Activation of ER-regulated Fusion Proteins In Vivo   

Abstract

Materials and Reagents

1. Corn oil (Sigma-Aldrich, catalog number: C8267 )
   
2. 200-proof ethanol (Sigma-Aldrich, catalog number: E7023 )
   
3. 4OHT (Sigma-Aldrich, catalog number: H7904 )
   

Equipment

1. Sonic dismembrator (Thermo Fisher Scientific, model: 100 )
   
2. Centrifuges
   
3. Hybridization oven
   

Procedure

1. Dissolve the 4OHT in ethanol (67 mg/ml) by heating to 48 °C for 10 min.
   
2. Mix 4OHT solution with pre-heated (48 °C) sterile corn oil (7.5 mg/ml).
   
3. Sonicate 20 sec, at 5 volts then place on ice for 1 min. Repeat 3 times.
   
4. Aliquot solution to avoid freeze/thaw cycles and store at -20 °C for up to 1 month.
   
5. Prior to i.p. injection, 4OHT should be thawed, sonicated again (3x, 20 sec, at 3 volts) and injected immediately.
   Note: If sustained activity is required daily injections must be performed.
   

References

1. Metzger, D. and Chambon, P. (2001). Site- and time-specific gene targeting in the mouse. Methods 24(1): 71-80.
   
2. Reuter, J. A. and Khavari, P. A. (2006). Use of conditionally active ras fusion proteins to study epidermal growth, differentiation, and neoplasia. Methods Enzymol 407: 691-702.