHEN. Construction of Chicken Embryo Fibroblast Cell Line with TET2 Gene Knockout Based on CRISPR-Cas9 Technology[J]. Journal of Agricultural Science and Technology, 2023, 25(11): 227-233.

图/表 6

表1 sgRNA寡核苷酸序列

Table 1 Sequences of sgRNA oligonucleotide

sgRNA	方向 Orientation	序列Sequence（5’→3’）
sgRNA-1	正义链Forward	caccgTTGCCGTTGACTTCGACCTC
sgRNA-1	互补链Reverse	aaacGAGGTCGAAGTCAACGGCAAc
sgRNA-2	正义链Forward	caccgAAAGGTCAGGGCTAATGCGG
sgRNA-2	互补链Reverse	aaacCCGCATTAGCCCTGACCTTTc
sgRNA-3	正义链Forward	caccgCTGCATGCGTGCGGAAGCGA
sgRNA-3	互补链Reverse	aaacTCGCTTCCGCACGCATGCAGc
sgRNA-4	正义链Forward	caccgTGATGCATTAAAGGGGCCGT
sgRNA-4	互补链Reverse	aaacACGGCCCCTTTAATGCATCAc

图1 T7E1酶切分析sgRNA切割活性注：M—DL2000 Marker；泳道1~4分别为转染sgRNA-1、sgRNA-2、sgRNA-3、sgRNA-4的细胞；泳道5和6为未转染sgRNA的细胞。

Fig. 1 Analysis of sgRNA activity by T7E1 cleavage of mismatched fragmentNote： M—DL2000 Marker；Lane 1~4 indicate samples transfected with sgRNA-1， sgRNA-2， sgRNA-3， and sgRNA-4， respectively； both lane 5 and 6 indicate samples transfected without sgRNA.

图2 TET2基因敲除的单克隆细胞DNA测序结果

Fig. 2 DNA sequencing results of monoclonal cells with TET2 gene knockout

图3 野生型细胞和TET2敲除细胞形态学比较

Fig. 3 Comparison of cell morphology between wild type and TET2 knockout cells

图4 Western blot检测野生型和TET2敲除细胞TET2蛋白表达

Fig. 4 Western blot detected the levels of expression of TET2 protein in wild type and TET2 knockout cells

图5 Dot blot检测野生型和TET2敲除细胞的羟甲基化水平

Fig. 5 Dot blot detected levels of hydroxymethylation （5hmC） in wild type and TET2 knockout cells

参考文献 22

1	WU X J, ZHANG Y. TET-mediated active DNA demethylation: mechanism, function and beyond [J]. Nat. Rev. Genet., 2017, 18(9): 517-534.
2	RASMUSSEN K D, JIA G S, JOHANSEN J V, et al.. Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis [J]. Genes Dev., 2015, 29(9): 910-922.
3	ZHONG C, ZHU J F. Tet2: breaking down barriers to T cell cytokine expression [J]. Immunity, 2015, 42(4): 593-595.
4	ICHIYAMA K, CHEN T T, WANG X H, et al.. The methylcytosine dioxygenase Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells [J]. Immunity, 2015, 42(4): 613-626.
5	LIO C W, ZHANG J Y, GONZALEZ-AVALOS E, et al.. Tet2 and Tet3 cooperate with B-lineage transcription factors to regulate DNA modification and chromatin accessibility [J/OL]. Elife, 2016, 5:e18290 [2022-05-12]. .
6	ZHANG Q, ZHAO K, SHEN Q C, et al.. Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6 [J]. Nature, 2015, 525(7569): 389-393.
7	MA S X, WAN X L, DENG Z H, et al.. Epigenetic regulator CXXC5 recruits DNA demethylase Tet2 to regulate TLR7/9-elicited IFN response in pDCs [J]. J. Exp. Med., 2017, 214(5): 1471-1491.
8	CHEN S H, HU X M, CUI I H, et al.. An endogenous retroviral element exerts an antiviral innate immune function via the derived lncRNA lnc-ALVE1-AS1 [J/OL]. Antivir. Res., 2019, 170: 104571 [2022-05-12]. .
9	HIMLY M, FOSTER D N, BOTTOLI I, et al.. The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses [J]. Virology, 1998, 248(2): 295-304.
10	TRAN N T, SOMMERMANN T, GRAF R, et al.. Efficient CRISPR/Cas9-mediated gene knockin in mouse hematopoietic stem and progenitor cells [J]. Cell Rep., 2019, 28(13): 3510-3522.
11	CHEN S H, WANG D D, LIU Y Y, et al.. Targeting the histone methyltransferase disruptor of telomeric silencing 1-like restricts avian leukosis virus subgroup J replication by restoring the innate immune response in chicken macrophages [J/OL]. Front. Microbiol., 2020, 11: 603131 [2022-05-12]. .
12	MA L F, DONG C H, YU M H, et al.. Nanomedicine enable efficient CRISPR-Cas9 genome editing for disease treatment [J]. Sci. Bull., 2022, 67(6): 572-576.
13	庄重, 赵龙, 白皓, 等. 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.
	ZHUANG Z, ZHAO L, BAI H, et al.. Development of CRISPR/Cas9 and its application in poultry [J]. J. Agric. Sci. Technol., 2022, 24(1): 14-23.
14	RASMUSSEN K D, HELIN K. Role of TET enzymes in DNA methylation, development, and cancer [J]. Genes Dev., 2016, 30(7): 733-750.
15	HUISMAN C, WIJST M G, SCHOKKER M, et al.. Re-expression of selected epigenetically silenced candidate tumor suppressor genes in cervical cancer by TET2-directed demethylation [J]. Mol. Ther., 2016, 24(3): 536-547.
16	LI X L, LI G H, FU J, et al.. Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression [J]. Nucleic Acids Res., 2018, 46(19): 10195-10215.
17	GANDHI S, PIACENTINO M L, VIECELI F M, et al.. Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo [J]. Dev. Biol., 2017, 432(1): 86-97.
18	MARTINEZ L M, TORRES R R. CRISPR-Cas9 technology: applications and human disease modelling [J]. Brief. Funct. Genomics, 2017, 16(1): 4-12.
19	CHENG Y Q, LUN M X, LIU Y X, et al.. CRISPR/Cas9-mediated chicken tbk1 gene knockout and its essential role in sting-mediated IFN-beta induction in chicken cells [J/OL]. Front. Immunol., 2018, 9: 3010 [2022-05-12]. .
20	LINSCOTT M L, CHUNG W C. TET1 regulates fibroblast growth factor 8 transcription in gonadotropin releasing hormone neurons [J/OL]. PLoS One, 2019, 14(7): e0220530 [2022-05-12]. .
21	杨雷, 叶洲杰, 李兆龙, 等. 利用电转的方法对T细胞TET2基因敲除并探讨TET2对T细胞增殖的影响[J]. 生物技术通报, 2020, 36(1): 229-237.
	YANG L, YE Z J, LI Z L, et al.. Effects of TET2 on T cell proliferation by electroporation [J]. Biol. Bull., 2020, 36(1): 229-237.
22	SHI K, LU Y L, CHEN X L, et al.. Effects of ten-eleven translocation-2 (Tet2) on myogenic differentiation of chicken myoblasts [J/OL]. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 2021, 252: 110540 [2022-05-12]. .

[1]	赵宁, 李星, 江勇, 王志秀, 毕瑜林, 陈国宏, 白皓, 常国斌. 图像识别技术在鸡养殖领域的应用[J]. 中国农业科技导报, 2023, 25(9): 13-22.
[2]	李宏博, 陈月月, 杨玉洁, 徐琦琦, 秦蕾, 蔡鑫, 夏利宁. 新疆伊犁昭苏健康鸡源大肠杆菌耐药性及耐药基因分析[J]. 中国农业科技导报, 2023, 25(11): 123-131.
[3]	卢奎, 李福伟, 朱丽慧, 和笑笑, 卫利春, 朱永豪, 严华祥. 饲粮中过量镁对新杨黑羽蛋鸡鸡蛋暗斑和蛋壳微生物组成的影响[J]. 中国农业科技导报, 2023, 25(1): 197-205.
[4]	张傲, 白皓, 毕瑜林, 黄应权, 王志秀, 江勇, 陈国宏, 常国斌. 鸡体核温度影响因素研究进展[J]. 中国农业科技导报, 2023, 25(1): 26-34.
[5]	吴月, 王育南, 宋哈楠, 关伟军, 李楠. 固始鸡脐带间充质干细胞原代培养及其诱导分化潜能研究[J]. 中国农业科技导报, 2022, 24(9): 79-87.
[6]	周蕾, 陈兆兰, 严玉波, 李桥. 鸡粪生物炭吸附固定铅的研究[J]. 中国农业科技导报, 2022, 24(11): 199-207.
[7]	石雷, 孙研研, 李云雷, 白皓, 陈继兰. 鸡交叉喙研究进展[J]. 中国农业科技导报, 2022, 24(11): 35-42.
[8]	何孟纤, 汪俊跃, 孙玲伟, 徐皆欢, 吴彩凤, 张树山, 戴建军, 杨凯旋, 张德福. 6种鸡精液冷冻稀释液以及冻后保存条件比较[J]. 中国农业科技导报, 2022, 24(10): 53-61.
[9]	苏雨萌§,张旭婷§,特日格乐,田敏,尚晓蕊,李国婧,王瑞刚*. 高通量测序鉴定中间锦鸡儿干旱条件下的microRNA[J]. 中国农业科技导报, 2021, 23(3): 51-57.
[10]	张智, 乔艳, 陈云峰, 胡诚, 刘东海, 李双来. 不同菌剂对鸡粪堆肥过程中有害气体排放的影响[J]. 中国农业科技导报, 2021, 23(12): 145-150.
[11]	胡博,闫伟*,刘宇,郝艳玲. 三种桑生理特性对盐胁迫的响应[J]. 中国农业科技导报, 2020, 22(4): 61-67.
[12]	梁玉刚1,李静怡1,王丹2,余政军1,黄璜1,陈灿1. 垄作稻鱼鸡共生对水稻群体生长特性及产量形成的影响[J]. 中国农业科技导报, 2020, 22(11): 165-175.
[13]	马建华1,朱晓菲1,何程相1,丁龙梅1,向明剑1,黄良伟1,姜良珍2,谢松林1*. 鸡爪槭（Acer palmatum Thunb.）品种‘赤枫’组培再生体系的建立[J]. 中国农业科技导报, 2020, 22(1): 171-178.
[14]	董晓宇1,郭月峰1*,姚云峰1,秦富仓1,祁伟2,王欣3,王慧1. 柠条锦鸡儿根长与游离氨基酸分布特征研究[J]. 中国农业科技导报, 2019, 21(10): 66-73.
[15]	刘龙1,姚云峰1,郭月峰1*,祁伟1,2,温健1,高玉寒1,尉迟文思1,韩兆敏1. 准格尔旗砒砂岩区三种典型造林树种蒸腾耗水研究[J]. 中国农业科技导报, 2018, 20(3): 124-131.