背景:SHARPIN(SHANK相关的RH结构域相互作用因子)是调节NF-κB信号通路的线性泛素化复合物的组成部分。为了更好地理解SHARPIN的功能,我们试图使用CRISPR/Cas9系统建立一种新型的SHARPIN破坏的基因工程叙利亚仓鼠.
方法:设计并构建了靶向SHARPIN基因外显子1的单向导核糖核酸。将通过Cas9/gRNA核糖核蛋白的细胞质注射产生的受精卵转移到假性仓鼠中。通过基因分型鉴定新生儿突变体。使用蛋白质印迹测定法检测SHARPIN蛋白表达。脾,肠系膜淋巴结(MLN),测量胸腺重量,并计算了器官系数。脾脏的组织病理学检查,肝脏,肺,小肠,食道由病理学家独立进行。使用逆转录酶定量聚合酶链反应评估淋巴细胞标志物和细胞因子的表达。
结果:所有后代都带有种系传播的SHARPIN突变。与野生型仓鼠相比,SHARPIN蛋白在SHARPIN-/-仓鼠中检测不到。在SHARPIN-/-仓鼠中发现脾脏增大和脾系数升高,随着MLN和胸腺的下降。Further,脾脏中嗜酸性粒细胞浸润和结构改变,肝脏,肺,小肠,SHARPIN缺失后食管明显。值得注意的是,基因敲除(KO)动物脾脏中CD94和CD22的表达下调。尽管如此,CCR3,CCL11,Il4和Il13的表达在食管中上调。在SHARPIN-/-动物中,NF-κB的表达以及NF-κB和IκB蛋白的磷酸化显着降低。
结论:使用CRISPR/Cas9系统成功建立了新型SHARPINKO仓鼠。次级淋巴器官的异常发育和多个器官中嗜酸性粒细胞浸润揭示了其描绘SHARPIN功能和慢性炎症的潜力。
SHARPIN (SHANK-associated RH domain interactor) is a component of the linear ubiquitination complex that regulates the NF-κB signaling pathway. To better understand the function of SHARPIN, we sought to establish a novel genetically engineered Syrian hamster with
SHARPIN disruption using the CRISPR/Cas9 system.
A single-guide ribonucleic acid targeting exon 1 of SHARPIN gene was designed and constructed. The zygotes generated by cytoplasmic injection of the Cas9/gRNA ribonucleoprotein were transferred into pseudopregnant hamsters. Neonatal mutants were identified by genotyping.
SHARPIN protein expression was detected using Western blotting assay. Splenic, mesenteric lymph nodes (MLNs), and thymic weights were measured, and organ coefficients were calculated. Histopathological examination of the spleen, liver, lung, small intestine, and esophagus was performed independently by a pathologist. The expression of lymphocytic markers and cytokines was evaluated using reverse transcriptase-quantitative polymerase chain reaction.
All the offspring harbored germline-transmitted
SHARPIN mutations. Compared with wild-type hamsters,
SHARPIN protein was undetectable in
SHARPIN-/- hamsters. Spleen enlargement and splenic coefficient elevation were spotted in SHARPIN-/- hamsters, with the descent of MLNs and thymuses. Further, eosinophil infiltration and structural alteration in spleens, livers, lungs, small intestines, and esophagi were obvious after the deletion of SHARPIN. Notably, the expression of CD94 and CD22 was downregulated in the spleens of knockout (KO) animals. Nonetheless, the expression of CCR3, CCL11, Il4, and Il13 was upregulated in the esophagi. The expression of NF-κB and phosphorylation of NF-κB and IκB protein significantly diminished in SHARPIN-/- animals.
A novel SHARPIN KO hamster was successfully established using the CRISPR/Cas9 system. Abnormal development of secondary lymphoid organs and eosinophil infiltration in multiple organs reveal its potential in delineating SHARPIN function and chronic inflammation.