生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 273-280.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1564
王海杰(), 王成稷, 郭洋, 王云, 陈艳娟, 梁敏, 王珏, 龚慧, 沈如凌()
收稿日期:
2022-02-21
出版日期:
2022-10-26
发布日期:
2022-11-11
作者简介:
王海杰,男,实验师,研究方向:模式生物表型;E-mail: WANG Hai-jie(), WANG Cheng-ji, GUO Yang, WANG Yun, CHEN Yan-juan, LIANG Min, WANG Jue, GONG Hui, SHEN Ru-ling()
Received:
2022-02-21
Published:
2022-10-26
Online:
2022-11-11
摘要:
基于CRISPR/Cas9技术构建凝血因子8(F8)基因敲除小鼠模型并对其表型进行初步验证。针对F8基因外显子1非编码区和外显子26下游序列设计sgRNA靶位点,体外转录获得sgRNA,与编码Cas9的mRNA混合后通过受精卵显微注射方法获得F0代阳性敲除小鼠,通过繁育及基因型鉴定获得F8基因敲除纯合子小鼠(F8 -/-小鼠);通过逆转录PCR(RT-PCR)检测主要组织中F8基因在mRNA水平的表达情况,通过酶联免疫吸附测定(ELISA)检测血浆中F8基因在蛋白水平的表达情况;通过活化部分凝血活酶时间检测(APTT)和纤维蛋白原含量(FIB)测定实验检测F8基因敲除后小鼠凝血功能情况。PCR及测序结果表明F8基因在小鼠基因组中被成功敲除;RT-PCR 和ELISA检测结果显示,F8 -/-小鼠中F8在mRNA和蛋白水平上表达均显著低于野生型小鼠(WT小鼠)(P<0.000 1,P<0.01);APTT、FIB和滴血实验检测结果显示,F8 -/-小鼠血浆凝固时间显著高于WT小鼠(P<0.05),F8 -/-小鼠存在严重凝血障碍表型,给予人凝血因子Ⅷ后凝血可恢复到正常水平。利用 CRISPR/Cas9技术成功构建F8基因敲除小鼠模型并初步验证其表型,证实该小鼠F8基因的缺失可以导致凝血功能障碍。
王海杰, 王成稷, 郭洋, 王云, 陈艳娟, 梁敏, 王珏, 龚慧, 沈如凌. 基于CRSIPR/Cas9技术构建凝血因子8基因敲除小鼠模型及表型验证[J]. 生物技术通报, 2022, 38(10): 273-280.
WANG Hai-jie, WANG Cheng-ji, GUO Yang, WANG Yun, CHEN Yan-juan, LIANG Min, WANG Jue, GONG Hui, SHEN Ru-ling. Construction of Coagulation Factor 8 Gene Knockout Mouse Model Based on CRSIPR/Cas9 Technique and Verification of Phenotype[J]. Biotechnology Bulletin, 2022, 38(10): 273-280.
sgRNA靶序列 sgRNA target sequence | 序列信息 Sequence information(5'-3') |
---|---|
sgRNA 1 | AGGCTTAACCCATTTCCTGC TGG |
sgRNA 2 | TCTGGATCCACAGATATAGC AGG |
表1 sgRNA序列信息
Table 1 sgRNA sequence information
sgRNA靶序列 sgRNA target sequence | 序列信息 Sequence information(5'-3') |
---|---|
sgRNA 1 | AGGCTTAACCCATTTCCTGC TGG |
sgRNA 2 | TCTGGATCCACAGATATAGC AGG |
寡核苷酸OligoDNA | 序列信息Sequence information(5'-3') |
---|---|
sgRNA 1 正义链 | ACTTATTAGTTTGCAAAACG |
sgRNA 1 反义链 | GTGGCCTTCGATTAGGCGGA |
sgRNA 2 正义链 | CGGTCAGAGCTGCACATACA |
sgRNA 2 反义链 | GCCAAATTGTGATCTCAGTT |
表2 寡核苷酸链序列信息
Table 2 Oligonucleotide chain sequence information
寡核苷酸OligoDNA | 序列信息Sequence information(5'-3') |
---|---|
sgRNA 1 正义链 | ACTTATTAGTTTGCAAAACG |
sgRNA 1 反义链 | GTGGCCTTCGATTAGGCGGA |
sgRNA 2 正义链 | CGGTCAGAGCTGCACATACA |
sgRNA 2 反义链 | GCCAAATTGTGATCTCAGTT |
引物Primer | 序列信息Sequence information(5'-3') |
---|---|
P1 | TTTTGGCTTTCTAACAGGTATCG |
P2 | CCAGAAAAGGGCATCAGGT |
表3 引物序列信息
Table 3 Primer sequence information
引物Primer | 序列信息Sequence information(5'-3') |
---|---|
P1 | TTTTGGCTTTCTAACAGGTATCG |
P2 | CCAGAAAAGGGCATCAGGT |
基因 Gene | 上游引物信息Upstream primer information(5'-3') | 下游引物信息Downstream primer information(5'-3') |
---|---|---|
F8 | AGGCCTCATTGGAGCTCTGCTA | CCATTGGGATTCCTCAGGGCA |
β-actin | CGTTGACATCCGTAAAGACC | AACAGTCCGCCTAGAAGCAC |
表4 逆转录PCR引物序列
Table 4 Reverse transcription PCR primer sequences
基因 Gene | 上游引物信息Upstream primer information(5'-3') | 下游引物信息Downstream primer information(5'-3') |
---|---|---|
F8 | AGGCCTCATTGGAGCTCTGCTA | CCATTGGGATTCCTCAGGGCA |
β-actin | CGTTGACATCCGTAAAGACC | AACAGTCCGCCTAGAAGCAC |
图1 F8 -/-小鼠基因敲除策略 Cas9/sgRNA复合体通过识别外显子1-26(exon1-26)区域靶点将F8基因相应片段切除
Fig.1 Knockout strategy of F8 -/- mice The Cas9/sgRNA complex removed the corresponding fragments of the F8 gene by recognizing targets in the exon1-26 region
图2 F8-/-基因敲除小鼠基因型鉴定结果 (a)F8-/-基因敲除小鼠的鉴定策略,通过两对引物P1/P2和P3/P4进行PCR鉴定,确定小鼠基因型,引物位置图;(b)和(c)为基因型鉴定电泳图,(b)中利用P1/P2引物对进行PCR检测,WT小鼠不能获得条带,F8-/+杂合子小鼠与F8-/-纯合子小鼠获得单一772bp片段,(c)中P3/P4引物对进行PCR检测,WT小鼠与F8-/+杂合子小鼠可获得357bp片段,F8-/-纯合子小鼠不能获得条带。经电泳鉴定,(b)和(c)中WT小鼠编号为3,5,6,11,F8-/+杂合子小鼠编号为1,2,7,8,9,10,14,16,18,19,F8-/-纯合子小鼠编号为4,12,13,15,17,20。WT:野生型;M:1kb DNA maker
Fig.2 Genotype results of F8-/- gene knockout mice (a)The identification strategy of F8-/- knockout mice. Two pairs of primers P1/P2 and P3/P4 were used for PCR identification to determine the mice genotype. The primer positions were shown in the figure.(b)and(c)were genotype identification electrophoresis diagram,with P1/P2 primer pair for PCR detection in(b),bands were obtained in WT mice,F8-/+ heterozygous mice and F8-/- homozygous mice obtained a single 772 bp fragment.(c)The P3/P4 primer pair was used for the PCR detection,WT mice and F8-/+ heterozygous mice could obtain a 357 bp fragment,F8-/- homozygous mice could not obtain a band. According to electrophoresis identification,WT mice in(b)and(c)were numbered 3,5,6,11,and F8-/+ heterozygous mice were numbered 1,2,7,8,9,10,14,16,18,19,F8-/- homozygous mice were numbered 4,12,13,15,17,20. WT:Wild type mice. M:1 kb DNA maker
类型Index | 序列信息Sequence information(5'-3') |
---|---|
野生型小鼠 WT mice | AGGATTCAAACTTGTTAGGATGCACCCAGCAGGA- AATGGGTTAAGCCTTAGCTCAGCCACTCTTCCTA- TTCCAGTT……GAGAAGTTGCTGAGAGTTCTATA- TCTGGATCCACAGATATAGCAGGAAGAGAAAGA- CACTGGGACTGACTTGGG |
F8-/-小鼠序列 F8-/-mice | AGGATTCAAACTTGTTAGGATGCACCCAGCAGGAA …(207293 bp缺失)…GAGAAAGACACTGGGAC- TGACTTGGG |
表5 WT小鼠和F8-/-敲除小鼠的序列信息
Table 5 Sequence information of wild type and F8-/-mice genotype
类型Index | 序列信息Sequence information(5'-3') |
---|---|
野生型小鼠 WT mice | AGGATTCAAACTTGTTAGGATGCACCCAGCAGGA- AATGGGTTAAGCCTTAGCTCAGCCACTCTTCCTA- TTCCAGTT……GAGAAGTTGCTGAGAGTTCTATA- TCTGGATCCACAGATATAGCAGGAAGAGAAAGA- CACTGGGACTGACTTGGG |
F8-/-小鼠序列 F8-/-mice | AGGATTCAAACTTGTTAGGATGCACCCAGCAGGAA …(207293 bp缺失)…GAGAAAGACACTGGGAC- TGACTTGGG |
图3 F8 -/-小鼠F8 mRNA 相对表达水平 (a)F8基因在WT小鼠各个主要组织中的表达情况;(b)F8 -/-和WT小鼠心、肝、脾、肺和肾组织中F8 mRNA相对表达水平检测,每组3 只小鼠(****P <0.0001);(c)RT-PCR代表性电泳鉴定结果(肝组织);WT-野生型,F8 -/+-杂合子,F8 -/--纯合子
Fig.3 Analysis of relative mRNA expression in F8-/- mice (a)F8 gene expression in multiple tissues of WT mice.(b)F8 mRNA relative expression level detection in heart,liver,spleen,lung and kidney tissues of F8 -/- and WT mice,3 mice per group(****P <0.0001).(c)RT-PCR representative identification result of electrophoresis(liver tissue). WT:Wild type mice,F8 -/+-heterozygous,F8 -/--homozygous mice
图4 F8-/-小鼠F8蛋白相对表达水平 F8 -/-小鼠和WT小鼠血浆中F8蛋白的含量结果,每组5 只小鼠(**P <0.01),WT-野生型,F8 -/--纯合子
Fig.4 Relative expression of F8 protein in F8-/- mice F8 -/- protein content in plasma of F8-/- and WT mice,5 mice per group(**P <0.01),WT-wild type,F8-/-- homozygous
图5 F8 -/-小鼠的APTT A:小鼠凝血功能检测给药过程示意图;B:F8 -/-小鼠与WT小鼠血浆中FIB含量比较;C:F8 -/-小鼠与WT小鼠给予人凝血因子Ⅷ前后APTT值比较;每组5 只小鼠(*P <0.05,****P <0.0001),NS-尾静脉注射生理盐水,APTT-尾静脉注射人凝血因子Ⅷ,WT-野生型,F8 -/--纯合子
Fig.5 APTT results of F8-/- mice A:Schematic diagram of the administration process of blood coagulation in mice. B:Comparison of FIB in plasma of F8 -/- and WT mice. C:Comparison of APTT values before and after administration of human coagulation factor Ⅷ between F8 -/- and WT mice,5 mice per group(*P <0.05,****P <0.0001),to NS-tail vein injected with saline,to APTT-tail vein injected with human coagulation factor Ⅷ,WT-wild type,and F8 -/-- homozygous
图6 F8-/-小鼠滴血实验 A:F8 -/-小鼠注射生理盐水;B:F8 -/-小鼠注射凝血因子
Fig. 6 F8 -/- mice blood drop test A:F8 -/-mice injection of saline. B:F8 -/-mice injection of clotting factor
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