Introduction and expression of a fetal human globin gene in mouse fibroblasts

Abstract

An 8.5-kilobase segment of cloned human DNA including the complete G gamma-globin gene was introduced into LMTK- cells by the calcium phosphate precipitation method in the presence or absence of carrier DNA. Transfectants containing one or more copies of intact G gamma-globin genes were obtained either by ligation of the human DNA segment to a plasmid containing the herpes simplex virus thymidine kinase gene or by nonligated cotransfer. The integrity of the integrated gamma-globin gene was established by Southern blotting experiments. Expression of the herpes simplex virus thymidine kinase and human gamma-globin genes was evaluated by Northern blotting and solution hybridization. Of 23 transfectants analyzed, 21 produced a 9S gamma-globin RNA migrating like authentic gamma-globin mRNA on denaturing agarose gels. The gamma-globin RNA is polyadenylated and present in the cytoplasm of the transfected cells; it accumulates to a level 10 times that of thymidine kinase mRNA, or about 5 to 50 molecules per transfected cell. By using plasmids in which the gamma-gene is inserted in either transcriptional orientation with respect to the thymidine kinase gene, it was possible to show that transcription occurred from the gamma-gene promoter.