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. Author manuscript; available in PMC: 2010 Jul 16.
Published in final edited form as: Neuron. 2009 Jul 16;63(1):27–39. doi: 10.1016/j.neuron.2009.06.014

Figure 1. Generation of transgenic mice with inducible expression of HA-hM3Dq.

Figure 1

A) Pronuclear injection of murine oocytes with the 2.36 kb XhoI restriction digest fragment containing HA-hM3Dq downstream of the Ptight TRE promoter produced a tet-responsive mouse line. When crossed with a CaMKIIα-tTA tet-driver line, tTA protein, produced TRE promoter to activate transcription of HA-hM3Dq; tTA binding to TRE is inhibited by doxycycline. B) Ethidium bromide-stained agarose gel of DNA amplified from tail clips of a single-transgenic (TRE-hM3Dq) mouse (Lane 1), water (Lane 2), and a double-transgenic mouse (Lane 3) reveals presence of CaMKIIα-tTA transgene (450 bp), TRE-hM3Dq transgene (250 bp), and murine genomic DNA control band (200 bp). C) Immunoprecipitation followed by immunoblot does not detect any transgene in single-transgenic mice (TRE-hM3Dq transgene only, Lane 1) or double transgenic (hM3Dq) mice maintained on 200 mg/kg doxycycline (Lane 3), in contrast to hM3Dq mice maintained on normal chow in which HA protein is detectable (Lane 2). β-actin was detected as a loading control. Mouse brains (with cerebellum removed) were homogenized and the membrane-containing fraction was isolated through differential centrifugation. HA-affinity matrix immunoprecipitated HA-tagged proteins which were then separated by SDS-PAGE and detected by Western blot with anti-HA antibody.