title: Preparation of high-molecular-weight DNA for use in DNA transfection : secondary transfections for cloning active genes by direct phenotypic selection.
abstract: Calcium phosphate mediated transfection of genomic DNA samples into mammalian recipient cells can be used to isolate specific genes of interest. In theory, for any phenotype for which there is a suitable selection in cell culture, a gene sequence encoding that phenotype can be transferred and will complement the selection. If the donor sequences are derived from a different species of origin, the presence of the donor DNA in the recipient cell can be confirmed by Southern blotting, using a repeat DNA sequence probe from the donor species. Wigler and coworkers (1) found that in each cycle of transfection, the donor sequences represent approx 0.1% of the recipient cell's DNA. Therefore, after a second cycle of transfection using DNA from the first-cycle transfectant, the selected recipient cell will contain essentially a single gene and adjacent sequences.
authors.affiliation: M.D.Anderson Cancer Center, University of Texas, Houstom, TX.
authors.forename: M A
authors.initials: MA
authors.lastname: Tainsky