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

doi:10.3969/j.issn.1004-1524.2022.09.04

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (9): 1849-1855.DOI: 10.3969/j.issn.1004-1524.2022.09.04

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S828

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.

Figures/Tables 8

References 25

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

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

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

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

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

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Figures/Tables 8

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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

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