Targeted disruption of a human interferon-inducible gene detected by secretion of human growth hormone.
A new method is described for the sib-selection of 'targeted' mammalian cells that have undergone homologous recombination (HR) with a transfected DNA construct. This method has been used to disrupt the 6-16 gene, an interferon (IFN)-inducible gene of unknown function, in two different human cell lines. Disruption was caused by integration of a targeting construct containing a promoterless gene for human growth hormone (hGH) which was expressed after HR with the 6-16 gene. Homologous recombinants were detected in pools of non-homologous recombinants by the appearance of hGH in the growth medium after the addition of IFN. Secondary and tertiary rounds of hGH assays were used to sib-select 9 homologous recombinants that were shown to have 1, 2 or 3 copies of the targeting construct integrated at the 6-16 locus. The method, which should be applicable to other transcribed targets, provides an alternative to selection methods, and offers advantages over other screening methods in being simple, rapid, sensitive and reliable.
Itzhaki
J E
JE
Department of Biochemistry, University of Oxford, UK.
Porter
A C
AC
D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D005354:Fibrosarcoma; D005786:Gene Expression Regulation; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D013006:Growth Hormone; D004247:DNA; D007372:Interferons