Cotransfer of circular and linear prokaryotic and eukaryotic DNA sequences into mouse cells.

N. Hsiung, H. Warrick, J. K. deRiel, Dorothy Tuan Lo, B. G. Forget, A. Skoultchi, R. Kucherlapati

Research output: Contribution to journalArticle

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Abstract

We have attempted to introduce some eukaryotic and prokaryotic DNA sequences into mouse fibroblasts. Purified herpes thymidine kinase gene (tk) was introduced into mouse cells. The presence of the herpes tk gene was established by gel electrophoresis, sensitivity to the purine analog acyloguanosine, and Southern blot hybridization. We utilized two different methods to introduce nonselectable markers into mouse cells. Bacterial plasmid pBR322 was ligated to herpes tk and used for transfection. All cells that were TK+ also contained the plasmid sequences. In the second method, pBR322 DNA was mixed with herpes tk DNA and presented to mouse cells. TK+ cells were tested for pBR322 sequences by blot hydridization. The frequency of unlinked cotransfer was greater than 40%. When the circular plasmid containing pBR322 and tk was used for transfection, each of the resulting transfectants acquired several copies of the plasmid. Most of the copies were associated with high molecular weight DNA in the cell. In addition, we found that some of the plasmid molecules may exist as free circular molecules. Using the nonligated cotransfer method, we introduced purified human beta-globin sequences into the recipient cells. We were unable to detect any transcripts of the human beta-globin gene at a level greater than or equal to 10 molecules per cell.

Original languageEnglish (US)
Pages (from-to)4852-4856
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume77
Issue number8
DOIs
StatePublished - Jan 1 1980

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Thymidine Kinase
Plasmids
Genes
beta-Globins
Transfection
DNA
Southern Blotting
Electrophoresis
Fibroblasts
Molecular Weight
Gels

ASJC Scopus subject areas

  • General

Cite this

Cotransfer of circular and linear prokaryotic and eukaryotic DNA sequences into mouse cells. / Hsiung, N.; Warrick, H.; deRiel, J. K.; Tuan Lo, Dorothy; Forget, B. G.; Skoultchi, A.; Kucherlapati, R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 77, No. 8, 01.01.1980, p. 4852-4856.

Research output: Contribution to journalArticle

Hsiung, N. ; Warrick, H. ; deRiel, J. K. ; Tuan Lo, Dorothy ; Forget, B. G. ; Skoultchi, A. ; Kucherlapati, R. / Cotransfer of circular and linear prokaryotic and eukaryotic DNA sequences into mouse cells. In: Proceedings of the National Academy of Sciences of the United States of America. 1980 ; Vol. 77, No. 8. pp. 4852-4856.
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