NOD/ShiLtJGpt-Prf1<sup>em9Cd161</sup></i>/Gpt
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Prf1（Perforin1）基因又称Pfn、Pfp，编码穿孔素蛋白。穿孔素（PRF，PRF1）是一种必需的成孔蛋白，由细胞毒性T淋巴细胞（CLs）细胞和CD8+T细胞表达并释放到靶细胞接触的位点，在那里低聚形成靶细胞膜上的孔，允许扩散一系列诱导细胞凋亡的丝氨酸蛋白酶、颗粒酶，进入靶细胞[1-2]。研究表明，穿孔素是参与细胞毒性细胞杀伤靶细胞的一个重要分子，在免疫监视、免疫调控中起着重要作用，其功能异常可导致多种疾病。国内外大量分子遗传学研究提示Prf1基因突变可引起家族性噬血细胞综合征（FHL）、噬血性淋巴组织细胞增生症（HLH）、Ⅰ型糖尿病、多发性硬化症和恶性血液肿瘤等疾病[3-5]。临床实验中，Prf1蛋白通过产生和输送CLs维持免疫稳态和肿瘤免疫监视[1]。Prf1基因突变纯合子病人的NK细胞、CD8+T细胞和CD56+T细胞功能正常，CLs功能减少，不能清除淋巴细胞性脉络丛脑膜炎病毒，消除纤维肉瘤细胞的效率降低。目前的研究主要集中在Prf1基因突变与FLH、HLH等疾病的关系，以期发现能够揭示FLH、HLH病因、临床诊治及预后有意义的分子生物学标志。我们构建针对mouse Prf1基因exon2的sgRNA，利用CRISPR/Cas9技术和囊胚注射技术，把特异性的sgRNA注射到受精卵中，sgRNA介导Cas9核酸内切酶切割靶向exon2，并导致DNA双链断裂，通过非同源末端重组的方式修复断裂的Prf1基因，最后Prf1基因在exon2上删除161bp碱基，导致移码而被破坏。通过PCR技术进行基因分型，然后进行序列分析，筛选的Prf1基因突变小鼠，将成为研究人类免疫缺陷疾病、糖尿病的动物模型。研发项目编号：XM001596，该项目获得两条line，此line通过表型分析验证。另一条line编号为T001181。
The Prf1 (Perforin 1) gene, also known as Pfn and Pfp, encodes the perforin protein. Perforin (PRF, PRF1) is an essential pore-forming protein that is expressed and released by cytotoxic T lymphocytes (CLs) and CD8+ T cells to the contact sites of target cells, where it oligomerizes to form pores on the target cell membrane, allowing the diffusion of a series of apoptosis-inducing serine proteases and granzymes into the target cells [1-2]. Studies have shown that perforin is a critical molecule involved in cytotoxic cell-mediated killing of target cells, playing an important role in immune surveillance and immune regulation, and its dysfunction can lead to various diseases. Numerous molecular genetic studies worldwide have suggested that Prf1 gene mutations can cause diseases including familial hemophagocytic lymphohistiocytosis (FHL), hemophagocytic lymphohistiocytosis (HLH), type 1 diabetes mellitus, multiple sclerosis, and malignant hematological neoplasms [3-5]. In clinical trials, Prf1 protein maintains immune homeostasis and tumor immune surveillance by producing and transporting CLs [1]. In patients with homozygous Prf1 gene mutations, NK cells, CD8+ T cells and CD56+ T cells exhibit normal functions, while CLs show reduced functionality, failing to clear lymphocytic choriomeningitis virus and exhibiting decreased efficiency in eliminating fibrosarcoma cells. Current research primarily focuses on the association between Prf1 gene mutations and diseases such as FHL and HLH, aiming to identify valuable molecular biomarkers that can reveal the etiology, clinical diagnosis, treatment and prognosis of FHL and HLH. We constructed sgRNAs targeting exon 2 of the mouse Prf1 gene. Using CRISPR/Cas9 technology and blastocyst injection technology, we injected the specific sgRNA into fertilized eggs. The sgRNA guides the Cas9 endonuclease to cleave the targeted exon 2, inducing double-strand DNA breaks, and the broken Prf1 gene is repaired via non-homologous end joining (NHEJ). Finally, a 161 bp base deletion in exon 2 of the Prf1 gene causes frameshift mutations resulting in gene disruption. Genotyped via PCR technology and subjected to sequence analysis, the screened Prf1 gene mutant mice will serve as animal models for studying human immunodeficiency diseases and diabetes mellitus. R&D project number: XM001596. Two mouse lines were generated in this project, one of which was validated via phenotypic analysis. The other line is numbered T001181.
