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

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Abstract
An attempt was made to introduce some eukaryotic and prokaryotic DNA sequences into mouse fibroblasts. Purified herpes simplex virus 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 acycloguanosine, and Southern blot hybridization. Two different methods were used 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 2nd method, pBR322 DNA was mixed with herpes tk DNA and presented to mouse cells. TK+ cells were tested for pBR322 sequences by blot hybridization. The frequency of unlinked cotransfer was > 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-MW DNA in the cell. Some of the plasmid molecules may exist as free circular molecules. Using the nonligated cotransfer method, purified human .beta.-globin sequences was introduced into the recipient cells. Transcripts of the human .beta.-globin gene were not present at a level .gtoreq. 10 molecules per cell.