生物技术通报 ›› 2022, Vol. 38 ›› Issue (3): 173-180.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0647
燕炯1(), 冯晨毅1, 高学坤1, 许祥1, 杨佳敏1, 陈朝阳2()
收稿日期:
2021-05-19
出版日期:
2022-03-26
发布日期:
2022-04-06
作者简介:
燕炯,男,博士,副教授,研究方向:营养相关疾病分子学;E-mail: 基金资助:
YAN Jiong1(), FENG Chen-yi1, GAO Xue-kun1, XU Xiang1, YANG Jia-min1, CHEN Zhao-yang2()
Received:
2021-05-19
Published:
2022-03-26
Online:
2022-04-06
摘要:
利用CRISPR/Cas9技术构建纯合围脂滴蛋白(Plin1)基因敲除小鼠模型,并初步分析其表型。针对Plin1基因2号外显子前后序列设计sgRNA并构建表达载体,体外转录获得sgRNA后与Cas9蛋白混合,显微注射至小鼠受精卵中并进行胚胎移植。出生小鼠经测序及PCR基因型鉴定获得F0代阳性小鼠;令F0代小鼠与野生型小鼠杂交,获得F1代杂合子小鼠;通过F1代杂合小鼠近交,获得F2代纯合子小鼠模型。常规饲喂同窝别Plin1基因敲除纯合小鼠、杂合小鼠和野生型小鼠,并测量小鼠体重、身长等体格参数;定量实时聚合酶链反应(Q-RT-PCR)和蛋白质印迹(WB)分别检测每个组织中Plin1基因在mRNA和蛋白质水平的表达。敲除了小鼠Plin1基因741 bp的片段(包含了2号外显子在内);Plin1基因敲除小鼠PLIN1 mRNA和蛋白表达水平均显著降低(P < 0.05);常规喂养4周后,相比于野生型及杂合型小鼠,纯合型Plin1基因敲除小鼠体重显著降低(P < 0.05)。成功构建了Plin1基因敲除小鼠模型;初步表型分析发现,Plin1基因敲除小鼠与野生型小鼠相比较为瘦弱。
燕炯, 冯晨毅, 高学坤, 许祥, 杨佳敏, 陈朝阳. 基于CRISPR/Cas9技术构建Plin1基因敲除小鼠模型及表型分析[J]. 生物技术通报, 2022, 38(3): 173-180.
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.
图1 sgRNA敲除Plin1基因2号外显子的示意图 Plin1基因转录本为NM_001113471.1
Fig. 1 Schematic diagram of sgRNA knocking out exon 2 of Plin1 gene The Plin1 gene transcript is NM_001113471.1
名称 Name | 序列 Sequence(5'-3') | PAM序列 PAM sequence |
---|---|---|
sgRNA-Plin1-1 | TTGGAGGGCGAATGTTACAT | AGG |
sgRNA-Plin1-2 | GAACTTCAGGCGTATGACCC | TGG |
表1 sgRNA序列
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 |
图2 Plin1基因小鼠敲除基因型鉴定的策略 Plin1基因转录本为NM_001113471.1。
Fig. 2 Strategies for genotype identification of Plin1 knoc-kout mice The Plin1 gene transcript is NM_001113471.1
名称Name | 序列Sequence(5'-3') | 大小Size/bp |
---|---|---|
Plin1-F | CTGAGAGAAGGCTTAACCTTGCTGG | 25 |
Plin1-R1 | AGCTTTCCATCCTGCAAGTGAGTCAG | 26 |
Plin1-R2 | AGGTGGCAAGGACAGAGACAGTGAG | 25 |
表2 基因型鉴定的引物序列
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 |
图3 sgRNA表达载体构建与鉴定 A:sgRNA表达载体的酶切结果(2,4泳道:sgRNA-Plin1-1、2载体;1,3泳道:酶切片段;M:DNA marker);B:sgRNA表达载体测序结果;C:sgRNA体外转录片段电泳结果(1-3泳道:sgRNA-Plin1-1转录产物;4-6:sgRNA-Plin1-2转录产物;M:DNA marker)
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)
图4 F0和F1代小鼠基因型鉴定 A:F0代阳性小鼠Plin1-F1/R1扩增Plin1基因的电泳结果;1-12泳道:F0阳性小鼠;WT:野生型对照;M:DNA marker;B-C:F1代小鼠Plin1-F1/R1扩增Plin1基因的电泳结果。1-13泳道:F1代小鼠;WT:野生型对照;M:DNA marker;D:F0、F1代小鼠基因测序结果
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
图5 F2代小鼠基因型鉴定 A:Plin1-F1/R1扩增Plin1基因的电泳结果;B:Plin1-F1/R2扩增Plin1基因的电泳结果;1-17泳道:F2代小鼠Plin1基因片段;白色箭头:纯合型;黑色箭头:野生型
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 |
表3 各组小鼠体格参数
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 |
图6 小鼠各组织中PLIN1 mRNA的相对表达量 A:肝脏中PLIN1 mRNA相对表达量;B:骨骼肌中PLIN1 mRNA相对表达量;C:白色脂肪组织中PLIN1 mRNA相对表达量;**:相比于Plin1+/-,P < 0.05;*:相比于WT,P < 0.05,下同
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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