MLV-Gag-YFP (Plasmid 1813) and mCherry-hAlix (Plasmid 21504) constructs were obtained from Addgene51 (link), 52 (link). CHMP4B-myc was purchased from Origene (RC207637). Other cDNAs used in this study were previously described11 (link) and have been deposited with Addgene. For retroviral infection, VSV-G pseudotyped retroviruses were generated, and MEFs were infected and selected as previously described11 (link). Following selection, stable pools were used at a maximum of 6–8 passages to avoid clonal selection or drift.
Mcherry halix
Manufactured by Addgene
The MCherry-hAlix is a fluorescent protein product. It is a fusion of the mCherry fluorescent protein and the human Alix protein. The mCherry protein provides a red fluorescent signal, while the Alix protein is involved in various cellular processes.
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Lab products found in correlation
4 protocols using mcherry halix
1
Retroviral Infection of Mammalian Cells
2
Retroviral Infection of Mammalian Cells
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MLV-Gag-YFP (Plasmid 1813) and mCherry-hAlix (Plasmid 21504) constructs were obtained from Addgene51 (link), 52 (link). CHMP4B-myc was purchased from Origene (RC207637). Other cDNAs used in this study were previously described11 (link) and have been deposited with Addgene. For retroviral infection, VSV-G pseudotyped retroviruses were generated, and MEFs were infected and selected as previously described11 (link). Following selection, stable pools were used at a maximum of 6–8 passages to avoid clonal selection or drift.
3
CRISPR/Cas9-Mediated Knockdown and Rescue of Alix and Syntenin
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Single-guide RNAs (sgRNAs) targeting Alix or Syntenin were chosen from GenScript gRNA database and cloned into the pSpCas9(BB)-2A-Puro (pX459) V2.0 plasmid (62988, Addgene) to generate CRISPR/Cas9 systems. Two CRISPR constructs were designed to target human Alix gene (exon 8; guide sequence: 5′-CGGGGTAGATTTCACAAGTG-3') and Syntenin gene (exon7; guide sequence: 5′-ACAGAATGTCATTGGATTGA-3'),65 (link) respectively. HepAD38 cells were transfected with pX459 plasmids with the above gRNAs inserted using FuGENE HD Transfection Reagent (PRE2311, Promega, Madison, WI). Forty-eight hours post-transfection, cells were treated with medium containing puromycin (2.5 μg/mL, P9620, Sigma-Aldrich) for 2 weeks. Stable KO clones were isolated, amplified, and analyzed by immunoblotting. For Alix-rescues experiments, HepAD38 Alix KO cells (5 × 106 cells per 150-mm dish) were transiently transfected using linear polyethyleneimine (9002-98-6, Polysciences) with 20 μg of human full-length Alix expression vector (mCherry-hALIX, 21504, Addgene). Exosomes were isolated from the supernatant at 48 hours post-transfection as mentioned above.
4
Cloning mCherry-CD63 Fusion Protein
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Full-length human CD63 was subcloned from CD63-pEGFP C2 (a gift from Paul Luzio, Addgene plasmid #62964) to pcDNA™6/myc-His A vector (Invitrogen, Waltham, MA, USA) to produce CD63 without tag using KpnI–BamHI enzymes. Then, mCherry sequence was obtained by PCR from the plasmid mCherry-hALIX (a gift from James Hurley, Addgene plasmid # 21504 [46 (link)] using 5′-GGG TAC CAT GGT GAG CAA GGG CGA GGA G (forward) and 5′-GGC ATG GAC GAG CTG TAC AAG TGG TAC CCC (reverse) primers and cloned into the pcDNA™6/myc-His A vector (Invitrogen) in frame with CD63 to produce mCherry-CD63 using KpnI enzyme. The previously mentioned mCherry sequence was also cloned into pcDNA™6/myc-His A vector to produce mCherry protein alone.
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