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:2021-03-23Online:2022-09-25Published:2022-09-30 -
Contact:WANG Han
CLC Number:
Cite this article
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.
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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 |
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 |
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 |
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 |
Fig.1 Identification of recombinant vector pX459-sgRNA a, Target of pX459-sgRNA1; b, Target of pX459-sgRNA2.
Fig.1 Identification of recombinant vector pX459-sgRNA a, Target of pX459-sgRNA1; b, Target of pX459-sgRNA2.
Fig.2 PK15 cells treated with different concentrations of puromycin (96 h)
Fig.2 PK15 cells treated with different concentrations of puromycin (96 h)
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.
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.
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 |
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 |
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).
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).
Fig.5 Position relationship between edited target sequence and porcine miR-22 precursor sequence
Fig.5 Position relationship between edited target sequence and porcine miR-22 precursor sequence
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