The morpholinos targeting gna12, gna13a, and gna13b are previously published38 (link) and were a kind gift from Fang Lin (University of Iowa). Morpholinos were combined and diluted in sterile water supplemented 1:10 with phenol-red dye, and then injected at a final concentration of 1 ng each in a total volume of 1 nl. To control for adverse side-effects from the injection process, we also injected control siblings with an equal volume of phenol-red diluted 1:10 in water. Contstitutively active Rho (rhov14) was kindly provided by Lila Solnica-Krezel and was transcribed and injected as described previously38 (link). The full-length mouse Gpr56 cDNA clone was obtained from Open Biosystems (Clone ID: 3709247), linearized with NotI, transcribed using the mMESSAGE mMACHINE® SP6 ULTRA kit (Ambion), and injected at a final concentration of 200 pg in 2 nl. For transcription of pCS2+_ zf_gpr56, the plasmid was linearized with NotI and also transcribed using the mMESSAGE mMACHINE® SP6 ULTRA kit (Ambion) before injection of a total concentration of 50 pg in 3 nl.
Protocol detail
Morpholino-Mediated Knockdown of Gna12, Gna13a, and Gna13b in Zebrafish
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Corresponding Organization : Hope Center for Neurological Disorders
Other organizations : Washington University in St. Louis, Boston Children's Hospital, Harvard University
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Variable analysis
independent variables
- Gna12 morpholino
- Gna13a morpholino
- Gna13b morpholino
- Constitutively active Rho (rhov14)
- Full-length mouse Gpr56 cDNA
- PCS2+_zf_gpr56
- Not explicitly mentioned
- Phenol-red dye diluted 1:10 in water
- Positive control: Injection of constitutively active Rho (rhov14)
- Negative control: Injection of phenol-red diluted 1:10 in water
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