I infected MEF cells simultaneously with lentiviral vectors containing hygro and puro resistance and waited 2 days before adding hygromycin and puromycin together. However, after 1-2 days they all died so I'm thinking whether there's a additive selection effect if two or more antibiotics are added together.
Hi Jan,
I used to infect mESCs simultaneously with lentiviral vectors carrying hygro and puromycin resistance cassette. Double selection is not an issue and worked fine, so I guess there are not Puromycin/Hygromycin additive effects that prevent their combined use.
Regarding MEF I never performed double transduction but I can tell you that they did not like very much Hygromycin selection. Unless transduction efficiency was higher than 40/50%, cells were starting to die anyway. And this is just regarding Hygromycin. With Puromycin I have never experienced any issue.
To rule out trivial problems I would suggest to check if your lentivirus are working individually. You can still perform double transduction but try to split the cells and check drug resistance separately. In this way you can assess if one (and which) virus is not working.
Should both viral constructs work, you may want to perform drug selection sequentially. In this case I would start with Hygromycin for 4-5 days and than Puromyicin (which is generally faster in killing non-transduced cells) for additional 2-3 days in absence of Hygromycin. Once the selection is concluded (your untransduced controls are dead) you can safely remove drugs and expand your cells as much as possible, the whole process could last 1-2 passages.
Hope it helps, please let me know if you have more questions
Best
Francesco
Thank you for the comprehensive answer! I will try to do the sequential selection like you suggested and also save half of the flask in case something bad happens again.
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