Stable transfer and expression of exogenous human globin genes in human erythroleukemia (K562) cells.
To study the expression of globin genes in human cells, human epsilon-globin genes were transferred into a K562 cell line, Bos, which synthesizes very low amounts of epsilon-globin mRNA. A plasmid (pSV2neo-epsilon) containing a complete epsilon-globin gene and 2 kilobases (kb) of 5' flanking DNA as well as a neomycin-resistance gene and a simian virus 40 origin of replication was transfected into Bos cells; the compound G418, a neomycin analogue, was used to select transformed cells. The presence of unique bands by DNA restriction analysis shows that 11 of 14 of the G418-resistant clones have at least one copy of an integrated epsilon-globin gene. RNA expression measured by RNA blotting shows significantly more epsilon-globin mRNA sequences than in untransfected Bos cells in 10 of 11 lines; in most lines, epsilon-globin mRNA was additionally increased in the presence of hemin. In two lines, epsilon-globin mRNA expression with hemin was comparable to that of a high epsilon-globin producing cell line, K562 clone 2. The one G418-resistant line without epsilon-globin genes had no epsilon-mRNA expression. The high epsilon-mRNA expression in several of the lines suggests that exogenous epsilon-globin genes with only 2-kb 5' flanking DNA may be sufficient to be appropriately expressed in these homologous erythroid cells. These results have implications for the potential success of transfer of normal human genes to human bone marrow cells as an approach to the treatment of inherited anemias.
D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D004262:DNA Restriction Enzymes; D005796:Genes / Q000187:drug effects; D005914:Globins / Q000235:genetics*; D006418:Heme / Q000494:pharmacology; D006801:Humans; D015470:Leukemia, Myeloid, Acute; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D006418:Heme; D005914:Globins; D004262:DNA Restriction Enzymes