Generation of Diverse DPR Fusion Proteins

To generate myc-BioID-DPR_x100 plasmids, sequences encoding poly-GR, -PR, and -GA (with alternating codons to avoid RNA repeats) were cloned into the pcDNA3.1 mycBioID plasmid (Addgene plasmid #35700) [80 (link)] by replacing the TDP-CTF BamHI/HindIII fragment in pcDNA3.1 mycBioID TDP-CTF [12 (link)]. Resulting plasmids encode myc-BioID-(GGGS)_x3-(DPR)_x100 fusion proteins. EGFP and mCherry-tagged DPR _x100 plasmids were generated by replacing the BioID NdeI/BamHI fragment in myc-BioID-(GGGS)_x3-(DPR)_x100 with EGFP or mCherry. 2xHA-poly-GA was cloned by replacing the PvuI/BamHI fragment of EGFP in EGFP-poly-GA with a double HA tag (MGYPYDVPDYAGGYPYDVPDYA). Expression constructs for EGFP-tagged SQSTM1, UBXN6, VCP, and HSPA8 (accession numbers #38277 [35 (link)], #86464 [75 (link)], # 23971 [55 (link)], #19487 [30 (link)], respectively), as well as mCherry-Calreticulin-N-16 (#55006) were obtained from Addgene. EGFP-tagged Calreticulin was cloned by replacing mCherry AgeI/NotI fragment in mCherry-Calreticulin-N-16 with EGFP from the pEGFP-N1 vector (Clontech).

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Liu F., Morderer D., Wren M.C., Vettleson-Trutza S.A., Wang Y., Rabichow B.E., Salemi M.R., Phinney B.S., Oskarsson B., Dickson D.W, & Rossoll W. (2022). Proximity proteomics of C9orf72 dipeptide repeat proteins identifies molecular chaperones as modifiers of poly-GA aggregation. Acta Neuropathologica Communications, 10, 22.

Publication 2022

pcDNA3.1 mycBioID plasmid pEGFP-N1 vector

Calreticulin Codons Ctf 1 Plasmids Poly a Proteins Vector

Corresponding Organization :

Other organizations : Mayo Clinic in Florida, University of California, Davis, WinnMed

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Variable analysis

independent variables

Sequences encoding poly-GR, -PR, and -GA (with alternating codons to avoid RNA repeats)

dependent variables

Expression of myc-BioID-(GGGS)x3-(DPR)x100 fusion proteins
Expression of EGFP and mCherry-tagged DPRx100 proteins
Expression of 2xHA-poly-GA protein
Expression of EGFP-tagged SQSTM1, UBXN6, VCP, and HSPA8 proteins
Expression of mCherry-Calreticulin-N-16 and EGFP-Calreticulin proteins

control variables

PcDNA3.1 mycBioID plasmid (Addgene plasmid #35700)
Myc-BioID-TDP-CTF plasmid

positive controls

None specified

negative controls

None specified

Annotations

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