Structure and expression of human globin genes introduced into mouse fibroblasts

• Published in: The Journal of biological chemistry 1981-09, Vol.256 (18), p.9680-9683
• Main Authors: Chen, M J, Nienhuis, A W
• Format: Article
• Language: English
• Subjects: Animals; Bacteriophage lambda - genetics; Cell Transformation, Viral; Cloning, Molecular; DNA Restriction Enzymes; DNA, Recombinant - metabolism; Fibroblasts - metabolism; Genes; Globins - genetics; Humans; Mice; Nucleic Acid Hybridization; Plasmids; Simplexvirus - genetics; Thymidine Kinase - deficiency
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)68816-2

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.