Stable transfer of genes into 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, so a dominant selectable marker is used to permit isolation of stable transfectants. Appropriate selection conditions for the parental cell line are first determined, and the gene to be studied is then transfected into that cell line with a gene that expresses a selectable marker. The cells are allowed to grow under selection for approximately 10 doublings before individual colonies are picked and expanded into cell lines. This unit includes conditions for six markers commonly used for selection of mammalian cells. An alternative, specialized procedure for transferring immunoglobulin chains into lymphoid cells is presented in Unit 10.17B.