Pce bifc vc155

Manufactured by Addgene

The PCE-BiFC-VC155 is a plasmid available from Addgene. It is designed for use in bimolecular fluorescence complementation (BiFC) experiments.

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Lab products found in correlation

Pce bifc vn173, by Addgene (4 mentions) Quikchange multi site directed mutagenesis kit, by Agilent Technologies (1 mentions) Lenticrispr v2, by Addgene (1 mentions) Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)

4 protocols using pce bifc vc155

Bimolecular Fluorescence Complementation in C. elegans

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cDNA sequences encoding N- and C-terminal fragments of Venus were amplified from pCe-BiFC-VN173 (Addgene) and pCe-BiFC-VC155 (Addgene) plasmids, respectively. The cDNA fragments encoding N- and C-termini of Venus were subsequently subcloned into modified C. elegans pMC10 expression vectors containing ceh-36Δp::odr-3 and hsp-16.2p::ric-8 sequences, respectively, using NEBuilder HiFi assembly or standard restriction enzyme cloning. The N-terminal Venus fragment was flanked with SGGGGS linkers on 5’ and 3’ ends and inserted between Gly118 and Glu119 of ODR-3. The plasmids were verified by Sanger sequencing.

Campagna C.M., McMahon H, & Nechipurenko I. (2023). The G protein alpha Chaperone and Guanine-Nucleotide Exchange Factor RIC-8 Regulates Cilia Morphogenesis in Caenorhabditis elegans Sensory Neurons. bioRxiv.

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Visualizing Protein Interactions in Worm Cilia

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The Venus-based BiFC assay was developed to detect the protein interactions in living worm cilia. For this purpose, we replaced the heat-shock promoter in worm BiFC vectors pCE-BiFC-VN173 and pCE-BiFC-VC155 (Obtained from Addgene, deposited by Dr. Chang-Deng Hu’s lab) with the worm ciliated-cell-specific promoter of arl-13 using SphI and XmaI enzyme sites. To visualize BBS-4 and BBS-5 in cilia, worm BBS-4 cDNA was sub-cloned into the VN173 vector, and BBS-5 cDNA into the VC155 vector, respectively. Then, the plasmids were co-injected along with the co-injection marker pRF4 [rol-6 (su1006)] into wild-type worms (15 ng/L for each BiFC plasmid and 100 ng/L pRF4). Fluorescent signals were visualized using a YFP filter.

Xu Q., Zhang Y., Wei Q., Huang Y., Li Y., Ling K, & Hu J. (2015). BBS4 and BBS5 show functional redundancy in the BBSome to regulate the degradative sorting of ciliary sensory receptors. Scientific Reports, 5, 11855.

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Generation and Characterization of p62, DAXX Mutants

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Plasmids generated in this study are listed in Supplementary Table 1. Flag-DAXX (#27974), pCE-BiFC-VC155 (#22020), pCE-BiFC-VN173 (#22019) and LentiCRISPRv2 (#52961) were from Addgene. HA-DAXX was a kind gift from Dr Hsiu-Ming Shih. pMXs-puro-GFP-p62 K7A D69A was from Addgene (Plasmid #38281) gifted by N. Mizushima. p62 or DAXX point mutants were generated with the Quikchange Multi Site-directed Mutagenesis kit according to manufacturing instruction (Agilent Technologies, #200514).

Yang Y., Willis T.L., Button R.W., Strang C.J., Fu Y., Wen X., Grayson P.R., Evans T., Sipthorpe R.J., Roberts S.L., Hu B., Zhang J., Lu B, & Luo S. (2019). Cytoplasmic DAXX drives SQSTM1/p62 phase condensation to activate Nrf2-mediated stress response. Nature Communications, 10, 3759.

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BiFC Plasmids for C. elegans

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Two C. elegans BiFC plasmids, pCE-BiFC-VN173 and pCE-BiFC-VC155, were obtained from AddGene. All site-directed mutagenesis reactions were performed by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, 210518). These plasmids were used in Figures 3C and 4A.

Hsu T.Y., Zhang B., L'Etoile N.D, & Juang B.T. (2021). C. elegans orthologs MUT-7/CeWRN-1 of Werner syndrome protein regulate neuronal plasticity. eLife, 10, e62449.

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