淋巴细胞活化基因-3敲除(Lag-3-/-)小鼠构建及初步表型分析

Construction and preliminary phenotypic analysis of Lag-3-/- mice

目的构建淋巴细胞活化基因-3(Lag-3)基因敲除小鼠,初步分析Lag-3基因敲除对小鼠表型影响,为后续Lag-3体内功能研究提供动物模型。方法利用CRISPR/Cas9技术结合受精卵显微注射构建敲除小鼠,通过分子鉴定筛选基因敲除阳性小鼠,利用RT-PCR和流式检测方法进一步验证Lag-3基因敲除小鼠体内Lag-3基因敲除效果;通过建立Con A诱导的肝损伤模型研究Lag-3基因在体内的功能。结果PCR和产物测序结果显示,获得了exon 2缺失的Lag-3基因敲除小鼠;该基因敲除纯合子小鼠(Lag-3-/-)经刀豆蛋白(Con A)刺激后,小鼠心脏、脾、肺、巨噬细胞和淋巴结中仅能检测到Lag-3 mRNA本底表达信号,小鼠巨噬细胞、脾细胞和淋巴结中仅能检测到非常低数量的Lag-3阳性细胞。表型分析发现,Lag-3基因的缺失显著减少了Con A诱导的小鼠外周血、骨髓和脾CD3+T细胞的数量,减轻了Con A诱发的急性肝损伤情况。结论成功构建Lag-3基因敲除小鼠模型,肝损伤过程中的体内功能研究显示,Lag-3缺失可以显著缓解Con A引发的肝损伤的发生。

Objective To construct lymphocyte activation gene-3(Lag-3)-knockout mice and analyze the influence of Lag-3 knockout on mouse phenotypes to provide an animal model for subsequent Lag-3 in vivo function research.Methods Knockout mice were constructed via CRISPR/Cas9 technology combined with microinjection of fertilized eggs.The Lag-3-knockout mice were screened via molecular identification and further verified via RT-PCR and flow cytometry.Lag-3 gene function in vivo was studied by establishing a Con A-induced liver injury model.Results PCR and product sequencing showed that exon 2-deleted Lag-3-knockout mice were obtained.The heart,spleen,lung and macrophages from Con A-induced homozygous mice were evaluated with only Lag-3 mRNA background expression signals detected in phagocytes and lymph nodes and only very low numbers of Lag-3-positive cells detected in mouse macrophages,spleen cells and lymph nodes.Phenotypic analysis revealed that deleting the Lag-3 gene significantly reduced the number of Con A-induced CD3+T cells in the peripheral blood,bone marrow and spleen as well as Con A-induced acute liver injury.Conclusions A Lag-3-knockout mouse model is successfully constructed.In vivo functional studies during liver injury show that Lag-3 deficiency significantly alleviated Con A-induced liver damage.