Construction of ICAT interference lentivirus expression vector and establishment of stable HL 60 cell lin / 国际检验医学杂志

ABSTRACT

Objective To construct the ICAT gene interference lentivirus expression vector targeting and to establish stable transfected cell line HL60 .Methods The interference sequence targeted at human ICAT gene was designed and synthesized ,after annealing ,which was connected to PGLV3 interference vector and with PG‐p1‐VSVG ,PG‐p1‐REV ,PG‐p1‐RRE were co‐transfected into 293T cells The lentivirus particles were packaged and generated .The virus titer was detected .HL60 cells were transfected for establishing the stable cell line ;RT‐PCR and Western Blot techniques were used to detect ICAT gene and protein expression in sta‐ble HL60 cells ,then the results were compared with those in the control group .Results The lentivirus expression vector targeted at ICAT was successfully constructed and the virus titer was 2 × 108 U/mL .Stable transfected HL60 cell line was established .The effective interference verification revealed that shICAT could significantly reduce the mRNA and protein level of ICAT ( P<0 .001) .Conclusion The shRNA lentiviral expression vector of ICAT gene is successfully constructed and the HL 60 cell line stably interfering ICAT expression is established .