I am making CRISPR knockouts in MCF10A cells and Blasticidin is used as a selection marker. When I add Blasticidin, some cells die which is expected as they haven't received my sgRNA. However, when I passage the cells (without the drug) only very few attach to the dish. This also varies for different sgRNA constructs designed for different genes. (For example, the cells NF2 and SMAD4 knoclout are fine but the cells transduced with hrosa26 die after passaging.) Hence, I am unable to maintain cells in Blasticidin for continuous passages.
Another problem is that it takes 7-8 days for selection at that concentration. Is it ok to change the media (with Blasticidin) in between (after 3-4 days)?
Furthermore, I confirmed the knockout (knockdown rather) of a SMAD4 gene by Western Blot. However, when I recently passaged these cells in Blasticidin, all the cells died in the presence of Blasticidin. Please note, I used Alfa Aeser Blasticidin before and changed to Sigma Aldrich later. However, both the drugs were used at the same concentration. Could that be a cause of this?
Is blasticidin not a good selectable marker?
Before you dive into more hurdles with selection you should make a Blasticidin kill curve with MCF10A cells. This will help you and you will know what is the optimal Blast concentration for your cells. Blast selection is not as fast as Puro, so 7-8 days seems ok, no need to worry about that. If your cells behave this way it might be that the Blast concentration is simply too high for them.
Hi Nina Kozlova Thanks a lot for you response. Do you mean a kill curve for the cells in which the knockouts have been created? I have performed a kill curve for MCF10A parental cell lines and determined 5ug to be optimum. Thank you!
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts