髓系特异性Abro1基因敲除小鼠构建和表型初步分析

Construction and preliminary phenotype analysis of myeloid-specific Abro1 knockout mice

目的:构建髓系特异性Abro1(Abraxas brother 1)基因敲除小鼠,并初步分析其对小鼠造血系统及LPS诱导败血症的影响。方法:采用CRISPR/Cas9基因编辑技术和Cre/LoxP重组系统构建Abro1^(flox/+)小鼠,将Abro1^(flox/+)雌雄小鼠自交,子代得到Abro1^(flox/flox)基因型小鼠。Abro1^(flox/flox)与Lyz2-Cre^(+)小鼠交配,子代得到Abro1^(flox/+)/Lyz2-Cre^(+)小鼠,再将其与Abro1^(flox/flox)交配,最终得到的Abro1^(flox/flox)/Lyz2-Cre^(+)小鼠为髓系特异性Abro1基因敲除小鼠,即MKO小鼠;Abro1^(flox/flox)/Lyz2-Cre^(−)小鼠作为野生型小鼠,即WT小鼠。提取WT和MKO小鼠尾基因组DNA,PCR扩增后琼脂糖凝胶电泳鉴定其基因型。提取WT和MKO小鼠免疫细胞蛋白,Western blot检测ABRO1蛋白表达。流式细胞术分析小鼠骨髓和外周血免疫细胞组成,检测MKO小鼠对造血系统的影响。LPS处理WT和MKO小鼠3 h,检测血清中相关生化指标变化以及炎症因子分泌情况。结果:PCR和Western blot结果显示髓系特异性Abro1基因敲除小鼠构建成功。流式细胞术结果显示WT和MKO小鼠骨髓和外周血中髓系细胞(CD11b^(+)、CD11b^(+)Ly6G^(+)、CD11b^(+)Ly6C^(+))和淋巴细胞组成(CD3^(+)、B220^(+))无差异。LPS诱导败血症实验中,MKO小鼠相关生化指标和炎症因子水平均低于WT小鼠,表明MKO小鼠可抵抗LPS诱导的败血症。结论:成功构建ABRO1 MKO敲除小鼠,发现MKO小鼠造血系统正常,但可抵抗LPS诱导的败血症,ABRO1 MKO小鼠的建立为揭示ABRO1在免疫细胞亚群中的差异性调控机制提供了良好的动物模型。

Objective:To construct myeloid-specific Abro1(Abraxas Brother 1)knockout mice and to preliminary analyze its influence on hematopoietic system and LPS-induced sepsis in mice.Methods:Abro1^(flox/+) mice were constructed by CRISPR/Cas9 gene editing technology and Cre/LoxP recombination system.Abro1^(flox/+) male and female mice were self bred,and offspring obtained Abro1^(flox/flox) genotype mice.Abro1^(flox/flox) mice were mated with Lyz2-Cre^(+) mice,and offspring obtained Abro1^(flox/+)/Lyz2-Cre^(+) mice.Mating it with Abro1^(flox/flox) mice,and obtained Abro1^(flox/flox)/Lyz2-Cre^(+) mice as myeloid-specific Abro1 knockout mice,namely MKO mice;Abro1^(flox/flox)/Lyz2-Cre^(−) mice as wild type mice,namely WT mice.Caudal genetic DNA of WT and MKO mice were extracted,and PCR and AGE to analyze genotypes.Proteins of immune cell of WT and MKO mice were extracted,and Western blot to detect expression of ABRO1,and flow cytometry to detect immune cells composition of bone marrow and peripheral blood.Effect of MKO mice on hemo-poietic system was detected.Changes in serum biochemical indicators and secretion of inflammatory factors were detected after 3 h of LPS treatment of WT and MKO mice.Results:PCR and Western blot results showed myeloid-specific Abro1 knockout mice were successfully constructed.Flow cytometry results showed that there was no difference in composition of myeloid cells(CD11b^(+),CD11b^(+)Ly6G^(+),CD11b^(+)Ly6C^(+))and lymphocyte cells(CD3^(+),B220^(+))in bone marrow and peripheral blood of WT and MKO mice.In experiment of LPS induced sepsis,serum biochemical indexes and inflammatory factors of MKO mice were lower than those of WT mice,which indicated that MKO mice were resistant to LPS induced sepsis.Conclusion:ABRO1 MKO knockout mice was sucessfully contracted,which had no influence on hematopoietic system,but were resistant to LPS-induced sepsis.Successful construction of ABRO1 MKO mice also provides a good animal model for revealing differential regulation mechanism of ABRO1 in immune cell subsets.