猪miR-22前体上游序列突变的PK15细胞系构建

doi:10.3969/j.issn.1004-1524.2022.09.04

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (9): 1849-1855.DOI: 10.3969/j.issn.1004-1524.2022.09.04

猪miR-22前体上游序列突变的PK15细胞系构建

丁兆雪(), 王佳洁, 沈中浩, 周晓龙, 杨松柏, 金航峰, 赵阿勇, 汪涵()

浙江农林大学动物科技学院·动物医学院,浙江省畜禽绿色生态健康养殖应用技术研究重点实验室,浙江杭州 311300

收稿日期:2021-03-23 出版日期:2022-09-25 发布日期:2022-09-30
通讯作者: 汪涵
作者简介:*汪涵,E-mail: wanghan1990@zafu.edu.cn
丁兆雪(1999—),女,福建福州人,本科生,主要从事动物遗传育种研究。E-mail: 201827010302@stu.zafu.edu.cn
基金资助:
国家自然科学基金(31802030);浙江农林大学国家级大学生创新创业训练计划(202010341041)

Construction of PK15 cells with porcine miR-22 upstream sequence mutation

DING Zhaoxue(), WANG Jiajie, SHEN Zhonghao, ZHOU Xiaolong, YANG Songbai, JIN Hangfeng, ZHAO Ayong, WANG Han()

Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, College of Animal Science and Technology · College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China

Received:2021-03-23 Online:2022-09-25 Published:2022-09-30
Contact: WANG Han

摘要/Abstract

摘要：

猪miR-22前体上游序列片段突变可能对miR-22的表达起到重要调控作用,为进一步探究其功能,基于CRISPR/Cas9基因编辑技术,通过针对猪miR-22前体上游序列片段设计2个靶标sgRNAs(short guide RNAs),构建重组表达载体pX459-sgRNA1和pX459-sgRNA2;随后将重组质粒转染猪肾上皮细胞系(PK15),经嘌呤霉素初步筛选后,提取基因组DNA,通过PCR及测序鉴定编辑效果;最后,通过实时荧光定量 PCR(qRT-PCR)检测编辑前后miR-22相对表达量的变化。结果显示,81个阳性克隆中,共产生6种突变类型,突变率达60.49%;qRT-PCR检测显示,编辑后miR-22的表达量显著下调约50%。本研究成功获得了miR-22前体上游序列突变的猪PK15细胞模型,为今后猪miR-22的功能研究提供了新的可应用研究对象。

关键词: 猪, 微小RNA, 突变, CRISPR/Cas9技术, PK15细胞系

Abstract:

The previous study found that the mutation on the fragment of upstream sequence of porcine miR-22 precursor might play an important role in regulating the expression of miR-22. In order to further explore its function, this study designed two target RNAs (short guide RNAs) for porcine miR-22 upstream sequence fragments, constructed a recombinant expression vector pX459-sgRNA1 and pX459-sgRNA2. Subsequently, the recombinant plasmid was transfected into porcine kidney epithelial cell line (PK15). After preliminary screening with puromycin, genomic DNA was extracted, and the editing effect was identified by PCR and sequencing. Finally, quantitative real-time PCR (qRT-PCR) was used to detect the relative expression of miR-22 before and after editing. The results showed that out of 81 positive clones, 6 mutation types were produced, with a mutation rate of 60.49%. The qRT-PCR test showed that the expression of miR-22 was significantly down-regulated by about 50% after editing. These results indicated that this study had successfully obtained a porcine PK15 cell model with mutations in the upstream sequence of the miR-22 precursor, which provided a new applicable research object for future functional studies on porcine miR-22.

Key words: pig, microRNA, mutation, CRISPR/Cas9 technology, PK15 cell lines

中图分类号:

S828

丁兆雪, 王佳洁, 沈中浩, 周晓龙, 杨松柏, 金航峰, 赵阿勇, 汪涵. 猪miR-22前体上游序列突变的PK15细胞系构建[J]. 浙江农业学报, 2022, 34(9): 1849-1855.

DING Zhaoxue, WANG Jiajie, SHEN Zhonghao, ZHOU Xiaolong, YANG Songbai, JIN Hangfeng, ZHAO Ayong, WANG Han. Construction of PK15 cells with porcine miR-22 upstream sequence mutation[J]. Acta Agriculturae Zhejiangensis, 2022, 34(9): 1849-1855.

图/表 8

表1 sgRNA序列信息

Table 1 Sequence information of sgRNA

编号 Code	sgRNA序列 sgRNA sequence(5'→3')
sgRNA1	F: CACCGTTTTCTCTCACTGAAGGCCC
	R: AAACGGGCCTTCAGTGAGAGAAAAC
sgRNA2	F: CACCGATTGAACATCTGCTGGGGC
	R: AAACGCCCCAGCAGATGTTCAATC

表2 miR-22茎环RT-PCR相关引物

Table 2 Related primers of miR-22 stem loop RT-PCR

引物名称Primer name	引物序列Primer sequence(5'→3')	基因编号Gene code
miR-22反转录茎环引物	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAGTTC	100498778
Reverse transcription stem loop primer of miR-22
miR-22上游引物Upstream primer of miR-22	CAGGAAGCTGCCAGTTGAA	100498778
miR-22下游引物Downstream primer of miR-22	TCAACTGGTGTCGTGGAGTC
U6上游引物Upstream primer of U6	GCTTCGGCAGCACATATACT	100522884
U6下游引物Downstream primer of U6	TTCACGAATTTGCGTGTCAT

图1 重组载体pX459-sgRNA的鉴定 a, pX459-sgRNA1靶点; b, pX459-sgRNA2靶点。

Fig.1 Identification of recombinant vector pX459-sgRNA a, Target of pX459-sgRNA1; b, Target of pX459-sgRNA2.

图2 不同浓度嘌呤霉素处理PK15细胞(96 h)

Fig.2 PK15 cells treated with different concentrations of puromycin (96 h)

图3 基因编辑后PK15细胞miR-22前体上游序列测序分析红色为Cas9识别位点PAM,紫色为突变碱基。1~6代表突变类型。

Fig.3 Sequencing of miR-22 precursor up-stream sequence of PK15 cells after gene editing Red was Cas9 recognition site PAM, purple was mutant base.1-6 were mutations.

表3 基因编辑后PK15细胞miR-22前体上游序列突变比率

Table 3 Mutation rate of upstream sequence of miR-22 precursor in PK15 cells after gene editing

LB培养基编号 No. of LB medium	挑选的阳性克隆数 Number of positive clones selected	突变个数 Number of mutations	突变比率 Mutation ratio/%
1	5	2	40
2	4	2	50
3	5	4	80
4	5	3	60
5	5	3	60
6	5	1	20
7	6	3	50
8	3	3	100
9	5	2	40
10	5	3	60
11	6	5	83.33
12	4	1	25
13	5	3	60
14	5	5	100
15	5	3	60
16	4	2	50
17	4	4	100
总计Total	81	49	60.49

图4 qRT-PCR检测基因编辑后PK15细胞中miR-22的表达量 A, miR-22相对表达量; B, miR-22熔解曲线; C, U6熔解曲线。** 表示差异极显著(P<0.01)。

Fig.4 Expression of miR-22 was detected by qRT-PCR after gene editing in PK15 cells A, Relative expression of miR-22; B, Dissociation curve of miR-22; C, Dissociation curve of U6. ** indicated the difference was significant (P<0.01).

图5 编辑的靶向序列与猪miR-22前体序列位置关系

Fig.5 Position relationship between edited target sequence and porcine miR-22 precursor sequence

参考文献 25

[1]	冉茂良, 陈斌, 尹杰, 等. 猪microRNA组学研究进展[J]. 遗传, 2014, 36(10): 974-984.
	RAN M L, CHEN B, YIN J, et al. Advances in porcine miRNAome[J]. Hereditas, 2014, 36(10): 974-984. (in Chinese with English abstract)
[2]	ZHANG J, LIU Y L. MicroRNA in skeletal muscle: its crucial roles in signal proteins, mus cle fiber type, and muscle protein synthesis[J]. Current Protein & Peptide Science, 2017, 18(6): 579-588.
[3]	HONG Q D, XU G L, HOU L J, et al. MicroRNA-22 inhibits proliferation and promotes differentiation of satellite cells in porcine skeletal muscle[J]. Journal of Integrative Agriculture, 2020, 19(1): 225-233. DOI
[4]	汪涵. MIR-696和MIR-22调控骨骼肌细胞增殖与分化的机制研究[D]. 南京: 南京农业大学, 2017.
	WANG H. Mechanism of MiR-696 and MiR-22 in regulating skeletal muscle cell proliferation and differentiation[D]. Nanjing: Nanjing Agricultural University, 2017. (in Chinese with English abstract)
[5]	黄瑞华, 张倩, 李平华, 等. 一种与苏淮猪肉色性状相关的SNP标记及其引物和应用: CN107937552B[P]. 2020-01-21.
[6]	BI Y Z, HUA Z D, LIU X M, et al. Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP[J]. Scientific Reports, 2016, 6: 31729. DOI PMID
[7]	WANG K K, OUYANG H S, XIE Z C, et al. Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system[J]. Scientific Reports, 2015, 5: 16623. DOI PMID
[8]	WU J Q, MEI G, LIU Z G, et al. Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system[J]. Hereditas, 2015, 37(1): 55-62.
[9]	MALI P, ESVELT K M, CHURCH G M. Cas9 as a versatile tool for engineering biology[J]. Nature Methods, 2013, 10(10): 957-963. DOI PMID
[10]	BONAFONT J, MENCÍA Á, GARCÍA M, et al. Clinically relevant correction of recessive dystrophic epidermolysis bullosa by dual sgRNA CRISPR/Cas9-mediated gene editing[J]. Molecular Therapy, 2019, 27(5): 986-998. DOI PMID
[11]	CHEN X Y, XU F, ZHU C M, et al. Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans[J]. Scientific Reports, 2014, 4: 7581. DOI URL
[12]	GHASSEMI B, SHAMSARA M, SOLEIMANI M, et al. Pipeline for the generation of gene knockout mice using dual sgRNA CRISPR/Cas9-mediated gene editing[J]. Analytical Biochemistry, 2019, 568: 31-40. DOI PMID
[13]	GEORGES M, COPPIETERS W, CHARLIER C. Polymorphic miRNA-mediated gene regulation: contribution to phenotypic variation and disease[J]. Current Opinion in Genetics & Development, 2007, 17(3): 166-176.
[14]	SLABY O, BIENERTOVA-VASKU J, SVOBODA M, et al. Genetic polymorphisms and microRNAs: new direction in molecular epidemiology of solid cancer[J]. Journal of Cellular and Molecular Medicine, 2012, 16(1): 8-21. DOI PMID
[15]	LEI B, GAO S, LUO L F, et al. A SNP in the miR-27a gene is associated with litter size in pigs[J]. Molecular Biology Reports, 2011, 38(6): 3725-3729. DOI PMID
[16]	LEE J S, KIM J M, LIM K S, et al. Effects of polymorphisms in the porcine microRNA MIR206/MIR133B cluster on muscle fiber and meat quality traits[J]. Animal Genetics, 2013, 44(1): 101-106. DOI URL
[17]	KRIZEK B A. Intronic sequences are required for AINTEGUMENTA-LIKE6 expression in Arabidopsis flowers[J]. BMC Research Notes, 2015, 8(1): 1-9. DOI URL
[18]	MOHAPATRA C, BARMAN H K. Identification of promoter within the first intron of Plzf gene expressed in carp spermatogonial stem cells[J]. Molecular Biology Reports, 2014, 41(10): 6433-6440. DOI URL
[19]	PEREIRA G B, MENG F X, KOCKARA N T, et al. Targeted deletion of the antisilencer/enhancer (ASE) element from intron 1 of the myelin proteolipid protein gene (Plp1) in mouse reveals that the element is dispensable for Plp1 expression in brain during development and remyelination[J]. Journal of Neurochemistry, 2013, 124(4): 454-465. DOI PMID
[20]	OLDRIDGE D A, WOOD A C, WEICHERT-LEAHEY N, et al. Genetic predisposition to neuroblastoma mediated by a LMO1 super-enhancer polymorphism[J]. Nature, 2015, 528(7582): 418-421. DOI URL
[21]	SRIBUDIANI Y, METZGER M, OSINGA J, et al. Variants in RET associated with hirschsprung’s disease affect binding of transcription factors and gene expression[J]. Gastroenterology, 2011, 140(2): 572-582.e2. DOI URL
[22]	VISSER M, KAYSER M, PALSTRA R J. HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter[J]. Genome Research, 2012, 22(3): 446-455. DOI PMID
[23]	VISSER M, PALSTRA R J, KAYSER M. Allele-specific transcriptional regulation of IRF₄ in melanocytes is mediated by chromatin looping of the intronic rs12203592 enhancer to the IRF4 promoter[J]. Human Molecular Genetics, 2015, 24(9): 2649-2661. DOI URL
[24]	KUO C H, GOLDBERG M D, LIN S L, et al. Identify intronic microRNA with bioinformatics[M]// YING S Y. MicroRNA protocols. Totowa, New Jersey, US: Humana Press, 2013: 77-82.
[25]	BURATTI E, BRINDISI A, PAGANI F, et al. Nuclear factor TDP-43 binds to the polymorphic TG repeats in CFTR intron 8 and causes skipping of exon 9: a functional link with disease penetrance[J]. American Journal of Human Genetics, 2004, 74(6): 1322-1325. DOI PMID

猪miR-22前体上游序列突变的PK15细胞系构建

Construction of PK15 cells with porcine miR-22 upstream sequence mutation

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 25

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王晨, 张敏, 王振旗, 钱晓雍, 徐昶, 倪远之, 李金文, 沈根祥. 长期施用猪粪稻田的重金属迁移规律与累积风险[J]. 浙江农业学报, 2022, 34(9): 1985-1994.
[2]	詹佳飞, 徐魁, 张磊, 夏介英, 洪杨, 董涵, 刘洋露, 周静, 袁明铭, 王永金, 鄢良春. 毛蕊花糖苷抑制2型猪链球菌的溶血素蛋白活性而降低其小鼠致病性[J]. 浙江农业学报, 2022, 34(8): 1609-1616.
[3]	许申平, 张燕, 梁芳, 蒋素华, 牛苏燕, 崔波, 袁秀云. 蝴蝶兰PhaSEP3基因的克隆及其在突变体中的表达[J]. 浙江农业学报, 2022, 34(8): 1703-1712.
[4]	张亮, 柴捷, 潘红梅, 郭宗义. 氯前列醇钠与卡贝缩宫素对荣昌猪和涪陵黑猪母猪分娩时间的影响[J]. 浙江农业学报, 2022, 34(5): 915-922.
[5]	李景上, 章啸君, 陈胜昌, 蒋锦华, 项云, 屠平光, 楼芳芳, 杨华, 肖英平. 金华猪肌肉和血清氨基酸谱的发育性变化及其与肌肉生长的相关性[J]. 浙江农业学报, 2022, 34(4): 687-694.
[6]	牟泓晔, 周小杰, 杨永春, 王晓杜, 周莹珊, 宋厚辉. 猪圆环病毒2型和3型双重荧光定量PCR检测方法的建立[J]. 浙江农业学报, 2022, 34(3): 457-463.
[7]	许金根, 靳二辉, 王重龙, 顾有方, 李庆岗. 猪CAST基因多态性与生物信息学分析[J]. 浙江农业学报, 2022, 34(1): 17-23.
[8]	欧秀琼, 李睿, 张晓春, 钟正泽, 李星, 景绍红, 郭宗义, 李兴桂. 肌纤维类型组成对猪肌肉品质与能量代谢的影响研究进展[J]. 浙江农业学报, 2022, 34(1): 196-203.
[9]	朱志伟, 陈晓宇, 于福先, 张樑, 黄菁, 王志刚, 赖建兵, 沈顺新, 殷文彬, 潘建治. 精准定时输精对后备母猪早期胚胎发育与繁殖性能的影响[J]. 浙江农业学报, 2021, 33(5): 794-800.
[10]	刘君雯, 王迪, 朱艳艳, 邢刚, 占松鹤, 刘晓露, 魏建忠, 孙裴, 刘雪兰, 李郁. 猪丹毒丝菌CbpB基因的克隆表达及其间接ELISA抗体检测方法的建立与应用[J]. 浙江农业学报, 2021, 33(5): 816-824.
[11]	涂藤, 尹清清, 张鹏飞, 王印, 杨泽晓, 姚学萍, 罗燕. 基于毛细管电泳的7种猪源性疫病的多重PCR检测方法的建立[J]. 浙江农业学报, 2021, 33(4): 618-631.
[12]	金俪雯, 刘增金, 刘爱军. 猪肉销售商可追溯体系参与行为及其影响因素——基于北京、上海、济南3市636位销售商的实证分析[J]. 浙江农业学报, 2021, 33(3): 541-552.
[13]	吴俊静, 乔木, 周佳伟, 梅书棋, 彭先文. 猪长链非编码RNA lnc-000649在PRRSV感染增殖中的作用[J]. 浙江农业学报, 2021, 33(2): 223-229.
[14]	张茂, 赵鑫, 蔡更元, 杨化强. 精原干细胞体外培养体系的建立[J]. 浙江农业学报, 2021, 33(12): 2254-2263.
[15]	李红英, 高延武, 于茹恩, 王政博, 李雪萍, 刘龙昌. 利用CRISPR_Cas9技术创建拟南芥Argonaute2基因缺失突变体[J]. 浙江农业学报, 2021, 33(11): 2001-2008.