Hi all,
I generated regulatable knockdown 4T1 mouse cell lines with TRIPZ doxyclycline-inducible shRNA vectors (scrambled sequence and targeted one). Unfortunalety, doxycyline (doxy) leads to tumor regression by itself even on wild type 4T1 (4T1_WT).
Effect that is even more striking in vivo with 2g/L of doxy + 10% of sucrose. I waited 8 days after the tumor cells (scrambled, targeted and 4T1_WT ones) inoculation to add doxy in the water but the tumor start regressing after 13 days of doxy exposure. Some of the mice are even free from 4T1 tumors (scrambled and WT ones)...clearly, doxy can control 4T1-tumor growth.
Therefore, do you have any alternative to induce the promoter without killing the 4T1 cells? I mean, is it possible to use a tetracycline "inactive form" that will activate the promoter while preventing 4T1 cell death? Can we use a lower dose of doxy in vivo? I saw a paper where they used 0.2g/L of doxy in 10% of sucrose and same thing, 4T1 tumor regressed...I am open to any suggestions.
Thanks for your help
Dear Claire Isabelle Vanpouille-Box,
in my view the amount of Dox you use for the cell culture experiment could be reduced by a factor of 10. If you look on original publications using improved versions of the reverse transactivator (such as Urlinger et al. PNAS 2000, Figure 3), you will realize that 100 ng/µl is enough for maximal induction (and even 50ng should be enough). This certainly depends also on the type of cells which are used, but I would try the experiment with this lowered concentration. An alternative approach could be the use of 9-tert-Butyl Doxycycline HCl:
https://www.echelon-inc.com/index.php?module=Products&func=detail&id=855
(for references see link). This compound hast been used in mice for induction of the rtTA-M2 version and should be functional with your rtTA3, too. However, if this inducer has an effect on tumor growth I do not know.
best regards,
Kai
Thank you Kai,
I will definitively titrate the doxy and if the KO is not strong enough, I will use the tetracycline analog you mentioned.
Again, thanks for your help !
Claire, I just saw this so please excuse the delayed response. We have 9TB Dox HCl in our catalog and its used in cases where expression is needed in lipophilic cellular environs-ie the brain and lungs- and it is non-antibacterial.
Contact me at Echelon if you need further information on uits use in the Tet On/Off system
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