p53基因敲除大鼠模型的构建及表型分析

Construction and Phenotypic Analysis of Gene p53 Knockout Rat Model

使用TALEN技术制作p53基因敲除大鼠模型,建立种群并分析大鼠表型。设计特异性识别p53基因外显子2的TALEN蛋白并构建相应载体,将体外转录的TALEN m RNA注射SD大鼠受精卵,出生后从仔鼠中通过测序筛选靶位点正确剪切的阳性小鼠。结果显示,注射得到11只新生仔鼠,通过测序发现其中有10只靶位点发生剪切。选取其中4只作为首建鼠进行繁殖。p53基因敲除纯合子表现出肿瘤易发性,主要自发性肿瘤类型为恶性纤维组织肉瘤。此外,p53基因敲除纯合子大鼠表现出眼睛发育异常,视网膜变性及晶状体纤维排列紊乱。通过TALEN技术高效地获得了p53基因敲除大鼠模型,纯合子敲除大鼠除了易发肿瘤并伴有眼发育异常,因而该敲除大鼠模型除了可用于研究肿瘤发生机制外,也可用于研究p53基因在眼发育过程中的功能。

The objectives of this work are to construct rat model with gone p53 knockout using TALEN, to establish colony, and to analyze the phenotypes of the rats. TALEN protein specifically recognizing exon 2 of gone p53 was designed and the corresponding vectors were constructed. In vitro transcribed TALEN mRNA was injected into fertilized eggs of SD rats. New born rats with right splicing at the target site were determined by sequencing. Together l lnew-born rats were obtained after injection and 10 of them were having right splicing at target site by sequencing. Four rats were selected as founder to establish Ihe colony. The homozygotes with gene p53 knockout presented tumor susceptibility and the main type of spontaneous tumor was malignant fibrous histioeytoma. In addition, the homozygous rats with gene p53 knockout also had eye developmental defects, such as retina degeneration and disorganized fiber of lens. In conclusion, rat model with p53 knockout is efficiently constructed by TALEN, the model exhibits tumor susceptibility and eye developmental defects, indicating that the model can be used for studying the mechanisms of tumorigenesis and the funetion of gene p53 in eye development.