Preparation of High-Molecular-Weight DNA for Use in DNA Transfection : Secondary Transfections for Cloning Active Genes by Direct Phenotypic Selection

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.