鸡胚胎干细胞多能性相关基因cNanog和cPouV干扰载体的构建及筛选

生物技术通报 ›› 2014, Vol. 0 ›› Issue (5): 117-121.

鸡胚胎干细胞多能性相关基因cNanog和cPouV干扰载体的构建及筛选

彭特^1，2 王丙云² 李东升^1，2 陈志胜² 陈胜锋² 计慧琴² 陈金顶¹

(1.华南农业大学兽医学院，广州 510642；2.佛山科学技术学院生命科学学院，佛山 528231)

收稿日期:2013-11-13 出版日期:2014-05-23 发布日期:2014-05-24
作者简介:彭特，女，硕士研究生，研究方向：预防兽医学和鸡胚胎干细胞； E-mail: pte072@163.com
基金资助:
国家自然科学基金项目(31072101)，广东省自然科学基金项目(S201101004876)

Construction and Screening of Recombinant Interference Vector of Pluripotency Associated Genes cNanog and cPouV in Chicken Embryonic Stem Cells

Peng Te^1，2 Wang Bingyun² Li Dongsheng^1，2 Chen Zhisheng² Chen Shengfeng² Ji Huiqin² Chen Jinding¹

(1. College of Veterinary Medicine，South China Agricultural University，Guangzhou 510642；2. School of Life Science，Foshan University，Foshan 528231)

Received:2013-11-13 Published:2014-05-23 Online:2014-05-24

摘要/Abstract

摘要： 旨在构建靶向鸡胚胎干细胞多能性相关基因cNanog和cPouV的siRNA表达载体，并在体外检测其对cNanog和cPouV表达的抑制作用。分别选择cNanog和cPouV中3个区域合成寡聚核苷酸构建siRNA表达载体，同时构建cNanog和cPouV全基因表达载体来检测基因表达下调的效果。经Western blotting检测显示，所构建的siRNA表达载体可在293T细胞内表达siRNA或shRNA，诱发RNA干扰效应，从而抑制目的基因cNanog和cPouV的表达。不同位点的寡核苷酸序列抑制效率不同，其中pS3-cNanog和pS3-cPouV重组质粒的干扰效果和抑制效率最好。研究结果为后续得到稳定干扰cNanog 和cPouV基因的重组载体和进一步研究cNanog 和cPouV基因功能奠定了基础。

关键词: 鸡, 胚胎干细胞, cNanog, cPouV, 干扰载体

Abstract: The study was to construct the siRNA(small interfering RNA)expression vectors targeting cNanog and cPouV of Chicken Embryonic Stem cells(CES cells), and evaluate its inhibitory effect on cNanog and cPouV in 293T cells. Three regions of cNanog and cPouV gene were respectively selected to synthesize oligonucleotides, and construct interference vectors. These vectors were respectively transfected into 293T cells as whole-gene expression vectors were transduced simultaneously. Western blotting analysis showed target genes expression were downregulated after RNAi and the oligonucleotide sequence S3 was found to be the most efficient one in interfering effect. The results of this experiment indicate siRNA or shRNA(short hairping RNA)were translated into cells and suppress the expression of target genes.

Key words: Chicken Embryonic, Stem, Cells, CNanog, CPouV Interference vector

彭特, 王丙云, 李东升, 陈志胜, 陈胜锋, 计慧琴, 陈金顶. 鸡胚胎干细胞多能性相关基因cNanog和cPouV干扰载体的构建及筛选[J]. 生物技术通报, 2014, 0(5): 117-121.

Peng Te, Wang Bingyun, Li Dongsheng, Chen Zhisheng, Chen Shengfeng, Ji Huiqin, Chen Jinding. Construction and Screening of Recombinant Interference Vector of Pluripotency Associated Genes cNanog and cPouV in Chicken Embryonic Stem Cells[J]. Biotechnology Bulletin, 2014, 0(5): 117-121.

参考文献

[1] Rodda DJ, Chew JL, Lim LH, et al. Transcriptional regulation of nanog by OCT4 and SOX2[J]. J Biol Chem, 2005, 280(26)：24731-24737.
[2] Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[J]. Cell, 2003, 113(5)：631-642.
[3] Pan G, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency[J]. Cell Res, 2007, 17(1)：42-49.
[4] Soodeen-Karamath S, Gibbins AM. Apparent absence of oct 3/4 from the chicken genome[J]. Mol Reprod Dev, 2001, 58(2)：137-148.
[5] Lavial F, Pain B. Chicken embryonic stem cells as a non-mammalian embryonic stem cell model[J]. Dev Growth Differ, 2010, 52(1)：101-114.
[6] 刘本杰, 陈志胜, 陈胜锋, 等. 全能性相关基因cNanog、cPouV和Sox2在鸡早期胚胎中的表达[J]. 中国组织工程研究, 2012(41)：7733-7736.
[7] 王丙云, 陈胜锋, 刘本杰, 等. 全能性相关基因在鸡胚胎干细胞中分化过程中的表达[C]. 南京：中国畜牧兽医学会动物生理生化学分会, 2012.
[8] 陈志胜, 刘本杰, 王丙云, 等. 全能性相关基因在早期鸡胚发育过程中的表达[C]. 南京：中国畜牧兽医学会动物生理生化学分会, 2012.
[9] Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391(6669)：806-811.
[10] Bosher JM, Labouesse M. RNA interference：genetic wand and genetic watchdog[J]. Nat Cell Biol, 2000, 2(2)：E31-E36.
[11] Sharp PA. RNA interference--2001[J]. Genes Dev, 2001, 15(5)：485-490.
[12] Tabara H, Grishok A, Mello CC. RNAi in C. elegans：soaking in the genome sequence[J]. Science, 1998, 282(5388)：430-431.
[13] Chang HS, Lin CH, Chen YC, et al. Using siRNA technique to gen-erate transgenic animals with spatiotemporal and conditional gene knockdown[J]. Am J Pathol, 2004, 165(5)：1535-1541.
[14] 张曼玉, 陈志胜, 计慧琴, 等. 鸡胚胎干细胞分离方法和培养体系的优化[J]. 中国组织工程研究与临床康复, 2011(45)：8429-8433.
[15] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells[J]. Science, 2002, 296(5567)：550-553.
[16] 庄淑珍, 窦忠英, 杨炜峰, 等. ES-D3细胞nanog基因干扰载体的构建及其干涉效果[J]. 农业生物技术学报, 2005(6)：754-758.