You Position: Home > Paper
Construction of Subtractive cDNA Library of K562 Cells
( views:185, downloads:0 )
- Author:
- No author available
- Journal Title:
- JOURNAL OF CHINESE PHYSICIAN
- Issue:
- 1
- DOI:
- 10.3760/cma.j.issn.1008-1372.2003.01.002
- Key Word:
- K562细胞;抑制性消减杂交;cDNA文库
Abstract： 目的构建氯化血红素诱导性K562细胞抑制消减cDNA文库,以期鉴定出K562细胞中氯化血红素诱导性表达的珠蛋白合成调节因子cDNA基因.方法采用不同浓度的氯化血红素诱导培养K562细胞,经联苯胺阳性细胞百分率及血红蛋白浓度分析,选择其最佳诱导浓度.分别提取氯化血红素诱导培养的K562细胞(tester, 检测子)和未加诱导剂培养的K562细胞(driver, 驱赶子)mRNA,逆转录合成双链cDNA.经两轮消减杂交,两轮PCR扩增后,正向消减的PCR产物与T载体连接,构建K562细胞抑制消减cDNA文库,文库经蓝-白筛选后,纯化阳性克隆质粒,EcoRⅠ酶切及PCR扩增插入片段.结果以50μmol/L 氯化血红素诱导K562细胞,其联苯胺阳性细胞百分率及血红蛋白浓度均达到最大值,故选择50μmol/L 氯化血红素诱导培养K562细胞并构建K562细胞抑制消减cDNA文库.文库鉴定证实其阳性克隆中分别含有不同长度的插入片段.结论成功构建了氯化血红素诱导性K562细胞抑制消减cDNA文库,该消减cDNA文库结合生物信息学(Bioinformatics) 分析可用于深入研究K562细胞内氯化血红素诱导性表达基因的结构和功能.
- [1]王吉伟.两种维甲类药物协同诱导白血病细胞HOXA1基因的表达[J].2000,(4).
- [2]Luda Diatchenko,Yun-Fai Chris Lau,Aaron P. Campbell.Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries[J].1996,93(12).
- [3]P, Liang,A B, Pardee.Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.[J].1992,257(5072).
- [4]M L, Freedman,J, Rosman.A rabbit reticulocyte model for the role of hemin-controlled repressor in hypochromic anemias.[J].1976,57(3).
- [5]Weigel RJ,Brown PO,Ross DT,etc.Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.[J].1999,27(6).
- [6]J S, Wan,S J, Sharp,G M, Poirier,etc.Cloning differentially expressed mRNAs.[J].1996,14.
- [7]M, Hubank,D G, Schatz.Identifying differences in mRNA expression by representational difference analysis of cDNA.[J].1994,22(25).