Expression of human tissue-type plasminogen activator in human cells transfected by the activator gene

Abstract

Human tissue-type plasminogen activator (t-PA) gene was expressed in human cell lines, into which the gene was transfected.
HeLa cells were transfected with a plasmid carrying neo, dhfr and t-PA gene which was controlled by β-actin promotor or SV40 early promoter. The clones grown in media with G-418were analyzed for evaluating the activities of both promoters. On regard to the expression of t-PA gene in HeLa cells, β-actin promoter exerted more efficacious than SV40 early promoter.
Difference was observed in average t-PA levels in the cultured media of G-418-resistant clones from three cell lines ( HeLa, WI-38 VA13 and KMS-5) transfected with t-PA-expression plasmid with β-actin promoter. Southern blotanalysis revealed that the copy number of t-PA gene in each clone was likely responsible for the amount of t-PA produced.
G-418-resistant HeLa clone was cultured in media with increasing concentration of methotrexate (MTX). MTX-treatment succeeded to evolve a high t-PA producing clone with high copy number of t-PA gene, and t-PA producing level of a resulting clone was kept constantly for 5 months.