Hey guys,
Is there anyone who tried to generated stable protein expression with the flp-in T-rex system in MDA-MB-231 cell line? I selected a clone with blasticidin after insertion of the flp-in and verified insertion and location in genome. Then, I inserted my gene of interest and selected with hygromycin. I didn't do clonal selection since I did it with blasticidin. However, I cannot induce the expression of my protein with tetracycline (up to 10ug/mL). I tried with flp-in Trex HEK 293 (Thermofisher) transfected with the same gene of interest and it works perfectly so it seems it is a problem of cell line. Any idea?
Hi Mathieu,
I would like to suggest to select with hygromycin after cotransfection of the recombinase and your gene.
Best,
Piero
Hi Mat,
I performed protein expression manipulations in HEK 293 cells. I found stable increased protein expression by > 2fold by altering cell culture conditions, especially, temp from 37 C to 33 C. Its just an idea!!..if it works for you.
Good Luck Mat!!
Hi Mathieu,
have you already solved your problem?
As a first step to Flp-In T-rex cell lines, you have to create the so called host cell line, which is generated through stable random integration of two plasmids pFRT/lacZeo (introducing the FRT site for targeted integration) and pcDNA6/TR (encoding Tet repressor). The host cell line should be Zeocin- and Blasticidin-resistent. The subsequent co-transfection of the host cell line with pOG44 plasmid (encoding Flp recombinase) and with pcDNA5/FRT/TO+GOI will introduce your target gene into the FRT position. The expression cell line should be now Hygromycin-resistent, but Zeocin-sensitive.
Here you can find more details:
https://www.thermofisher.com/de/de/home/references/protocols/proteins-expression-isolation-and-analysis/protein-expression-protocol/generation-of-isogenic-expression-cell-lines-using-flp-in-t-rex-system.html#prot3
Since you didn't mention Zeocin selection, I was wondering if you have considered to introduce FRT site which is absolutely required for downstream integration?
Good luck!
Hi Mathieu,
Can you please tell me what concentration of the Blasticidin antibiotic you used for the MDA MB-231 for stable cell generation.? Since I need to generate a stable cell line in the same cell line.
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