| 摘要: |
| 目的 通过制备Rtn4-A/B基因敲除小鼠,探索Rtn4-B基因的生物学功能。 方法 用细菌人工染色体(BAC)载体构建Rtn4-A/B基因打靶载体并使其线性化,通过电转化法将其转入129SvEv品系雄性小鼠胚胎干细胞(embryonic stem cell,ES细胞)。将正确同源重组的ES细胞注射入C57BL/6J小鼠囊胚腔,繁育出嵌合体小鼠后,进一步繁殖以获得杂合子小鼠。抽提小鼠尾尖组织DNA,采用PCR法鉴定小鼠的基因型。 结果 基因打靶后,得到14个发生双臂正确同源重组ES细胞克隆。利用阳性ES细胞克隆进行囊胚内显微注射,得到5只嵌合率大于50%的雄鼠,最终繁育得到4只Rtn4-A+/-B+/-杂合子小鼠。 结论 利用ES细胞基因打靶、同源重组等方法,成功获得Rtn4-A/B基因敲除杂合子小鼠。 |
| 关键词: Rtn4 基因打靶 基因敲除小鼠 胚胎干细胞 同源重组 |
| DOI： |
| 投稿时间：2013-01-06修订日期：2013-04-19 |
| 基金项目:国家自然科学基金面上项目(81170060/H011),国家自然科学基金青年项目(81200030/H0108). |
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| Construction of Rtn4-A/B knockout mouse model |
| ZHU Ying1,XU Wu-jian1,NING Yun-ye1,WU Lin2,LU Jian3,LI Qiang1* |
(1. Department of Respiratory Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
2. Department of Gastroenterology, General Hospital, PLA Nanjing Military Area Command, Nanjing 200001, Jiangsu, China
3. Department of Pathophysiology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China
*Corresponding author.) |
| Abstract: |
| Objective To generate Rtn4-A/B knockout mouse model and to explore the biological function of the Rtn4-B gene. Methods The targeting construct for inactivating Rtn4-A/B gene was prepared by bacterial artificial chromosome (BAC). The vector was linearized and electroporated into 129SvEv mouse embryonic stem cells (ES cells). Then the Rtn4-A/B knockout ES cells were microinjected into blastula of C57BL/6J mice after superovulation. F1 hybrid mice were bred to obtain mouse aggregation chimeras, and were identified by PCR amplification of tail genomic DNA. Results Fourteen clones of gene-targeted ES cells were identified after gene knockout and five male chimeras with a higher than 50 chimeric ratio were produced after microinjection into the blastula. Finally four Rtn4-A/B hybrid mice were obtained. Conclusion A Rtn4-A/B deficient mouse strain has been successfully generated by homologous recombination using genetically modified ES cells. |
| Key words: Rtn4 gene targeting knockout mice embryonic stem cells homologous recombination |
