Construction of Subtractive cDNA Library of K562 Cells / 中国医师杂志

ABSTRACT

Objective To construct the suppression subtractive library of hemin-inducing K562 cells for identifying the cDNA genes of globin synthesis regulatory factors expressed in K562 cells induced by hemin. Methods K562 cells were cultured under different concentration of hemin, both the percentage of positive benzidine staining cells and hemoglobin content were measured, the most reasonable concentration of hemin was chosen for inducing incubation of K562 cells. The poly (A) positive RNA (mRNA) was isolated from the hemin-induced K562 cells (tester) and non-induced K562 cells (driver) respectively, and double-strand cDNA molecules were synthesized by reverse transcription. After two times subtractive hybridization followed by two times polymerase chain reaction (PCR) amplification, the forward subtracted PCR products were ligated with pGEM T-Easy vector and the subtracted cDNA library was constructed. The library clones were selected by blue-white screening. The plasmid DNAs of the single positive colony were purified and digested by EcoRⅠ, and the inserts in plasmid were amplified by PCR. Results The maximum of positive benzidine staining cells percentage and hemoglobin content of K562 cells were obtained in 50?mol/L of hemin inducing condition. The suppression subtractive library of hemin-inducing K562 cells was constructed after K562 cells were induced by 50?mol/L of hemin. The library identification showed that the positive clones contain inserts in different length respectively. Conclusions The suppression subtracted cDNA library of hemin-inducing K562 cells were successfully constructed. This subtracted cDNA library combined with bioinformatics analysis can be used to explore structures and functions of hemin-inducing expression genes in K562 cells.