利用TALENs技术制备瘦素基因敲除的大鼠  

Leptin knockout rats generated by microinjection of TALENs

作　　者：关菲菲[1] 张旭[1] 陈炜[1] 董伟[1] 高凯[1] 张连峰[1] 

机构地区：[1]中国医学科学院北京协和医学院医学实验动物研究所,卫生部人类疾病比较医学重点实验室,北京100021

出　　处：《中国比较医学杂志》2013年第8期38-43,F0003,共7页Chinese Journal of Comparative Medicine

基　　金：国家科技支撑计划课题(2012BA139B02);国家"重大新药创新"科技重大专项课题(2011ZX09307-302)

摘　　要：目的瘦素(leptin)是一种调节机体摄食和能量代谢的细胞因子,也参与神经细胞发育、免疫、糖脂代谢等,由于大鼠在代谢和神经研究等方面的优点,研制leptin基因敲除大鼠(leptin-/-),为这几方面的深入研究提供模型。方法利用类转录激活因子效应因子核酸酶(transcription activator-like effector nucleases,TALENs)技术,制备leptin-/-大鼠。利用称量方法测定不同时间点的体重变化,利用磁共振成像观测大鼠脂肪积累及分布,利用病理学方法观察胰腺的病理变化,并进行血生化、腹腔糖耐受实验及葡萄糖刺激下的血清胰岛素含量测定。结果获得一个在leptin基因编码区418～425位有8个碱基缺失的首建鼠,由于产生了TGA终止密码子,使该蛋白C末端缺失了28个氨基酸。Leptin-/-大鼠从5周龄开始出现显著的体重增加,到4月龄时体重比野生SD大鼠(leptin+/+)增加了52%。2月龄MRI结果显示leptin-/-大鼠与leptin+/+大鼠相比,在腹腔和皮下均有更多的脂肪积累,同时病理观察也发现leptin-/-大鼠胰腺中胰岛细胞显著增生。4月龄时,leptin-/-大鼠的血清中甘油三酯和胆固醇含量分别是leptin+/+大鼠的8.4倍和1.8倍,同时出现了明显的糖耐受受损和高胰岛素血症。结论利用TALENs技术成功建立了瘦素基因敲除大鼠,并表现出明显的肥胖、高胰岛素血、高脂血和糖代谢异常。结果表明:该敲除大鼠可作为代谢、神经、免疫等研究的潜在模型。Objective Leptin is a cytokine that is involved in the regulation of energy intake and expenditure, neurogenesis, immunity, and lipid and glucose metabolism. Because the characteristics of metabolism and physiology in rats are more close to those of human beings than those of mice, leptin -/- rat provided a good model to assess those effects. Methods Transcription activator-like effector nucleases （TALENs） was used to disrupt the rat leptin locus, creating heritable mutations that eliminate ]eptin function. The body weight of leptin -/- rat was recorded at 4 - 16 weeks of age. The subcutaneous and visceral adipose tissue was measured by MRI and histopathology of the pancreas was observed by light microscopy. Serum biochemicals, intraperitoneal glucose tolerance test （IPGTT） and glucose stimulated insulin secretion were determined in 4-months-old leptin - / - rats. Results A F0 rat with the 8-bp deletion in leptin gene ranging from 418 to 425 base pairs was gained, and C-terminal 28 amino acids of ]eptin were deleted. The body weight of leptin -/- rats increased significantly from 5 weeks of age, and at 4-month-old increased by 52% , compared with that of leptin ＋ / ＋ rats. MRI revealed that the increased body weight was mainly caused by excessive accumulation of subcutaneous and visceral adipose tissue. Histopathological observation showed that the total islet volume of the leptin - / - rats was higher than that of leptin ＋ / ＋ rats at 2-months of age. Triglyceride and cholesterol levels in the leptin -/- rats were 8.4 and 1.8 times as high as those of the leptin ＋/＋ rats at 4 months of age. Hyperinsulinemia and glucose intolerance was observed in the leptin - / - rats at the same age. Conclusion Leptin - / - rats are successfully created using TALENs. The leptin -/- rats are obese, with hyperinsulinemia, hyperlipidemia and glucose intolerance. The leptin -/- rats could be used as a model for research of lipid and glucose metabolism, neurogenesis and immunity.

关 键 词：TALENs技术 瘦素 基因敲除大鼠 肥胖 脂质代谢 糖代谢 

分 类 号：R33[医药卫生—人体生理学]