pSP64T-MyoDGR and pSP64T-MyoDER are subclones of S. Hollenberg's original MyoD-GR and MyoD-ER constructs (Hollenberg et al (1993) PNAS 90:8028 ) into pSP64T (Krieg and Melton (1984) Nucleic Acids Res 12:7057-70). When transcribed with SP6 (after linearization with BamHI) RNA encoding an efficiently translated, dexamethasone or b-estradiol inducible MyoD is produced ( Kolm and Sive (1995) Dev. Biol. 171:267). We have found that the GR ligand binding domain gives the tightest regulation in Xenopus embryos. (See the Hormone-Inducible Fusion Protein Construction Information below for more details).
The GR ligand binding domain (lbd) can be isolated from this construct by PCR. Primers which allow in-frame fusion with your protein can be designed with the appropriate restriction sites (for example: GR1 and GR2 which introduce BamHI sites into the protein). We have also successfully used primers with SacI, StuI, PstI, AvaII, and ApaI sites, among others. After ligation into the gene of interest, the insert can be sequenced using the remaining indicated primers (GR3-GR9) to ensure no errors were introduced by PCR. We have not as yet used this approach to clone out the ER ligand binding domain, but it should work similarly.There is a good review of fusion protein construction in : Mattioni et al. (1994) Methods in Cell Biology. vol 43 (Pt A): p 335-352.
- Making Hormone-Inducible Fusion Proteins from the CSH Early Development of Xenopus laevis: A Laboratory Manual
- Dexamethasone and other steriod hormones - Concentrations and technical information
- Cloning Information
- Maps of pSP64T-MyoDGR and pSP64T-MyoDER
- GR cloning and sequencing primers
- GR ligand binding domain sequence
- Sequence map and primer locations in GR lbd
- Restriction sites which are absent from the human GR lbd
- Restriction sites which are unique in the human GR lbd
- Restriction sites which are absent or unique in the human ER lbd
- Papers using hormone-inducible protein in Xenopus: