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 rati...

Full description

Saved in:
Bibliographic Details
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
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)68816-2