Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (3): 173-180.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0647
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YAN Jiong1(), FENG Chen-yi1, GAO Xue-kun1, XU Xiang1, YANG Jia-min1, CHEN Zhao-yang2()
Received:
2021-05-19
Online:
2022-03-26
Published:
2022-04-06
Contact:
CHEN Zhao-yang
E-mail:yanjiong@126.com;ccytycn@163.com
YAN Jiong, FENG Chen-yi, GAO Xue-kun, XU Xiang, YANG Jia-min, CHEN Zhao-yang. Construction of Homozygous Plin1-knockout Mouse Model and Phenotype Analysis Based on CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2022, 38(3): 173-180.
名称 Name | 序列 Sequence(5'-3') | PAM序列 PAM sequence |
---|---|---|
sgRNA-Plin1-1 | TTGGAGGGCGAATGTTACAT | AGG |
sgRNA-Plin1-2 | GAACTTCAGGCGTATGACCC | TGG |
Table 1 sgRNA sequence
名称 Name | 序列 Sequence(5'-3') | PAM序列 PAM sequence |
---|---|---|
sgRNA-Plin1-1 | TTGGAGGGCGAATGTTACAT | AGG |
sgRNA-Plin1-2 | GAACTTCAGGCGTATGACCC | TGG |
名称Name | 序列Sequence(5'-3') | 大小Size/bp |
---|---|---|
Plin1-F | CTGAGAGAAGGCTTAACCTTGCTGG | 25 |
Plin1-R1 | AGCTTTCCATCCTGCAAGTGAGTCAG | 26 |
Plin1-R2 | AGGTGGCAAGGACAGAGACAGTGAG | 25 |
Table 2 Primer sequence for genotyping
名称Name | 序列Sequence(5'-3') | 大小Size/bp |
---|---|---|
Plin1-F | CTGAGAGAAGGCTTAACCTTGCTGG | 25 |
Plin1-R1 | AGCTTTCCATCCTGCAAGTGAGTCAG | 26 |
Plin1-R2 | AGGTGGCAAGGACAGAGACAGTGAG | 25 |
Fig. 3 Construction and identification of sgRNA expression vector A:Digestion results of sgRNA expression vector(Lane 2 and 4:sgRNA-Plin1-1,2 vectors. Lane 1 and 3:Enzyme fragment. M:DNA marker). B:Sequencing results of sgRNA expression vector. C:In vitro transcriptional fragment electrophoresis results of sgRNA(Lane 1-3:sgRNA-Plin1-1 transcription products. Lane 4-6 lane:sgRNA-Plin1-2 transcription products. M:DNA marker)
Fig. 4 Genotype identification of F0 and F1 generation mice A:Electrophoretic results of Plin1-F1/R1 amplified from F0 generation positive mice;lane 1-12:F0 positive mice;WT:wild type control;M. DNA marker. B-C:Electrophoretic results of Plin1-F1/R1 amplified from F1 generation mice;lane 1-13:F1 generation mice;WT:wild type control. M:DNA marker. D:gene sequencing results of F0 and F1 generation mice
Fig. 5 Genotype identification of F2 generation mice A:Electrophoretic results of Plin1 gene amplified by Plin1-F1/R1. B:Electrophoretic results of Plin1 gene amplified by Plin1-F1/R2. Lane 1-17:Plin1 gene fragment of F2 generation mice. White arrow:Homozygous. Black arrow:Wild type
组别/指标 Group/index | 体重 Body weight/g | 体长 Body length/cm | 摄食量 Food intake/g |
---|---|---|---|
野生型WT | 22.95±0.55 | 8.88±2.17 | 16.26±2.13 |
杂合型Plin1+/- | 23.20±1.57 | 9.31±3.49 | 15.43±3.03 |
纯合型Plin1-/- | 18.34±0.35* | 8.72±1.34 | 15.67±2.59 |
Table 3 Physical parameters of mice in each group
组别/指标 Group/index | 体重 Body weight/g | 体长 Body length/cm | 摄食量 Food intake/g |
---|---|---|---|
野生型WT | 22.95±0.55 | 8.88±2.17 | 16.26±2.13 |
杂合型Plin1+/- | 23.20±1.57 | 9.31±3.49 | 15.43±3.03 |
纯合型Plin1-/- | 18.34±0.35* | 8.72±1.34 | 15.67±2.59 |
Fig. 6 Relative expressions of PLIN1 mRNA in the various tissues of mice A:Relative level of PLINI mRNA in liver. B:Relative level of PLINI mRNA in skeletal muscle. C:Relative level of PLINI mRNA in white adipose tissue. **:Com-pared with Plin1+/-. P < 0.05. *:Compared with WT,P < 0.05. The same below
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