Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading.

Abstract

Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection of mammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418 (Geneticin) or on acquisition of the ability to utilize the salvage pathway of pyrimidine biosynthesis. These loading methods can be employed to generate cell lines that express the gene product of the transfected DNA molecules both transiently and stably. Optimal transfection is observed when the DNA is added to cells in physiological saline lacking divalent cations and containing K+ in place of Na+. DNA molecules 7.1 to 30 kilobases long have been introduced by the scrape loading procedure. In addition, the scrape loading procedure has been employed for cotransfection and subsequent expression of nonselectable genes encoded on DNA molecules added in a mixture with DNA molecules whose expression is selected. Cell lines expressing oncogenes or proteins that are important for regulation of cell growth and division have been obtained by this procedure. The scrape loading procedure is also useful for studies of the cellular changes that occur upon expression of an exogenous gene. As many as 80% of cells scrape loaded with the plasmid pC6, which encodes the simian virus 40 large tumor antigen, contained this protein in the nucleus between 1 and 5 days after transfection. Thus, scrape loading and sonication loading are simple, economical, and reproducible methods for introduction of DNA molecules into adherent and nonadherent cells, and these methods may be useful in the future for experimentation at both fundamental and applied levels.