肿瘤坏死因子受体超家族成员4(TNFRSF4)敲除和人源化小鼠模型的建立

Establishment of TNFRSF4 knockout and humanized mice

目的建立肿瘤坏死因子受体超家族成员4(TNFRSF4)基因人源化小鼠模型,同时建立TNFRSF4基因敲除小鼠模型,为针对TNFRSF4靶点的肿瘤治疗性抗体研发和筛选提供有效的小鼠模型,并为研究TNFRSF4在免疫过程中的作用机制提供小鼠模型。方法利用CRISPR/Cas9技术,建立TNFRSF4基因人源化和敲除小鼠模型,利用PCR、RT-PCR、免疫组织化学、流式细胞术等方法鉴定及比较分析人源化小鼠、敲除小鼠和野生型小鼠的差异。结果获得表达人源TNFRSF4基因小鼠和TNFRSF4基因敲除小鼠。在TNFRSF4人源化小鼠中稳定表达人源TNFRSF4,未检测到小鼠TNFRSF4表达。在TNFRSF4敲除小鼠中未检测到TNFRSF4表达。TNFRSF4人源化及敲除小鼠出生后6个月内均正常存活,组织病理学表型及免疫系统未见明显异常。结论利用CRISPR/Cas9技术构建TNFRSF4人源化小鼠及敲除小鼠,可用于筛选及评估与TNFRSF4基因相关的治疗性抗体及药物或研究TNFRSF4在免疫过程中作用机制。

Objective To establish tumor necrosis factor receptor superfamily member 4(TNFRSF4)knockout and humanized mice for TNFRSF4-targeted cancer therapeutic antibodies development,and to study the function of TNFRSF4 in the immune response.Methods The TNFRSF4 knockout and humanized mice were established with the CRISPR/Cas9 method.The mice were characterized by PCR,RT-PCR,HE staining and fluorescence-activated cell sorting.Results We successfully generated mice expressing the human TNFRSF4 gene,as well as TNFRSF4 gene knockout mice.TNFRSF4 humanized mice expressed human TNFRSF4,but not murine TNFRSF4.No TNFRSF4 expression was detected in TNFRSF4 knockout mice.TNFRSF4 humanized mice and knockout mice survived normally within 6 months after birth,and there were no obvious abnormalities in histopathology and immune system.Conclusions TNFRSF4 humanized mice and knockout mice produced using CRISPR/Cas9 technology can be used as animal models for screening and evaluating of TNFRSF4-targeted cancer therapeutic antibodies or for studying the role of TNFRSF4 in immune processes.