Mmessage machine sp6 transcription kit

Plasmids containing the cDNA coding for CAAX-GFP (membrane marker), H2B-RFP (nuclei marker) and the dominant negative form of Suppressor of Hairless (DN-Su(H)) [32 (link)] have been linearized and mRNAs have been synthesized using the mMessage Machine SP6 transcription kit from Ambion. mRNAs were injected either at the one-cell stage for ubiquitous expression or into one blastomere between the 16- to 64-cell stages for mosaic labelling of neural progenitor cells. The mRNA was injected at 10–150 pg per embryo and did not exceed half the volume of a cell.

The pCS2+ BiFC constructs were linearized with Not1, and mRNA was transcribed using a mMessage Machine Sp6 Transcription kit (Thermo Fischer Scientific) according to manufacturer’s instructions. Microinjections were performed using a Picospritzer II (Parker Instruments) into the 1–2 cell stage embryo as previously demonstrated [38 , 67 –69 ]. For TDP-43 fading experiments a 1:1 ratio of complementary mRNA (20 pg) was injected with 100 pg H2B-mCerulean3 mRNA (n = 3). In control experiments assessing the nonspecific fluorescence reconstitution, an adjusted 1:5 ratio of fluorophore fragment control mRNA (CC155) and TDP-43-fused fragment (VN155-TDP-43) was used to adjust for different construct lengths (CC155 and VN155-TDP-43 are encoded by 303 and 1755 base pairs, respectively, three independent experiments, n = 36 per experiment). For Fus experiments, 100 pg Fus (or 50 pg of CC155-mKate2 in control experiments) and 400 pg H2B-mCerulean3 mRNA was injected (n = 3). Images were taken on either a M165FC fluorescent stereomicroscope (Leica) or TCS SP5 Confocal Microscope (Leica) with settings kept constant within experiments.