摘要
目的应用Loxp-Cre系统构建去泛素化酶Otud4基因敲除小鼠。方法利用Loxp-Cre系统原理,将Otud4基因中的4号外显子选为打靶序列,将两个Lox P位点分别设在E4的两端,经打靶载体转入ES细胞、阳性克隆筛选、囊胚注射等过程,得到嵌合体小鼠,再将嵌合体小鼠与C57BL/6小鼠交配繁育,最终得到杂合子小鼠并进行自交得到新生F1代小鼠。然后利用PCR进行基因型鉴定,使用免疫印迹(WB)技术从蛋白水平检测OTUD4在各组织器官中的表达,以明确基因敲除小鼠是否构建成功。对各基因型小鼠进行数量统计分析明确其是否符合孟德尔遗传定律,是否具有胚胎致死表型。解剖小鼠观察主要组织器官有无明显发育异常,利用HE染色观察是否具有自发的病理改变。通过葡萄糖耐受实验分析基因敲除小鼠是否具有代谢异常等。结果基因型鉴定和WB检测结果显示Otud4基因敲除小鼠模型成功建立。杂合子小鼠自交后各基因型比例符合孟德尔定律,提示Otud4基因敲除小鼠非胚胎致死。各组织器官大小无显著性差异,HE染色并无异常,且Otud4基因敲除后不影响小鼠的血糖稳态。结论应用Loxp-Cre系统成功构建了Otud4基因敲除小鼠,且小鼠正常出生,无任何胚胎发育缺陷,各器官无显著病理性变化,血糖代谢无异常。
Objective To construct deubiquitinase Otud4 gene knockout mice via Loxp-Cre system. Methods Using Loxp-Cre system,exon 4( E4) was set as the target sequence and the two Loxp sites were at the opposite ends of the E4,respectively,after targeting carrier to ES cells,screening positive clones and blastocyst injection process. The chimeric mice were primed and then mated with C57 BL/6 mice to generate heterozygous mice of a new generation. Then,the genotype was verified by PCR,and the expression of OTUD4 in various tissues was detected by Western blotting( WB) technology to verify the successful establishment of knockout mice. Mathematical statistical analysis was conducted to determine whether homozygous OTUD4 mice were born at the expected Mendelian frequency. By dissecting the mice,the main tissues and organs were analyzed,and the spontaneous pathological changes were analyzed by HE staining. Glucose tolerance test was also used to analyze whether the knockout mice had abnormal metabolism. Results Genotype identification and WB test results demonstrated that the Otud4 gene knockout mouse model was successfully established. The percentage of genotypes of heterozygous mice after self-inbreeding conformed to Mendel’s law,suggesting that Otud4 gene knockout mice were not embryonically leathal. There was no significant difference in the size of tissues or organs,no abnormality in HE staining,and the Otud4 gene knockout did not affect the blood glucose homeostasis of mice. Conclusion Otud4 gene knockout mice are successfully constructed using the Loxp-Cre system,and the mice are born normal without any embryonic development defects,obvious pathological changes in the organs,or abnormal glucose metabolism.
作者
秦书霞
刘雯
张令强
QIN Shu-xia;LIU Wen;ZHANG Ling-qiang(Graduate School, Anhui Medical University, Hefei 230032, China;Institute of Radiation Medicine, Academy ofMilitary Medical Sciences, Academy of Military Sciences, Beijing 100850, China;State Key Laboratory of ProteinSciences( Beijing) , Institute of Lifeomics, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing100850, China)
出处
《军事医学》
CAS
CSCD
北大核心
2018年第10期721-726,共6页
Military Medical Sciences
基金
国家重点研发计划基金资助项目(2017YFA0505602)
