Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (6): 325-334.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1398
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MA Yu-jing(), DUAN Chun-hui, HE Ming-yang, ZHANG Ying-jie, YANG Ruo-chen, WANG Yong, LIU Yue-qin()
Received:
2022-11-14
Online:
2023-06-26
Published:
2023-07-07
Contact:
LIU Yue-qin
E-mail:mayujing66@126.com;Liuyueqin66@126.com
MA Yu-jing, DUAN Chun-hui, HE Ming-yang, ZHANG Ying-jie, YANG Ruo-chen, WANG Yong, LIU Yue-qin. Effects of Knockout of G0S2 Gene in Ovarian Granulosa Cell Proliferation, Steroids Hormones and Related Gene Expression[J]. Biotechnology Bulletin, 2023, 39(6): 325-334.
引物名称Primer name | 序列Sequence(5'-3') |
---|---|
O-sp-G0S2-E1-1F | caccGTGCGAATGTACCTGCTGGG |
O-sp-G0S2-E1-1R | aaacCCCAGCAGGTACATTCGCAC |
Table 1 G0S2 sgRNA primer sequences
引物名称Primer name | 序列Sequence(5'-3') |
---|---|
O-sp-G0S2-E1-1F | caccGTGCGAATGTACCTGCTGGG |
O-sp-G0S2-E1-1R | aaacCCCAGCAGGTACATTCGCAC |
引物名称Primer name | 序列Sequence(5'-3') | 退火温度Annealing temperature/℃ | 片段大小Fragment length/bp |
---|---|---|---|
GAPDH | F: GGTCGGAGTGAACGGATTTG | 60 | 222 |
R: CTTGACTGTGCCGTGGAACTT | |||
G0S2 | F: GCGAAGCTGGTGCGAATGTAC | 60 | 154 |
R: CCTCCAGTGGCTTGCCTTGATC | |||
Bcl-2 | F: GATGACCGAGTACCTGAACCG | 60 | 120 |
R: GACAGCCAGGAGAAATCAAACA | |||
Caspase3 | F: GCTACAAGGTCCGTTATGCC | 60 | 128 |
R: GATGCTGCCGTATTCGTTCTC | |||
Bax | F: TGCTCACTGCCTCACTCACC | 60 | 179 |
R: CCCAAGACCACTCCTCCCTA | |||
StAR | F: ATTCAGGAGGCAAAGAGCAGC | 60 | 270 |
R: TCGGGTAAGGAAAATGGGTCA | |||
3β-HSD | F: CAGTCTATGTTGGCAATGTGGC | 60 | 283 |
R: CGGTTGAAGCAGGGGTGGTAT | |||
CYP11 | F: GTTTCGCTTTGCCTTTGAGTC | 60 | 120 |
R: ACAGTTCTGGAGGGAGGTTGA | |||
CYP19 | F: GCTTTTGGAAGTGCTGAACCC | 60 | 379 |
R: CATGCCGATGAACTGCAACC |
Table 2 RT-qCR primers’ sequences
引物名称Primer name | 序列Sequence(5'-3') | 退火温度Annealing temperature/℃ | 片段大小Fragment length/bp |
---|---|---|---|
GAPDH | F: GGTCGGAGTGAACGGATTTG | 60 | 222 |
R: CTTGACTGTGCCGTGGAACTT | |||
G0S2 | F: GCGAAGCTGGTGCGAATGTAC | 60 | 154 |
R: CCTCCAGTGGCTTGCCTTGATC | |||
Bcl-2 | F: GATGACCGAGTACCTGAACCG | 60 | 120 |
R: GACAGCCAGGAGAAATCAAACA | |||
Caspase3 | F: GCTACAAGGTCCGTTATGCC | 60 | 128 |
R: GATGCTGCCGTATTCGTTCTC | |||
Bax | F: TGCTCACTGCCTCACTCACC | 60 | 179 |
R: CCCAAGACCACTCCTCCCTA | |||
StAR | F: ATTCAGGAGGCAAAGAGCAGC | 60 | 270 |
R: TCGGGTAAGGAAAATGGGTCA | |||
3β-HSD | F: CAGTCTATGTTGGCAATGTGGC | 60 | 283 |
R: CGGTTGAAGCAGGGGTGGTAT | |||
CYP11 | F: GTTTCGCTTTGCCTTTGAGTC | 60 | 120 |
R: ACAGTTCTGGAGGGAGGTTGA | |||
CYP19 | F: GCTTTTGGAAGTGCTGAACCC | 60 | 379 |
R: CATGCCGATGAACTGCAACC |
Fig. 2 FSHR immunofluorescence staining of ovarian granulosa cells in sheep(400×) A: FSHR stained ovarian granulosa cells of sheep. B: DAPI stained ovarian granulosa cell nuclei of sheep. C: Overlay dyeing effect
Fig. 3 Effects of plasmid vector transfection of the ovarian granulosa cells in sheep(400×) A: Green fluorescence expression of ovarian granulosa cells transfected with blank PX458 plasmid. B: Green fluorescence expression of ovarian granulosa cells transfected with PX458-sgRNA-G0S2 recombinant plasmid. C: Untransfected blank ovarian granulosa cells in brightfield
Fig. 4 Effects of G0S2 gene knockout on the G0S2 mRNA and protein expression in ovarian granulosa cells A: Statistics of mRNA expression results after G0S2 knockout. B: Statistics of protein expression results after G0S2 knockout. C: Grayscale plot of protein expression after G0S2 knockout; * P<0.05, **P<0.01. The same below
Fig. 5 Effects of G0S2 gene knockout in ovarian granulosa cell proliferation viability and apoptosis A: Flow cytometry apoptosis assay of G0S2-Knockout group. B: Control group flow cytometry apoptosis assay map. C: Flow cytometry apoptosis result statistics. D: Cell proliferation viability result statistics
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