Plasmid Constructs for Studying Neurodegenerative Diseases

M337V human TDP-43 cDNA was cloned into pCMV6-AC-GFP (Origene) to generate a mutant TDP-43 construct C-terminally tagged with tGFP18 (link). HA-NFAT1(1-460)-GFP was a gift from Anjana Rao (Addgene plasmid #11107). Huntingtin protein-encoding constructs, pT-Rex-Htt46Q-Tc1-mCherry and pT-Rex-Htt25Q-Tc1-mCherry, were gifts from Dr. Danny Hatters (University of Melbourne, Australia)19 (link). pEGFP-SOD1, pEGFP-SOD1-A4V, pEGFP-SOD1-G93A were gifts from Dr. Brad Turner (The Florey Institute of Neuroscience and Mental Health, Australia)20 (link). SOD1-tdTomato constructs were created by replacing the GFP sequences in the SOD1-GFP plasmids with tdTomato (Genscript)18 (link). The expression vector pCMV6-AC-GFP containing FUS was obtained from Origene and site directed mutagenesis was performed by Genscript to create the R495X mutant18 (link). Plasmids containing sequences encoding wild type (WT) and temperature-sensitive double mutant (R188Q, R261Q; DM) firefly luciferase-eGFP were a gift from Prof Ulrich Hartl (Max Plank Institute, Germany)21 and were recloned into pcDNA4/TO (Life Technologies) for mammalian cell transfection.

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Whiten D.R., San Gil R., McAlary L., Yerbury J.J., Ecroyd H, & Wilson M.R. (2016). Rapid flow cytometric measurement of protein inclusions and nuclear trafficking. Scientific Reports, 6, 31138.

Publication 2016

pCMV6-AC-GFP

Cdna Cell Firefly luciferase Gifts Human Huntingtin protein Mammalian Mental health Plasmids Site directed mutagenesis Tdp 43 Tdtomato Transfection Vector

Corresponding Organization :

Other organizations : University of Wollongong, Illawarra Health and Medical Research Institute

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Variable analysis

independent variables

M337V human TDP-43 cDNA
HA-NFAT1(1-460)-GFP
Huntingtin protein-encoding constructs (pT-Rex-Htt46Q-Tc1-mCherry and pT-Rex-Htt25Q-Tc1-mCherry)
PEGFP-SOD1, pEGFP-SOD1-A4V, pEGFP-SOD1-G93A
SOD1-tdTomato constructs
PCMV6-AC-GFP containing FUS and R495X mutant
Plasmids containing wild type (WT) and temperature-sensitive double mutant (R188Q, R261Q; DM) firefly luciferase-eGFP

dependent variables

Outcomes or responses resulting from the expression of the various constructs

control variables

PCMV6-AC-GFP (Origene) vector
PcDNA4/TO (Life Technologies) vector

positive controls

Wild type (WT) firefly luciferase-eGFP plasmid

negative controls

Temperature-sensitive double mutant (R188Q, R261Q; DM) firefly luciferase-eGFP plasmid

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