Construction of Coagulation Factor 8 Gene Knockout Mouse Model Based on CRSIPR/Cas9 Technique and Verification of Phenotype

doi:10.13560/j.cnki.biotech.bull.1985.2021-1564

Abstract

Abstract:

A coagulation factor 8（F8）gene knockout mouse model was constructed based on CRISPR/Cas9 technique and its phenotype was verified. sgRNA target sites were designed according to exon 1 non-coding region to exon 26 downstream sequences of F8 gene，the sgRNA was obtained from in vitro transcription and mixed with mRNA of encoding Cas9. F0 generation positive knockout mice were obtained by microinjection of fertilized eggs. F8^-/- gene knockout homozygous mice（F8^-/- mice）were obtained by breeding and genotype identification. The expression of F8 gene at mRNA level in main tissues was detected by reverse transcription PCR（RT-PCR），the protein expression of F8 gene in plasma was detected by enzyme-linked immunosorbent assay（ELISA），and the coagulation function of F8 knockout mice was detected by activated partial thromboplastin time（APTT）and fibrinogen content（FIB）. PCR and sequencing results showed that F8 gene was knocked out successfully in mouse genome，and RT-PCR and ELISA results demonstrated that the expression of F8 in F8^-/- mice was significantly lower than that in wild-type mice（WT mice）（P < 0.0001，P<0.01）. APTT，FIB and drop blood test results revealed that the plasma coagulation time of F8^-/- mice was significantly longer than that of WT mice. F8^-/- mice had severe coagulation disorder phenotype，which restored to normal levels after administration of human factor VIII. The F8 gene knockout mice model is successfully constructed by CRISPR/Cas9 technique and its phenotype is preliminarily verified，which confirms that the deletion of F8 gene can lead to coagulation dysfunction.

Key words: coagulation factor Ⅷ（F8）, CRISPR/Cas9, gene knockout, hemophilia A

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.

Figures/Tables 12

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

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

Table 3 Primer sequence information

引物Primer	序列信息Sequence information（5'-3'）
P1	TTTTGGCTTTCTAACAGGTATCG
P2	CCAGAAAAGGGCATCAGGT

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

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

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

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

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

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

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

Fig. 6 F8 -/- mice blood drop test A：F8 -/-mice injection of saline. B：F8 -/-mice injection of clotting factor

Fig.7 Gray value of blood spot of F8-/- mice

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