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

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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

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