Construction and gene identification of myeloid specific Spi1 knockout mice
10.19405/j.cnki.issn1000-1492.2024.03.007
- Author:
Xuming Wu
1
;
Huihui Wang
1
;
Xiangling Zhu
1
;
Yuanyuan Zhou
1
;
Anqi Wang
1
;
Huiru Zhang
1
;
Chong Liu
1
;
Jiajie Tu
1
Author Information
1. Institute of Clinical Pharmacology of Anhui Medical University , Hefei, 230032
- Publication Type:Journal Article
- Keywords:
myeloid specific;
Spi1;
gene knockout;
CRISPR/Cas9;
Cre/Lox;
PCR;
Western blot
- From:
Acta Universitatis Medicinalis Anhui
2024;59(3):413-417
- CountryChina
- Language:Chinese
-
Abstract:
Objective :To construct myeloid specific Spi1 gene knockout mice and analyze their genotypes , so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets .
Methods :According to the principle of CRISPR/Cas9 technology and C re/LoxP system , sgRNA and Donor vectors were de signed and constructed . The transcript of Exon 2 ( Exon 2) was used as the knockout region , and Loxp elements were placed on both sides of Exon 2 . Cas9 protein , sgRNA and Donor vector were mixed and microinj ected into the fertilized eggs of C57BL/6J mice , the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice , and F0 generation was obtained after 19 ~ 20 days . Positive F0 mice were mated with C57BL/6J mice to ob tain stable F1 Spi1 flox/ + mice . Spi1 flox/ + mice of F1 generation were selfed to obtain Spi1 flox/flox mice . Spi1 flox/flox mated with Lyz2-Cre + mice to obtain Spi1 flox/ + /Lyz2-Cre + mice , and then mated with Spi1 flox/flox , the Spi1 flox/flox/Lyz2-Cre + mice were myeloid specific Spi1 gene knockout ( KO) mice . Spi1 flox/flox/Lyz2-cre - mice were used as wild type (WT) mice . DNA of WT and KO mice was extracted , and the genotypes were identified by agarose gel electro
phoresis after PCR amplification . Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene , Spi - 1 /purine rich box - 1(PU . 1) in immune cells of WT and KO mice .
Results:The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1 flox/flox homozygote , and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre + . Western blot showed that compared with WT group , the protein PU . 1 was not expressed in bone marrow derived macropha ges (BMDMs ) and peritoneal macrophages (PM) in KO group (P < 0.01) . There was no significant difference of statistics in the expression level of PU . 1 in T cells between KO mice and WT mice . The results of PCR and West ern blot showed that myeloid specific Spi1 KO mice were successfully constructed .
Conclusion :The myeloid spe cific Spi1 gene KO mice are successfully constructed and identified , which provides animal model basis for further revealing the potential mechanism of PU . 1 inimmune regulation .
- Full text:2024062610483079051髓系特异性Spi1基因敲除小鼠的构建和基因鉴定_吴旭铭.pdf
