In Vitro Study On The Leukemic Cell Lines Transfected By RbAp46 Gene

1.Establishment and characterization of leukemic cell lines stablyexpressing exogenous RbAp46 mediated by electroporationObjective To optimize transfection conditions for suspended cells and to establish leukemic cell lines transfected by RbAp46 gene. Methods The efficiency of gene transfer into leukemic cell lines in two different electroporation buffers were compared.Under conditions of low voltage and high capacitance, RbAp46 was transfected into U937 and K562 by electroporation .Selected with G418 for 3 weeks, individual clones were then isolated .The integration of and the protein levels of the exogenous RbAp46 in transfectants were determined by PCR and Western blot analysis, respectively. Results Stable clones were obtained when time constant (T) for electroporation ranged from 10msec to 40msec. The clones expressing high level of RbAp46 were established.Conclusion By optimal electroporation, leukemic cell lines stably expressing exogenous RbAp46 were established.2. In Vitro study on the impact of overexpression of RbAp46 in U937cell lineObjective To study the impact of overexpression of RbAp46 in monoblastic cell line U937. Methods Viability of transfected cells was assayed by trypan blue exclusion.Cell number was counted daily to determine the growth rate.Cells at an initial concentration of 0.2x10 cells/ml were incubated with or without 50ng/ml TPA. After 72h,cell surface antigen CD11b,mRNA p21 and cell cycle distribution were assayed by FACS, RT-PCR,and FACS,respectively. Results The cell lines expressing exogenous RbAp46 demonstrated about 1-fold lower growth rate than the control. RbAp46 upregulated the expression of CD11b in U937 cell line on incubation with TPA or not. Furthermore,the expression of p21 in RbAp46 transfected cells was similar to control cells at mRNA level,while U937/RbAp46 treated with TPA exhibited a marked increase in p21 expression and arresting at G1 phase. Conclusion Overexpression of RbAp46 inhibits the growth of U937 and triggers the differentiation program. p21 may involve in the process.3. Establishment of expression of RbAp46 in U937 cell line controlledby the RevTet-On regulatory systemObjective To establish a tetracycline-controlled inducible gene expression system in U937 cell line. Methods To produce recombinant retrovirus,the package cell line PT67 was transfected with vectors pTet-On,pTRE-Luc and pTRE-RbAp46, respectively,by using liposome transfection reagent.U937 cell line was infected with recombinant retrovirus RevTet-On.The infected cells were selected in growth mediumcontaining G418.G418-resistant cells were then subcloned with limited dilution. 14 days later,all individual G418-resistant clones were screened by transient infection with retrovirus TRE-Luc for clones with low backgroud and high induction of luciferase in response to doxycycline.One U937 cell subline with high levels of induction and high gene expression levels was obtained.Then the celi line was infected with retrovirus TRE-RbAp46. Results One U937 cell line expressing RbAp46 regulated by the RevTet-On regulatory system was established. Conclusion The RevTet-On regulatory system could modulate the expression of RbAp46 by tetracycline and its derivatives, providing a useful model for research the function of RbAp46 in leukemic cells.