Keratinocyte transient transfections.
The goal of this chapter is to give the reader a concise and easy-to-follow guide to proven transient transfection techniques for primary strains and continuous lines of keratinocytes. The emphasis is on readily available and inexpensive resources that also allow for repeatability and adaptability to scale, experimental conditions, and sample replicates. In addition, basic cell culture techniques necessary to achieve optimal transfection results are provided. Although specified for keratinocytes, these techniques also work well for simple epithelial lines and common laboratory lines, such as COS-7 and CV-1. The standard precipitation protocol is suitable for many experimental designs, and the poly-L-ornithine protocol is intended for situations where calcium is contraindicated. The extended notes section provides necessary technical details to guide the novice user and reminders to the experienced scientist that success with the protocol will depend on several quantitative and qualitative factors including promoter strength, cell culture practices and plasmid isolation.
Flores
Anthony M
AM
Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, USA.
Aneskievich
Brian J
BJ
D002130:Calcium Phosphates / Q000737:chemistry*; D002478:Cells, Cultured; D017930:Genes, Reporter; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry*; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D010455:Peptides; C008973:polyornithine; C020243:calcium phosphate