WDR5真核表达载体的构建及其在LNCaP细胞中的稳定表达

段君君; 方峰; 张小玉; 王江; 贾霄; 何颖红

WDR5真核表达载体的构建及其在LNCaP细胞中的稳定表达

段君君^1,2,
方峰^1,2,
张小玉^1,2,
王江^1,2,
贾霄^1,2,
何颖红^1,2

1. 云南省高校细胞生物学重点实验室
2. 大理大学基础医学院

基金项目:

基金：国家自然科学基金资助项目 (31360288);

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出版历程
- 收稿日期: 2017-03-19

Construction of WDR5 Eukaryotic Expression Vector and Its Stable Expression in LNCaP Cells

Duan Jun Jun ^1,2,
Fang Feng ^1,2,
Zhang Xiao Yu ^1,2,
Wang Jiang ^1,2,
Jia Xiao ^1,2,
He Ying Hong ^1,2

Funds:

基金：国家自然科学基金资助项目 (31360288);

摘要

摘要: 目的构建人源WDR5全长结构基因真核表达载体, 建立稳定表达WDR5的LNCaP细胞株.方法PCR扩增WDR5基因, 将WDR5插入pCI-neo载体中, 酶切及测序鉴定重组质粒.利用脂质体转染法将重组质粒转染LNCaP细胞, Real Time-PCR检测转染细胞WDR5的m RNA表达水平.结果经酶切及测序证实真核表达载体pCI-neo-WDR5构建正确.重组质粒转染到LNCaP细胞后, 用G418筛选获得稳定表达的细胞株, Real Time-PCR方法检测到WDR5基因在转录水平的表达.结论 PCI-neo-WDR5成功转染LNCaP细胞株中并稳定表达, 为下一步WDR5的深入研究及应用奠定了基础.
- WDR5 /
- 真核表达 /
- G418抗性 /
- 脂质体转染
Abstract: Objective To construct a recombinant vector containing human WDR5 and establish LNCaP cell strain with stable expression of WDR5. Me thods WDR5 gene was amplified by PCR and was cloned into eukaryotic expression vector pCI-neo. The resulted pCI-neo-WDR5 plasmid was verified by restriction enzyme digestion and DNA sequencing. LNCaP cells were transfected with pCI-neo-WDR5 by liposome. The expression of WDR5 in the cells was examined by Real-time PCR. Re s ults Restriction enzyme digestion and sequencing analysis showed that WDR5 c DNA was correctly cloned into pCI-neo vector. After transfection of LNCaP cells with the recombinant plasmids, the monoclonal cell strain of stably expressed were obtained by G418 screening, which showed expressions of WDR5 m RNAs detected by Real Time-PCR. Conclus ion The recombinant of PCI-neo-WDR5 stably express in the transfected LNCaP cells, which laid a foundation for further study on WDR5.
- WDR5 /
- Eukaryotic expression /
- G418 resistance /
- Liposome tansfection

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参考文献(10)

[1]	[1]TRIEVEL R C, SHILAIFAR D A.WDR5, a complexed protein[J].Nat Struct Mol Biol, 2009, 16 (8) :678-680.
[2]	[2]AHN Z, GUO L, WANG L, et al.Structural basis for the specific recognition of methylatedhistone H3 lysine 4 by the WD-40 protein WDR5[J].Mol Cell.2006, 22 (1) :137-144.
[3]	[3]WYSOCKA J, SWIGUT T, MILNE T A, et al.WDR5associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development[J].Cell, 2005, 121 (8) :859-872.
[4]	[4]GE Z, SONG E J, KAWASAWA Y I, et al.WDR5 high expression and its effect on tumorigenesis in leukemia[J].Oncotarget, 2016, 7 (25) :37740-37754.
[5]	[5]WANG Y, LIU L, ZHONG B, et al.WDR5 is essential for assembly of the VISA-associated signaling complex and virus-triggered IRF3 and NF-kappa B activation[J].Proc Natl Acad Sci U S A, 2010, 107:815-820.
[6]	[6]ANG Y S, TSAIS Y, LEE D F, et al.Wdr5 mediates selfrenewal and reprogramming via the embryonic stem cell core transcriptional network[J].Cell, 2011, 145 (2) :183-197.
[7]	[7]CAO F, CHEN Y, CIERPICKI T, et al.An Ash2L/Rb BP5heterodimer stimulates the MLL1 methyltransferase activity through coordinated substrate interactions with the MLL1SET domain[J].PLo S One, 2010, 5 (11) :e14102.
[8]	[8]XU Z, GAO X, HE Y, et al.Synergistic Effect of SRY and Its Direct Target, WDR5, on Sox9 Expression[J].PLo S ONE, 2012, 7 (4) :e34327.
[9]	[9]XU Z, HE Y, JU J, et al.The role of WDR5 in silencing human fetal globin gene expression[J].Haematologica, 2012, 97 (11) :1632-1640.
[10]SAMBROOK J, RUSSELL D W.分子克隆实验指南[M].第3版.北京:科技出版社, 2002:1307-1308.

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