Transformation of mammalian cells with genes from procaryotes and eucaryotes

Wigler, M. H., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., Axel, R. (1979) Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell, 16 (4). pp. 777-785. ISSN 0092-8674

URL: http://www.ncbi.nlm.nih.gov/pubmed/222468
DOI: 10.1016/0092-8674(79)90093-X

Abstract

We have stably transformed mammalian cells with precisely defined procaryotic and eucaryotic genes for which no selective criteria exist. The addition of a purified viral thymidine kinase (tk) gene to mouse cells lacking this enzyme results in the appearance of stable transformants which can be selected by their ability to grow in HAT. These biochemical transformants may represent a subpopulation of competent cells which are likely to integrate other unlinked genes at frequencies higher than the general population. Co-transformation experiments were therefore performed with the viral tk gene and bacteriophage [Phi]X174, plasmid pBR322 or the cloned chromosomal rabbit [beta]-globin gene sequences. Tk+ transformants were cloned and analyzed for co-transfer of additional DNA sequences by blot hybridization. In this manner, we have identified mouse cell lines which contain multiple copies of 4)X, pBR322 and the rabbit [beta]-globin gene sequences. The [Phi]X co-transformants were studied in greatest detail. The frequency of co-transformation is high: 15 of 16 tk+ transformants contain the [Phi]X sequences. Selective pressure was required to identify co-transformants. From one to more than fifty [Phi]X sequences are integrated into high molecular weight nuclear DNA isolated from independent clones. Analysis of subclones demonstrates that the [Phi]X genotype is stable through many generations in culture. This co-transformation system should allow the introduction and stable integration of virtually any defined gene into cultured cells. Ligation to either viral vectors or selectable biochemical markers is not required.

Item Type: Paper
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene expression
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene transfer
CSHL Authors:
Communities: CSHL labs > Wigler lab
Depositing User: CSHL Librarian
Date: 1979
Date Deposited: 06 Apr 2012 17:53
Last Modified: 07 Feb 2014 20:16
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25956

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