人Lumican基因转基因鼠模型的建立  

Establishment of a mutant Lumican transgenic mouse model

作　　者：宋彦铮[1] 赵燕燕[1] 张丰菊[1] 于艳秋[2] 马玲[2] 

机构地区：[1]首都医科大学附属北京同仁医院北京同仁眼科中心北京市眼科学与视觉科学重点实验室,100730 [2]中国医科大学基础医学院病理生理教研室

出　　处：《中华眼科杂志》2014年第1期37-41,共5页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金（30872834,81271041）;北京市自然科学基金资助项目（7122041）

摘　　要：目的建立携带突变位点（cDNA596T〉C）的人Lumican（LUM）基因的转基因鼠模型。方法实验研究。利用基因重组技术，将人工合成的LUM基因第二外显子596位碱基T替换为C，再克隆到质粒pRP．Des3d的多克隆位点，构建pRP．EX3d—EFlA〉LUM／flag〉IRES／hrGFP质粒载体；然后用显微注射法将其转至BDFl小鼠受精卵雄原核中，存活的受精卵再移入美国癌症研究所（ICR）假孕雌鼠输卵管内，得到可表达LUM基因的F0代转基因小鼠C57一TgN（LUM）CCMU。经PCR、免疫印迹杂交法分析子代鼠中基因整合及表达的情况。结果得到31只新生仔鼠，PCR检测有6只LUM阳性转基因鼠，免疫印迹杂交法证明转基因阳性鼠转入的目的基因片段确实合成了相应蛋白质。稳定遗传3代，共获得128只仔鼠，经PCR检测其中有68只为LUM阳性，阳性率达53．13％。结论携带突变位点（cDNA596T〉C）的LUM基因的转基因鼠模型构建成功，为进一步探索LUM基因突变对病理性近视眼的发生发展及细胞外基质成分的影响提供了重要的研究平台。Objective Pathological myopia （PM） is a hereditary ocular disease leading to severe loss of visual acuity and blindness. Lumican gene （ LUM ） is one of those candidate genes of PM. The purpose of this study was to establish a mutant Lumiean transgenie mouse model, and to prepare for the further study of the pathogenesis of PM. Methods Experimental study. Mutation of LUM gene was created by site-directed mutagenesis. Recombinant DNA techniques were used for the construction of the pRP. EX3d-EF1A 〉 LUM/flag 〉 IRES/hrGFP transgene. The gene fragments were microinjected into the zygote male pronuclei of BDF1 mice, and then the zygote cells alive were transplanted into the oviduct of acceptor pregnant female ICR mice. The F0 generation transgenie mice obtained were named C57-TgN （LUM） CCMU. Genome DNA from mice tail was detected by PCR and Western blotting. Results Six of 31 F0 generation mice were positive transgenic mice. The western blotting study showed that the flag-tag was expressed in the mouse tail tissue. Sixty-eight of 128 mice （F1 to F3 generation） were positive transgenic mice, the positive rate is 53.13%. Conclusions The mutant Lumican （cDNA 596T 〉 C） transgenic mouse model has been established. This model will provide fundamental conditions for studies of the pathogenesis of PM. Also it will he the basis of further studies about the effect of Lumican mutation on the development of PM and structure and function of the extra cellular matrix.

关 键 词：硫酸角质素 蛋白硫酸软骨素类 小鼠 转基因 模型 动物 近视 退行性 

分 类 号：R-332[医药卫生] R778.11