Selection of transfected mammalian cells.
Analysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 10(4) cells in a transfection will stably integrate DNA (the efficiency can vary depending on the cell type). Therefore, a dominant, selectable marker is used to permit isolation of stable transfectants. In the first part of this unit, the procedure for determining selection conditions and the resulting stable transfection is presented and the most commonly used selectable markers are discussed. The second protocol includes conditions for thirteen markers commonly used for selection of mammalian cells. A third protocols describes selection of transfected cells from the total population soon after transfection with plasmids that express both the gene of interest and a selection tag. Optimization of transfection conditions can be facilitated by a simple staining assay detailed in a .
Mortensen
Richard
R
Brigham and Women's Hospital, Boston, Massachusetts, USA.
Chesnut
Jonathan D
JD
Hoeffler
James P
JP
Kingston
Robert E
RE
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D005784:Gene Amplification; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics; D018189:Immunomagnetic Separation; D008322:Mammals; D008863:Microspheres; D012641:Selection, Genetic; D014162:Transfection / Q000379:methods*
D003470:Culture Media; D005819:Genetic Markers