Prior to the introduction of transgene constructs into animals, it is useful first to determine the presence of a functional promoter and transcription unit. This can be achieved by transient transfection of the construct into heterologous cell lines and subsequent measurement of reporter enzyme activity in cell extracts. Following ligation of the chosen promotor sequence to the reporter enzyme coding region in a suitable plasmid vector, DNA is purified and transfected into an appropriate cell line. Popular methods of transfection are the calcium phosphate method, electroporation, and lipofection. The latter method, although relatively expensive, provides a quick and reliable method of transfection and is the method described in this chapter. This method of transfection relies on a positively charged lipid, DOTMA (N [1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), which forms liposomes that interact with DNA and RNA and carries them into mammalian cells in culture (1). DOTMA is available commercially and has been shown to be suitable for a variety of cell lines.
KeywordsTissue Culture Dish Transcription Unit Chloramphenicol Acetyl Transferase Functional Promoter Polystyrene Tube
- 2.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1987) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar