Construction of differentially expressed cDNA library in human leukemia HL-60 cells induced by diallyl disfuld with suppression subtractive hybridization / 中国药理学通报

ABSTRACT

AIM To construct differentiation subtractive cDNA library of human leukemia HL 60 cells induced by diallyl disfuld using suppression subtractive hybridization (SSH) technique and to clone differentiation associated genes in leukemia cells. METHODS Poly A + RNAs were isolated from HL 60 cells induced by dially disfuld and were reversely transcripted into double strand cDNAs (as tester). After the cDNAs were digested into short cDNAs with blunt ends, they were divided into two groups and were ligated to the special adaptor 1 and adaptor 2R, respectively. The tester cDNAs were then hybridized with driver cDNA from normal HL 60 cells and the products were amplified twice by nested PCR technique. The PCR products were ligated with pGEM T plasmid vectors and were transformed into E.coli JM109. The inserts of cDNAs were analyzed by restrictive enzyme EcoR I. RESULTS Subtractive cDNA library was constructed successfully and efficiently. Random analysis of 200 clones with enzyme restriction showed that about 84 5% clones contained 100～600 bp cDNA inserts. It can help clone novel genes associated with differentiation and explore the molecular mechanism of leukemia differentiation induced by diallyl disfuld. CONCLUSION DADS can induce human leukemia HL 60 cells differentiation, and suppression subtractive hybridization (SSH) technique is an effective method to isolate those differentially expressed genes.