MARCO基因敲除小鼠模型鉴定

Identification of MARCO gene knockout mouse model

目的通过基因分型、基因测序与蛋白免疫印迹法鉴定MARCO基因敲除小鼠模型。方法将经成簇的规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)技术获得16种碱基敲除的MARCO^(-/-)亲代C57BL/6小鼠(雌、雄鼠各8只)与MARCO^(+/+)的亲代C57BL/6雌性小鼠(8只)合笼繁殖,获得子一代MARCO^(+/-)小鼠;再以MARCO^(+/-)小鼠自交得到MARCO^(-/-)足够数量的纯合小鼠。采用基因测序对小鼠基因型进行鉴定,采用蛋白免疫印迹法检测MARCO蛋白相对表达水平。结果经过1年的合笼繁殖,共繁殖5代,有5种敲除不同碱基数(-11、-25、-36、-46、-61 bp)的MARCO^(-/-)基因型稳定遗传;MARCO^(-/-)、MARCO^(+/-)、MARCO^(+/+)基因型比例约为1∶2∶1,符合孟德尔遗传定律。以MARCO(-11 bp)为例,共获得子五代(F5代)MARCO^(-/-)小鼠42只,MARCO^(+/-)小鼠92只,MARCO^(+/+)小鼠48只;上述3种基因型F5代小鼠出生后第4、6和8周的体质量分别比较,差异无统计学意义(P>0.05)。与MARCO^(+/+)小鼠和MARCO^(-/-)(-36 bp)、MARCO^(-/-)(-61 bp)小鼠比较,MARCO^(-/-)(-11 bp)和MARCO^(-/-)(-46 bp)小鼠的MARCO蛋白相对表达水平均下调(P<0.05),选择该2种小鼠模型作为MARCO基因敲除小鼠模型。结论成功鉴定MARCO基因敲除小鼠模型,为深入研究MARCO基因在小鼠矽肺纤维化中的作用及调控机制奠定基础。

Objective To identify MARCO gene knockout mouse model by genotyping,sequencing and Western blotting.Methods A total of 16 base-knockout MARCO^-/-C57 BL/6 mice（ 8 female and 8 male） were obtained by clustered regularly interspaced short palindromic repeats（ CRISPR）/CRISPR associated protein 9（ Cas9） technique with MARCO^＋/＋C57 BL/6 mice（8 female）,and their offspring MARCO^0＋/-mice were obtained. Then MARCO^0＋/-mice were inter-crossed to get a sufficient number of MARCO^-/-homozygous mice. The genotypes of mice were identified by gene sequencing and the relative expression of MARCO protein was detected by Western blotting. Results After one year of breeding,a total of 5 generations were bred. There were 5 types of MARCO^-/-genotypes（-11,-25,-36,-46,-61 bp） stably inherited; MARCO^-/-∶ MARCO^0＋/-∶ MARCO^＋/＋= 1 ∶ 2 ∶ 1,which was consistent with Mendelian＇s law of heredity. Using MARCO（-11 bp） as an example,42 MARCO^-/-mice,92 MARCO^0＋/-mice and 48 MARCO^＋/＋mice were obtained from the 5 th generation（ F5 generation）; and there was no significant difference in body mass of the above 3 genotypes of F5 generation mice at the 4 th,the 6 th and the 8 th weeks after birth（ P 0. 05）. The relative expression of MARCO protein in MARCO^-/-（-11 bp） and MARCO^-/-（-46 bp） mice was significantly down-regulated,compared with that of MARCO^＋/＋,MARCO^-/-（-36 bp） and MARCO^-/-（-61 bp） mice（P 0. 05）. MARCO^-/-（-11 bp）and MARCO^-/-（-46 bp） mice were chosen as the MARCO gene knockout mice. Conclusion MARCO gene knockout mice were successfully identified,which laid a foundation for further study on the role and regulatory mechanism of MARCO gene in silicotic fibrosis in mice.