Mmessage mmachine sp6 ultra kit

The morpholinos targeting gna12, gna13a, and gna13b are previously published^{38 (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 previously^{38 (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.

TALEN constructs were assembled by the Hope Center for Neurological Disorders at Washington University using the Golden Gate TALEN assembly protocol ^{53 (link)}. The repeat variable diresidue sequences for the left and right TALENs were as follows: Left: NN NG NG NG HD NG HD HD HD NG NI NN NI HD NI NI NG and Right: NI NI HD NG NN NG NI NG HD NN HD NI NN HD HD NI NG HD NI NG. Synthetic TALEN mRNA was then transcribed using the mMESSAGE mMACHINE® SP6 ULTRA kit (Ambion), and RNA from each arm was combined and injected into 1-cell stage WT embryos (AB) at 100 pg quantities. To recover germ-line transmitted mutations, injected founders (F0s) were raised to adulthood, outcrossed to WT AB partners, and genomic DNA was extracted from individual F1 embryos for PCR amplification and restriction digest analysis of the targeted region. Genomic DNA from F1 embryos that showed disruption of the target site was then cloned using the TOPO® TA Cloning® kit (Invitrogen) and sequenced using Sanger sequencing.