Isolation Of Genes Related To K562 Cells Differentiation Using A Modified DDRT-PCR

Modified differential display reverse transcription PCR(DDRT-PCR)method was applied to isolate differentially expressed sequence tags(ESTs) related to K562 Cells Differentiation. DDRT-PCR invented By Liang and Pardee has been considered one of the important methods used to isolate novel functional genes.But it also has higher false positives and lower reproducibility .In this study, We used longer DD primers in combination with a two-step PCR protocol to overcome these two problems in term of modified methods of Martin and Pardee.K562 erythroleukemia cell differentiation induced by HEMIN or PMA for 36 hours was used as erythriod or megakaryocytic differentiation model in vitro.Total RNAs were extracted from induced and uninduced K562 cells and were applied to mRNA differential display analysis. 180 differentially expressed fragments were got from DDRT-PCR products by using 3 single-base anchored primers in combination with 6 random primers. 60 of 180 cDNA fragments were related to erythroid differentiation of K562 cells inducted by Hemin. Among them, 38 were upregulated and 22 downregulated; 120 cDNA fragments were related to megakaryocytic differentiation of K562 cells inducted by PMA. Among them, 46 were upregulated and 74 were downregulated. 40 remarkable differential ESTs related to erythroid differentiation and verified by reverse Northern assay were selected for cloning,sequencing and bioinformational analyzing.23 of the 40 differential ESTs were found tohave more than 95% homology to known GenBank sequences and 10 represented cDNA sequences with only dbEST database matches and 7 ESTs have no any database matches. 8 ESTs representing new sequence or providing functional clue were selected for Northern blot analysis and the results of 6 ESTs were shown to be consistent with the differential expressionof DDRT-PCR, suggesting that the modified DDRT-PCR method has effectively surmounted problems on the false positive. These ESTs lay a foundation on studying the molecular mechanisms of erythroid differentiation and megakaryocytic differentiation.Now we are secreening cDNA library in order to isolate full-length cDNA corresponding to these ESTs.