微小RNA簇miR-302s条件性基因打靶小鼠的制备和鉴定

doi:10.3969/j.issn.1674-5817.2016.02.002

实验动物与比较医学 ›› 2016, Vol. 36 ›› Issue (2): 87-92.DOI: 10.3969/j.issn.1674-5817.2016.02.002

微小RNA簇miR-302s条件性基因打靶小鼠的制备和鉴定

余飞¹, 钱丽萍¹, 丁利军²

南京大学医学院附属鼓楼医院1.动物实验中心, 2.生殖医学中心, 南京210008

收稿日期:2015-10-22 出版日期:2016-04-25 发布日期:2016-04-25
作者简介:余飞(1982-), 女, 硕士, 助理研究员, 主要研究方向: 实验动物学。E-mail: yufei0802@yeah.net
基金资助:
国家自然科学基金项目(30900847), 中央高校苗圃项目 (20620140707), 南京市卫生杰出青年人才项目(JQ2014004)

Generation and Identification of miR-302s Conditional Gene Targeting Mice

YU Fei¹, QIAN Li-ping¹, DING Li-jun²

1. Experimental Animal Center; 2. Reproductive Medicine Center, Nangjing Drum Tower Hospital The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China

Received:2015-10-22 Online:2016-04-25 Published:2016-04-25

摘要/Abstract

摘要： 目的制备微小RNA簇miR-302s条件性基因打靶小鼠。方法构建miR-302s打靶载体, 通过电穿孔的方法转染胚胎干细胞(ES细胞),用正负筛选法筛选阳性ES细胞并进行PCR鉴定和Southern分析。利用显微注射技术将正确同源重组的ES细胞注射至B6(Cg)-Tyr<sup>c-2J</sup>/J小鼠囊胚腔内,注射后的囊胚植入受体小鼠子宫。将得到的嵌合体小鼠与野生型雌鼠交配获得miR-302s-LoxP基因打靶小鼠。结果获得嵌合体小鼠11只, 其中4只嵌合雄鼠嵌合率>50%。利用嵌合体小鼠进行繁育交配,得到miR-302s基因打靶杂合子小鼠11只。结论成功建立miR-302s条件性基因打靶小鼠模型,为进一步研究miR-302s基因功能打下基础。

关键词: miR-302s, 条件性基因打靶小鼠, 胚胎干细胞, 同源重组

Abstract: Objective To generate microRNA miR-302s gene targeting mice. Methods The conditional gene targeting vector were generated by molecular cloning, and then the electroporation of embryonic stem (ES) cells were carried out. The targeted ES cells were screened by positive-negative selection and identified by PCR and Southern blot, and then the correct targeted ES cells were microinjected into blastula of B6(Cg)-Tyr<sup>c-2J</sup>/J mice. Chimerical mice were generated after transplantation of the injected blastula into the host mice, and the miR-302s-LoxP gene recombinat mice were obtained after breeding. Results Eleven miR-302s chimeric mice were obtained including four male chimeras with a higher than 50% chimeric ratio. Eleven miR-302s heterozygous gene recombinat mice were generated after outbred and inbred. Conclusion The miR-302s conditional gene targeting mice were successfully obtained. This animal model provided the foundation for further study of miR-302s in vivo.

Key words: miR-302s, Conditional gene targeting mice, Embryonic stem (ES) cell, Homologous recombination

中图分类号:

R-33Q95-33

余飞, 钱丽萍, 丁利军. 微小RNA簇miR-302s条件性基因打靶小鼠的制备和鉴定[J]. 实验动物与比较医学, 2016, 36(2): 87-92.

YU Fei, QIAN Li-ping, DING Li-jun. Generation and Identification of miR-302s Conditional Gene Targeting Mice[J]. Laboratory Animal and Comparative Medicine, 2016, 36(2): 87-92.

参考文献

[1] Bartel DP.MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2):281-297.
[2] Barroso-del Jesus A, Lucena-Aguilar G, Menendez P. The miR-302-367 cluster as a potential stemness regulator in ESCs[J]. Cell Cycle, 2009, 8(3):394-398.
[3] Rosa A, Spagnoli FM, Brivanlou AH.The miR-430/427/302 family controls mesendodermal fate specification via species-specific target selection[J]. Dev Cell, 2009, 16(4):517-527.
[4] Parchem RJ, Moore N, Fish JL, et al.miR-302 is required for timing of neural differentiation, neural tube glosure, and embryonic viability[J]. Cell Rep, 2015, 12(5):760-773.
[5] Wade SL, Langer LF, Ward JM, et al.MiRNA-mediated regulation of the SWI/SNF chromatin remodeling complex controls pluripotency and endodermal differentiation in human ESCs[J]. Stem Cells, 2015, 33(10):2925-2935.
[6] Lu Y, Loh YH, Li H, et al.Alternative splicing of MBD2 supports self-renewal in human pluripotent stem cells[J]. Cell Stem Cell, 2014, 15(1):92-101.
[7] 王宏, 田海滨, 陈娟, 等. Knockout血清替代品可提高C57BL/6J小鼠胚胎干细胞建系效率[J]. 细胞与分子免疫学杂志, 2007, 23(3):269-276.
[8] Lin SL, Chang DC, Chang-Lin S, et al.Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state[J]. RNA, 2008, 14(10):2115-2124.
[9] Lin SL, Chang DC, Lin CH, et al.Regulation of somatic cell reprogramming through inducible mir-302 expression[J]. Nucleic Acids Res, 2011, 39(3):1054-1065.
[10] Subramanyam D, Lamouille S, Judson RL, et al.Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells[J]. Nat Biotechnol, 2011, 29(5):443-448.
[11] Hu S, Wilson KD, Ghosh Z, et al.MicroRNA-302 increases reprogramming efficiency via repression of NR2F2[J]. Stem Cells, 2013, 31(2):259-268.
[12] Yan GJ, Yu F, Wang B, et al.MicroRNA miR-302 inhibits the tumorigenicity of endometrial cancer cells by suppression of Cyclin D1 and CDK1[J]. Cancer Lett, 2014, 345(1):39-47.

微小RNA簇miR-302s条件性基因打靶小鼠的制备和鉴定

Generation and Identification of miR-302s Conditional Gene Targeting Mice

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