We have created a stable cell line with CHO DG44 cells. I have a couple of questions.
1. Do we need to continue MTX selection during passaging after clonal selection?
2. Even after clonal selection we see inconsistent protein expression. Does this indicate a polyclonal culture and the further need of clonal selection?
Thanks!
You can use MTX whenever you don't like your expression level. If increasing MTX resistance stops improving your expression level, then it means some cells have de-coupled DHFR from your protein. For example this can happen if your vector randomly breaks between your gene and the DHFR. Yes clone picking can help you fix that, as long as you can find a clone where it hasn't happened yet.
I usually maintain selection in my media for most transgenes as gene silencing can occur if the high level of protein is disadvantageous. The inconsistent levels in a 'clonal' population may be an issue of expression/silencing even though everything is isogenic - try growing them in selective media and see if levels stabilize.
Inconsistencies in protein expression can be a result of other factors (apart from clonal selection related problems) also like medium composition, feeds, trace salts components in both, concentration of source of carbon like glucose and glutamine in your feed and medium, pH of the medium, temperate condition and stage at which you do temp shift (if applicable).
Also the time at which feeds are added and initial cell concentration of inoculum can drastically effect the amount of protein yield you get.
Hope this helped you! cheers!
Jitendra
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