I am trying to make a stable cell line that expresses my protein of interest. I have the gene of my protein in a vector that provides puromycin resistant, but I am not sure what protocol to use. Should I just transfect the DNA and then do the selection with the adequate concentration of antibiotic?
Dear Edna,
Here are my protocols:
Day 0: Seed target cells in 24-well plates. The number of seeding cells differs according to the cell proliferation rate.
Day 1: Transfect cells with virus. For polybrene accessible cells, mix the culture medium with proper concentrations of polybrene. Replace the medium completely with 0.5 ml polybrene-containing medium. For polybrene sensitive cells, this step can be skipped.
Day 2: Refresh the culture medium 24 hours post infection.
Day 3: Change to fresh medium with puromycin 48 hours post infection. The recommended concentration of puromycin ranges from 1 to 10μg/ml according to cell lines.
Additionally, the killing dose of puromycin vary from cell lines, 0.5-5 ug/mL in common. First, I suggest you use a couple of wells to titer the concentration for each cell line to find the optimal dose. The cells may die after screening for 2-3 days. Second, it works well to screen with puromycin when passaging.
Genemedi got a rich experience in lentivirus production and transfection, you could find more information on https://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
Do a dose test to see which lowest dose is capable of killing ALL untransfected 293T cells in 2-3 days and choose that dose. Use that dose for selecting transfected cells. See this paper which is on 293T cells
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3140840/pdf/10616_2011_Article_9357.pdf
Good information. I am working on creating a stable cell line infected with a GFP lentivirus construct. Usually 1 ug/ml works but I have been increasing it to see when selection is complete.
Dear Edna,
Here are my protocols:
Day 0: Seed target cells in 24-well plates. The number of seeding cells differs according to the cell proliferation rate.
Day 1: Transfect cells with virus. For polybrene accessible cells, mix the culture medium with proper concentrations of polybrene. Replace the medium completely with 0.5 ml polybrene-containing medium. For polybrene sensitive cells, this step can be skipped.
Day 2: Refresh the culture medium 24 hours post infection.
Day 3: Change to fresh medium with puromycin 48 hours post infection. The recommended concentration of puromycin ranges from 1 to 10μg/ml according to cell lines.
Additionally, the killing dose of puromycin vary from cell lines, 0.5-5 ug/mL in common. First, I suggest you use a couple of wells to titer the concentration for each cell line to find the optimal dose. The cells may die after screening for 2-3 days. Second, it works well to screen with puromycin when passaging.
Genemedi got a rich experience in lentivirus production and transfection, you could find more information on https://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
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