Targeting tgs1 in Zebrafish Embryos

Control (non-targeting) and tgs1 morpholino oligonucleotides (MOs) were purchased from (GeneTools, LLC) using the tgs1 XM_003197865.5 sequence as reference. MO sequences are reported in Supplementary Table S11. For TGS1 mRNA injection, human TGS1 cDNA (NM_001317902) was cloned into an N-terminal flag-pCS2 + mRNA expression vector. In vitro transcription of 5′-capped mRNAs was performed using the mMACHINE SP6 Transcription Kit (Ambion) following the manufacturer's protocol and as previously described in (33 (link),34 (link)). Embryos from TL/EK wild-type crossings were used to visualize the CaP-MN phenotype. Embryos were injected with the respective dose of MOs or mRNA in an aqueous solution containing 0.05% PhenolRed and 0.05% Rhodamine-Dextran (Sigma-Aldrich). At 6–7 h after injection, embryos were sorted according to homogeneity of the rhodamine fluorescence signal.

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Chen L., Roake C.M., Maccallini P., Bavasso F., Dehghannasiri R., Santonicola P., Mendoza-Ferreira N., Scatolini L., Rizzuti L., Esposito A., Gallotta I., Francia S., Cacchione S., Galati A., Palumbo V., Kobin M.A., Tartaglia G.G., Colantoni A., Proietti G., Wu Y., Hammerschmidt M., De Pittà C., Sales G., Salzman J., Pellizzoni L., Wirth B., Di Schiavi E., Gatti M., Artandi S.E, & Raffa G.D. (2022). TGS1 impacts snRNA 3′-end processing, ameliorates survival motor neuron-dependent neurological phenotypes in vivo and prevents neurodegeneration. Nucleic Acids Research, 50(21), 12400-12424.

Publication 2022

mMACHINE SP6 Transcription Kit PhenolRed Rhodamine-Dextran

Cdna Embryos Fluorescence Human Morpholino oligonucleotides Mrna Phenotype Rhodamine Rhodamine dextran Transcription Vector

Corresponding Organization : Sapienza University of Rome

Other organizations : Institute of Biosciences and Bioresources, University of Cologne, Institute of Genetics and Biophysics, FIRC Institute of Molecular Oncology, Istituto di Genetica Molecolare, Italian Institute of Technology, Howard Hughes Medical Institute, University of Padua, Columbia University, University Hospital Cologne, Institute of Molecular Biology and Pathology

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Variable analysis

independent variables

Control (non-targeting) morpholino oligonucleotides (MOs)
Tgs1 morpholino oligonucleotides (MOs)
Human TGS1 cDNA (NM_001317902)

dependent variables

CaP-MN phenotype

control variables

Embryos from TL/EK wild-type crossings

controls

Control (non-targeting) morpholino oligonucleotides (MOs)

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