Making a Stable Cell Line Expressing Your Favorite Gene

  1. Make a death curve. This is to determine the minimal concentration of antibiotic that kills cells that do not have the resistance gene.
  2. transfect the cells with the plasmid containing the gene that you want to express in the cells (see Amaxa protocol).
  3. incubate the cells for about 48 hours after transfection so that the gene is expressed, and the resistence gene is expressed.
  4. add the appropriate dose of antibiotic (this is the selection step). The cells that do not contain the resistance gene (i.e. that were not successfully transfected) will die. Change the medium every 2 or 3 days to remove dead cells. Replace medium with fresh medium containing the antibiotic.
  5. split the cells as needed. Use medium with antibiotic. Make some coverslips to see if the cells are expressing the gene of interest.

After ~2 weeks any cell without the transgene and selectable marker should have died. The remaining cells are a mixed population – different cells might express different levels of the transgene. These can be used for preliminary experiments.

Making a clonal cell line

  • A Clonal cell line contains cells that are derived from a single cell, and thus are a clone. They will all be genetically identical; this can be very helpful for experiments.
  • a. Trypsinize your 'mixed population' of cells; keep some growing in a flask and freeze some in case your cloning does not work the first time and you need to repeat the procedure.
  • b. take 50ul of the trypsinized cells, dilute again into 1 ml medium. Add 10 -150 ul of this dilute solution of cells to 100mm dishes containing growth medium. Look under the tissue culture scope. The cells should be very dilute – one or just a few cells per field of view. If they are too dense or dilute, adjust accordingly. You want each individual cell to land on the plastic dish and grow into a colony of cells that is separate from other colonies on the dish.
  • c. wait about 1 week for the colonies to grow.
  • d.isolate individual colonies. These are potential clonal cell lines.
  • Toss any cell lines that are not healthy or have too much or too little fluorescence.