Screening And Cloning Differently Expressed Genes In Human Leukamia HL-60 Cells Treated By Dially Disulfide

Object: To screen and clone different expressed genes in human leukamia HL-60 cells.Method: Different expressed genes in treated HL-60 cells by DADS were identified by using suppression subtractive hybridization (SSH), T/A cloning, sequenced and analyzed for homology in the Genbank database with BLAST. Results: Cellular size shrinked in HL-60 cells treated by DADS for 24h, as nuclear-to-cytoplasmic ratio was reduced, and coloring of nucleus weaked. Abstract of RNA:Total RNA abstracted by TRLzol reagent was detected by agrose gel electrophoresis. 28S RNA and 18S RNA displayed as two bands without trail. mRNA was showed as a distinct smear strap.Enzymatic results of Rsa I: Undigested cDNA manifest as a distinct strap of 0.5-1.0kb ,digested cDNA manifest as a distinct strap of 0.1-2kb.Analysis of ligation: Efficiency of adaptor-ligation with digested cDNA exceed 2 5%.The nest PCR analysis o f S SH: Fragments o f subtracted products 1 ess than unsubtracted products, which size about 100-600 bp.PCR anallysis of subtraction efficiency: Using G3PDH 3'primer and 5' primer subtract product and unsubtract product were amplified, respectively. G3PDH product can be seen after 28 cycles, and the former's abundance less than the latter's. It demonstration that subtraction efficiency is very high. T/A clone: The ratio of white and blue clones is about 2.Purified plasmid of positive clones exceed 3kb, Size of PCR products is about 200-300bp.Sequencing results showed that 89 clones were identified. After comparing with public database(Genbank/EMBL) and Expressed Sequence Tag(EST), 7 known genes were found in the two libaries. Data showed that those known genes represent a variety groups on the basis of their cellular functions and have 99-100% identities in sequences. In the forward substract-library, we found PRDX2, CNOT8, PQBP1 and RPS7 were up-regulated in HL-60 cells induced by DADS, while hRRM2, calcium/calmedulin-dependent protein kinase and Homo sapiens isolate C30 MK2 non-allergic IgE heavy chain were down-regulated in...