Chat
 
 
 
Home |  RNAi | MicroRNA-adapted Retroviral Ve | Regulated shRNAmir expression

Regulated shRNAmir expression
in vitro and in vivo
Expression of shRNAmir cassettes from the TRE-CMV promoter in TMP allows regulated gene knockdown in tet-on or tet-off configurations. The expression of shRNAmir toTrp53 was tightly and stably regulated both in culture and in mice by changing the level of doxycycline. This tight regulation also allowed switching of primary cells between active proliferation and senescence in culture and to promote or inhibit tumor growth in vivo by simply adding or removing antibiotic (Dickens et al 2005).  

Regulated shRNAmir expression from TMP-Rb.670 using a Tet-off system
Hela cells stably expressing the tetracycline transactivator protein tTA (tetracycline-off) were transduced with TMP-RB.670 at low MOI (<10% efficiency) and expanded in the absence of doxycycline. Single cell clones isolated from the population of transduced cells showed excellent RB1 knockdown showing that the TRE-CMV promoter can effectively drive shRNAmir expression at low copy. For all clones showing RB1 knockdown in the absence of doxycycline, RB1 levels returned to normal after approximately one week of treatment with doxycycline. Also in all cases GFP and RB1 levels were inversely corelated showing that in this system GFP expression serves as a marker for shRNA production.

Figure 1 (a) RB1 expression in homogenous cultures derived from single cell clones of Hela-tTA cells  infected at single copy with TMP-RB.670. Cells were cultured in normal doxycycline-free medium before collection. Con is uninfected Hela-tTA cells. (b) RB1 expression in Hela-tTa clone RB.670C cells over time in response to shifting into or out of doxycycline (Dox). Cells were cultured without Dox (left panel) or in 100ng/ml Dox (right panel) for 8 days before being shifted to 100ng/ml Dox or Dox-free medium.


Control of tumor growth in vivo by regulating Trp53 knock-down
Dickens et al  examined the requirement for Trp53 inactivation in tumors derived from WtT-p53.1224/Kras MEFs. These transformed cells were subcutaneously injected into the flanks of nude mice where they formed visible, rapidly growing and strongly GFP positive tumors after ~ 2 weeks. In mice treated with doxycycline in their drinking water, tumor GFP intensity was markedly and rapidly diminished compared with untreated mice and after 4 days most tumors were GFP-negative. Notably, tumor growth slowed upon doxycycline treatment and mice treated with Dox for 10 days showed tumor regression and eventually became tumor-free. See Dickens et al 2005 for more details.

a)b)

Figure 2: Regulated Trp53 knockdown in tumors. (a) GFP and standard imaging of representative tumor-bearing mice, with doxycycline treatment commencing at day 0 (lower panels). Untreated controls are shown (upper panels). (b) Representative tumor growth curves for WtT-p53.1224/Kras tumors in an untreated mouse (open squares) or a mouse treated for 10 days with doxycycline (Dox; filled circles indicate doxycycline treatment) commencing at day 0.


References:
Dickens NG et al (2005) "Probing tumor phenotypes using stable and regulated synthetic microRNA precursors" Nature Genetics Vol 37, No 11 1289-1295.

1-888-412-2225 | My Account | Privacy Policy | Terms & Conditions
© 2005-2007 Open Biosystems All Rights Reserved