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目的 采用CRISPR/Cas9技术构建Fermitin家族成员1(FERMT1)杂合缺失(FERMT1+/-)小鼠，完成品系繁育及基因型鉴定。方法 以C57BL/6J小鼠为遗传背景，基于CRISPR/Cas9体系，靶向敲除FERMT1(编码Kindlin-1蛋白)特定外显子片段，制备Kindlin-1蛋白部分功能缺失小鼠模型。采用Cas9 mRNA/sgRNA体外转录体系配合供体DNA，经原核显微注射获得可稳定遗传的F0代阳性小鼠。建立聚合酶链式反应(PCR)-琼脂糖凝胶电泳分型方法，区分野生型(WT)与杂合突变型小鼠；采用逆转录实时荧光定量 PCR(qRT-PCR)及蛋白质免疫印迹(WB)检测主动脉、心、肾、脑、肝、肺组织中FERMT1mRNA和蛋白表达，并利用WB分析各组织核因子κB(NF-κB)蛋白表达情况。结果 成功获得可稳定遗传的FERMT1+/-小鼠模型。基因型分析显示：纯合野生型个体呈现约400 bp单一扩增条带；杂合子个体同时显示400 bp及突变型442 bp条带。分子验证表明，与WT相比，FERMT1+/-小鼠在主动脉、心脏、肾、脑、肝及肺组织中，FERMT1mRNA水平均下调(均P<0.05)；同时，各检测组织的FERMT1蛋白表达量亦降低(均P<0.001)。FERMT1+/-小鼠主动脉、心脏、脑、肺及肝脏组织中，NF-κB蛋白表达水平均下调(均P<0.05)；然而，在肾脏组织中，FERMT1+/-小鼠与WT小鼠的NF-κB蛋白水平无统计学差异。结论 成功建立C57BL/6J背景的FERMT1+/-小鼠模型，构建了可靠的PCR基因分型体系，并通过qRT-PCR与WB双重验证手段确认了该小鼠模型敲除效率稳定、可稳定传代。该模型为FERMT1功能研究及相关疾病机制探索提供稳定可靠的实验基础。
Abstract：Objective To generate FERMT1 heterozygous knockout (FERMT1+/-) mice using CRISPR/Cas9 technology， and to establish a stable strain with reliable genotyping characterization. Methods Using C57BL/6J mice as the genetic background， specific exon fragments of FERMT1 (encoding Kindlin-1 protein) were targeted for knockout via the CRISPR/Cas9 system to establish a mouse model with partial loss of Kindlin-1 protein function. In vitro transcribed Cas9 mRNA/single-guide RNA (sgRNA) combined with donor DNA were microinjected into fertilized zygotes to obtain heritable F0-generation founder mice. A polymerase chain reaction (PCR)-agarose gel electrophoresis genotyping method was established to distinguish wild-type (WT) from FERMT1+/- mutant mice. Quantitative reverse-transcription PCR (qRT-PCR) and Western blot (WB) were performed to detect FERMT1 mRNA and protein expression in the aorta， heart， kidney， brain， liver， and lung. WB was also used to analyze nuclear factor-κB (NF-κB) protein expression in these tissues. Results A stably heritable FERMT1+/- mouse model was successfully established. Genotyping analysis showed that WT mice exhibited a single amplification band of approximately 400 bp， whereas heterozygous mice displayed both 400 bp and 442 bp mutant bands. Molecular validation revealed that FERMT1 mRNA levels were significantly downregulated in the aorta， heart， kidney， brain， liver， and lung of FERMT1+/- mice compared with WT mice (all P<0.05). Consistently， FERMT1 protein abundance markedly decreased in all tested tissues (all P<0.05). NF-κB protein expression was significantly downregulated in the aorta， heart， brain， lung， and liver of FERMT1+/- mice (all P<0.05). Nevertheless， no statistically significant difference in renal NF-κB protein level was observed between FERMT1+/- and WT mice. Conclusion The FERMT1+/- mouse model on the C57BL/6J background was successfully established， and a reliable PCR genotyping system was developed. Dual validation by qRT-PCR and WB confirmed stable knockout efficiency and heritability of this model， providing a solid experimental platform for investigating FERMT1 function and exploring the pathogenetic mechanisms of related diseases.
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基本信息:
中图分类号:R-332
引用信息:
[1]王立杰,夏木塞亚·亚尔买买提,阿斯亚·阿不得斯木,等.FERMT1杂合缺失小鼠的繁育、基因型鉴定及多组织表达验证[J].安徽医科大学学报().
基金信息:
国家自然科学基金项目（编号：82360090）
2026-09-01
2026-09-01
2026-09-01