Molecular cloning of ING4 gene and ING4-induced apoptosis in HeLa cells

AIM:To isolate a gene encoding mouse ING4,construct pcDNA3.0-ING4 recombinant eukaryotic expression plasmid and investigate its effects on HeLa cells in vitro.METHODS:The mouse ING4cDNA was amplified by RT-PCR from mouse liver.The eukaryotic expression vector pcDNA3.0-ING4 was constructed by DNA recombination technique.The recombinant plasmid pcDNA3.0-ING4 was identified by PCR,restriction enzyme digestion and DNA sequence analysis,then was transfected into HeLa cells by lipofectamine.The expression was determined by RT-PCR.Apoptosis was detected by fluorescence microscope with Hoechst33258 staining and laser scanning confocal microscope.Cell cycle distribution was measured with flow cytometry.RESULTS:RT-PCR product was about 750 bp specific fragment.Analysis by restricting enzyme digestion and PCR of pcDNA3.0-ING4 recombiant plasmid showed that results were about 750 bp,DNA sequencing revealed that ING4 cloning were successful.With Hoechst fluorescence staining,we found that the percentage of apoptotic rate in HeLa cells transfected with pcDNA3.0-ING4(21.25%)was higher than that in HeLa cells transfected with pcDNA3.0(8.91%,P0.01).Apoptosis was also detected by laser scanning confocal microscope.Cell cycle analysis reavealed the cell number in S phase of HeLa cells transfected with pcDNA3.0-ING4 increased.CONCLUSION:The gene encoding mouse ING4 and construction of pcDNA3.0-ING4 eukaryotic expression vector were successfully obtained,ING4 could enhance apoptosis in HeLa cells.