Structure and expression of human globin genes introduced into mouse fibroblasts.
The human delta- and beta-globin genes, contained in a recombinant bacteriophage (lambda H beta G1), were introduced into mouse fibroblasts by cotransformation with a plasmid (chi 1) containing the herpes simplex thymidine kinase gene using the calcium phosphate precipitation technique. A molar ratio of lambda H eta G1 to chi 1 DNA of 3:1 was used. Four of the eleven stable transformants obtained contained intact delta- and beta-globin genes as determined by Southern blot analysis. To assess methylation in the segment of human DNA introduced into mouse cells, digestion with Hpa II or Msp I alone or with a second restriction enzyme was performed. The sites examined near the human delta- and beta-globin genes in transformed cells were not methylated. RNA extracted from the transformed cells was analyzed by RNA-cDNA hybridization; no more than 100 copies of human beta-globin mRNA/cell were found. Although hypomethylation of sites surrounding expressed globin genes in erythroid cells has been described, this property is not sufficient to ensure a high level of expression in fibroblasts.
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005796:Genes; D005914:Globins / Q000235:genetics*; D006801:Humans; D051379:Mice; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D018139:Simplexvirus / Q000235:genetics; D013937:Thymidine Kinase / Q000172:deficiency
D004274:DNA, Recombinant; D005914:Globins; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes