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Regulatable protein fusions can be made by attaching the hormone binding domain of the estrogen receptor to the N or C-terminus of a protein of interest. Activation of such ER-fusion proteins in vivo can be achieved by daily administration of a synthetic ligand, 4-hydroxytamoxifen (4OHT), via intraperitoneal (i.p.) injection.

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[Bio101] Activation of ER-regulated Fusion Proteins In Vivo

Molecular Biology > Protein > Expression

[Abstract] Regulatable protein fusions can be made by attaching the hormone binding domain of the estrogen receptor to the N or C-terminus of a protein of interest. Activation of such ER-fusion proteins in vivo can be achieved by daily administration of a synthetic ligand, 4-hydroxytamoxifen (4OHT), via intraperitoneal (i.p.) injection.

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.


How to cite this protocol: Reuter, J. (2011). Activation of ER-regulated Fusion Proteins In Vivo. Bio-protocol Bio101: e164. DOI: 10.21769/BioProtoc.164; Full Text



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