Dicer1转基因小鼠模型的建立

2008年8月

第16卷　第4期中国实验动物学报ACT A LABORAT ORIUM ANI M A LIS SCIE NTIA SINICA August ,2008V ol.16　N o.4

研究报告

Dicer 1转基因小鼠模型的建立

郑志红1,高峰2,杨葳1,汪瑛1,于洋1,周生来1,李兆阳1

,

吕相川1,张梅英1,王禄增

1(1.中国医科大学实验动物部,沈阳　110001;2.中国医科大学附属第一医院,沈阳　110001) 【摘要】　目的　建立Dicer 1转基因小鼠模型。方法　构建pcDNA3112Dicer1转基因构件,经酶切、纯化后通过显微注射方法导入BDF1小鼠受精卵原核并移植到同期受孕的ICR 受体母鼠输卵管内。出生后仔鼠用PCR 和S outhern 方法检测鼠尾DNA 鉴定基因型,通过免疫组化检测Dicer 1基因表达。结果　显微注射172枚卵,移植119枚卵于3只受体输卵管中,2只怀孕,共产仔15只,经PCR 检测获得6只阳性鼠,S outhern 检测6只均为阳性。对S outhern 检测阳性转基因小鼠子代进行RT 2PCR 检测和免疫组化分析证明Dicer1基因在肝脏、肾脏、肺内均有表达。对腹腔肿胀的转基因阳性1号鼠解剖发现肝脏、脾脏明显增大,胚胎发育异常。结论　成功建立Dicer 1基因表达的转基因小鼠模型,该模型为进一步研究DICER 1基因功能及miRNA 的表达及功能等奠定基础。

【关键词】　Dicer 1;显微注射法;转基因小鼠

【中图分类号】R -33 【文献标识码】A 【文章编号】100524847(2008)0420258203

Establishment of a Dicer 1Transgenic Mouse Model

ZHE NG Zhi 2hong 1,G AO Feng 2,Y ANG Wei 1,W ANG Y ing 1,Y U Y ang 1,ZH OU Sheng 2lai 1,LI Zhao 2yang 1

,

LU X iang 2chuan 1,ZH ANG Mei 2ying 1,W ANGLu 2zeng 1(boratory Animal Center ,China Medical University ,Shenyang 110001,China ;

21The First A ffiliated H ospital of China Medical University ,Shenyang 110001,China )

【Abstract 】　Objective T o establish a Dicer 1transgenic m ouse m odel.Methods pcDNA3112Dicer1construct was constructed ,linearized ,purified and then injected into superovulated pronuclear zyg otes to produce transgenic mice.The injected zyg otes were transplanted into the oviduct of pseudopregnant mice.The genotype of transgenic founders were identified by PCR and S outhern blot.The expressions of human Dicer 1protein in the tissues of the transgenic mice were detected by immunohistochemistry.R esults 172zyg otes were injected and 119zyg ote cells were transplanted into oviducts of 3recipients.15viable offsprings were born from 2of the 3recipients.G enomic DNA from baby tails was extracted.PCR and S outhern blot were used to identify transgenic founders of Dicer 1,and showed 6of the 15offsprings were positive transgenic mice of Dicer 11Dicer 1was expressed in the liver ,kidney and lung.Conclusion Dicer 1transgenic mice have been established success fully.The m odels will contribute to the research of Dicer gene function and the expression of miRNA.

【K ey w ords 】　Dicer1;M icroinjection ;T ransgenic mice

[基金项目]国家自然科学基金:30571836;辽宁省重点实验室专项资金:辽科发[2005]36号。

[作者简介]郑志红(1969-),女,研究方向:实验动物转基因与

基因敲除。E -mail :zhihongzheng @1631com

[通讯作者]王禄增。E 2mail :wanglz @1631com DICER 1基因与表观遗传调控密切相关,是

2000年被克隆的,定位于人染色体14q32113,编码

蛋白属于RNA 酶Ⅲ家族[1],在许多组织中广泛表

达。其功能是将具有颈环结构的RNA 或双链RNA

剪切成长约21个碱基的成熟miRNA (或siRNA )[1]。

DICER 1蛋白是成熟miRNA 产生所必需的酶,

DICER 1基因异常可导致不同组织、不同发育阶段miRNA 表达异常,DICER 1基因敲除的小鼠在胚胎发育过程中就发生死亡[2],无法通过敲除来详细研究该基因的功能。由于DICER 1基因是发育过程中重要的调控基因,建立DICER 1转基因小鼠模型对研究该基因的功能具有重要的意义。1　材料方法

111　Dicer 1基因克隆及转基因构件制备按Dicer 1mRNA 序列设计正、反向引物,以