Recombinant Hsp104 Protein Injection in C. elegans

Two different HSP104 mRNAs were used in this study. One was derived from the coding region of pRS416GAL-HSP104^A503V (Jackrel and Shorter, 2014 (link)), which was subcloned into pRN3P (a gift from John Gurdon; Zernicka-Goetz et al., 1996 (link)) where the original GFP was substituted for mRed to make C-terminal RFP fusion, and contains 5′ and 3′ untranslated regions from ß-globin. The mutant Hsp104 was derived from pRS313- HSP104^K218;K620T (Patino et al. 1996 (link); Addgene), which was cloned into the modified pRN3P vector. Transcription templates were generated using PCR, and following purification (Qiagen), mRNA was generated from the PCR product using mMESSAGE mMACHINE T3 Transcription Kit (AM1348, Ambion), precipitated with LiCl, and diluted to 0.1 µg/µl in DEPC water for injection into adult C. elegans hermaphrodite germlines. Recombinant Hsp104 protein (LifeSpan BioSciences) was injected following dilution to 0.1 mg/ml in a buffer containing 20 mM Tris HCl, 100 mM NaCl, and 2 mM EDTA, pH 8.