Construction of YOD1 Knockout Mice on CRISPR/Cas9 Technology

doi:10.13523/j.cb.20180607

China Biotechnology

2018, Vol. 38

Issue (6): 52-57 DOI: 10.13523/j.cb.20180607

Construction of YOD1 Knockout Mice on CRISPR/Cas9 Technology

Hong-miao DAI,Ye-sheng FU,Ling-qiang ZHANG(

)

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center of Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 100850, China

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Abstract

Objective: Construct YOD1 gene knockout mice based on CRISPR/Cas9 technology. Methods: Design and synthesize single-guide RNA (sgRNA) according to the YOD1 sequence in Genbank. Cas9 and sgRNA are transcribed to RNA in vitro, these RNA are then microinjected into zygotes of mice. The genotype is analyzed by PCR and sequencing. After YOD1 heterozygotes self-crossing and analysis of genotype of live offspring at weaning, wild type(WT)and knockout genotype(KO)littermates of YOD1 gene are verified. It is recorded that quantity and ratio of each genotype of live offspring of YOD1 heterozygotes self-crossing. And it is evaluated whether the ratio is in agreement with Mendel’s law of segregation. Protein lysates are made from main organs of the WT and KO littermates. And western blotting is used to assay the expression of YOD1 protein of these tissues. Meanwhile, size and weight of main organs and tissues of KO and WT mice are compared. Then analyze pathological phenotype of liver by H.E. staining. The glucose tolerance test (GTT) are carried out on the male mice of 6 months old. Results: According to PCR analysis and sequencing results, it is chose that mouse with deletion mutation and frameshift mutation in exon 2 of YOD1 gene to breed. After YOD1 heterozygotes self-crossing, WT and KO littermates are generated. According to statistics results, it is in agreement with Mendel’s law of segregation that the ratio of live offspring. Therefore, it is suggested that YOD1 KO mice birth normally without embryonic lethality. Western blotting results show that the expression of YOD1 in main organs is knocked-out significantly. Liver of YOD1 KO mouse is smaller in size than of WT littermate. There is no significant pathological phenotype in liver of YOD1 KO mice. YOD1 KO mice have general glycemic control in a GTT as compared to the control mice. Conclusions: YOD1 gene knockout mice are constructed successfully on CRISPR/Cas9 technology. And YOD1 KO mice birth and live normally without embryonic lethality. Compared to the control mice, livers of YOD1 KO mice are smaller in size and YOD1 KO mice have general glycemic control.

Key words： CRISPR/Cas9 YOD1 Knockout mice

Received: 09 March 2018 Published: 06 July 2018

ZTFLH:

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Corresponding Authors: Ling-qiang ZHANG E-mail: zhanglq@nic.bmi.ac.cn

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Cite this article:

Hong-miao DAI,Ye-sheng FU,Ling-qiang ZHANG. Construction of YOD1 Knockout Mice on CRISPR/Cas9 Technology. China Biotechnology, 2018, 38(6): 52-57.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180607 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I6/52

Fig.1 YOD1 knockout strategy

Table 1 sgRNA targeting sequences

Table 2 Primer sequences

Fig.2 Genotype of YOD1 knockout mice (a)Mice breed strategy (b)Analysis of littermates genotype by PCR (c)Sequencing analysis of WT and KO mice

Table 3 Live offspring at weaning from HET× HET

Fig.3 The expression of YOD1 in tissues of KO mice is knocked-out significantly

Fig.4 Liver of YOD1 KO mouse is smaller in size (a)、(b)Livers of YOD1 KO mice are smaller in size(c)H.E. staining for liver of YOD1 KO mouse and WT littermate

Fig.5 Glucose tolerance test(n=3)


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