Construction and identification of shRNA lentiviral vectors targeting histone lysine transferase KAT6B gene / 实用肿瘤学杂志

ABSTRACT

Objective The aims of this study were to construct short hairpin RNA(shRNA)lentiviral vector in breast cancer T47D cells,to carry out RNA interference on lysine acetyltransferase 6B(KAT6B/MORF)gene,to down-regulate its expression and to explore its function.Methods Two pairs of single-stranded short hairpin RNA(shRNA5 and shRNA8)and the corresponding control sequences(Scramble5 and Scramble8)were synthesized based on the CDS of KAT6B gene.Polymerase chain reaction(PCR) was used to amplify double-stranded and ligated with the entry vector(pENTR/pSM2(CMV)GFP),which were subjected to a doub-le digestion(EcoRl and Xhol)linearization and homologous recombination with the entry vector(pENTR/pSM2(CMV)GFP)to obtain an entry clone containing the desired fragment.The target fragment was recombined onto the target vector(pLenti x1 puro DEST)via the LR cloning reaction of the Gateway system.The lentiviral packaging plasmids were co-transfected into HEK-293T cells with two pairs of target plasmids. The supernatant of HEK -293T cells was collected and transformed into T47D cells. The expression of KAT6B protein was detected in T47D cells by Western blot.Results The single colony obtained from the transformation was identi-fied by sequencing,which was consistent with the target sequence,indicating that the lentiviral vector had been successfully construc-ted.The expression of KAT6B protein was significantly lower in the shRNA KAT6B group than that in the control group,which indica-ted that the constructed gene silencing vector could play a role in the KAT6B gene in T47D cells.Conclusion The shRNA lentiviral gene silencing vectors of KAT6B were constructed and identified in T47D cells,which indicated that the foundation for further study