pbad33  (New England Biolabs)


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    Structured Review

    New England Biolabs pbad33
    Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the <t>pBAD33</t> multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).
    Pbad33, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae"

    Article Title: gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

    Journal: bioRxiv

    doi: 10.1101/2021.02.11.430729

    Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).
    Figure Legend Snippet: Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).

    Techniques Used: Molecular Cloning, Expressing, Clone Assay, Sequencing, Staining, Acrylamide Gel Assay, Western Blot, Produced, Molecular Weight, Plasmid Preparation

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    New England Biolabs mcherry plasmid
    EFF-1 expression in BWM enables VSVΔG-AFF-1 and VSVΔG-G spreading along fused muscles (A-L) Z-stack projections of wt-like (A-F) and Unc+Dpy animals (G-L) expressing myo-3p:: EFF-1 and myo-3p:: <t>mCherry</t> infected with VSVΔG-AFF-1 (35-63 IU red pins) or VSVΔG-G (3*10 5 IU; white pins). Insets and their corresponding images (yellow frames). Arrows, individual infected (cyan) BWMs. Dashed lines outline grouped BWMs that express myo-3p ::mCherry and myo-3p:: EFF-1 (magenta) and infected with virus (cyan) showing spreading of GFP. Scale bars, 100 µm. (M-N) Number of BWM cells/worm expressing EFF-1 (magenta cell), infected (cyan cell) or expressing EFF-1 and infected (magenta and cyan). wt-like (circles) and Unc+Dpy (triangles). Each point represents a single worm. (O-P) Quantitation of multinucleation of infected BWMs. Each dot represents an average number of nuclei/ GFP(+) BWM, calculated from 1-6 multinucleated BWMs of a single worm. (M and O) wt-like n=6 and Unc+Dpy n=10 animals. (N and P) wt-like n=9 and Unc+Dpy n=7 animals. Black horizontal lines, average ± SEM. Student’s t-test, *** p
    Mcherry Plasmid, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    EFF-1 expression in BWM enables VSVΔG-AFF-1 and VSVΔG-G spreading along fused muscles (A-L) Z-stack projections of wt-like (A-F) and Unc+Dpy animals (G-L) expressing myo-3p:: EFF-1 and myo-3p:: mCherry infected with VSVΔG-AFF-1 (35-63 IU red pins) or VSVΔG-G (3*10 5 IU; white pins). Insets and their corresponding images (yellow frames). Arrows, individual infected (cyan) BWMs. Dashed lines outline grouped BWMs that express myo-3p ::mCherry and myo-3p:: EFF-1 (magenta) and infected with virus (cyan) showing spreading of GFP. Scale bars, 100 µm. (M-N) Number of BWM cells/worm expressing EFF-1 (magenta cell), infected (cyan cell) or expressing EFF-1 and infected (magenta and cyan). wt-like (circles) and Unc+Dpy (triangles). Each point represents a single worm. (O-P) Quantitation of multinucleation of infected BWMs. Each dot represents an average number of nuclei/ GFP(+) BWM, calculated from 1-6 multinucleated BWMs of a single worm. (M and O) wt-like n=6 and Unc+Dpy n=10 animals. (N and P) wt-like n=9 and Unc+Dpy n=7 animals. Black horizontal lines, average ± SEM. Student’s t-test, *** p

    Journal: bioRxiv

    Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: EFF-1 expression in BWM enables VSVΔG-AFF-1 and VSVΔG-G spreading along fused muscles (A-L) Z-stack projections of wt-like (A-F) and Unc+Dpy animals (G-L) expressing myo-3p:: EFF-1 and myo-3p:: mCherry infected with VSVΔG-AFF-1 (35-63 IU red pins) or VSVΔG-G (3*10 5 IU; white pins). Insets and their corresponding images (yellow frames). Arrows, individual infected (cyan) BWMs. Dashed lines outline grouped BWMs that express myo-3p ::mCherry and myo-3p:: EFF-1 (magenta) and infected with virus (cyan) showing spreading of GFP. Scale bars, 100 µm. (M-N) Number of BWM cells/worm expressing EFF-1 (magenta cell), infected (cyan cell) or expressing EFF-1 and infected (magenta and cyan). wt-like (circles) and Unc+Dpy (triangles). Each point represents a single worm. (O-P) Quantitation of multinucleation of infected BWMs. Each dot represents an average number of nuclei/ GFP(+) BWM, calculated from 1-6 multinucleated BWMs of a single worm. (M and O) wt-like n=6 and Unc+Dpy n=10 animals. (N and P) wt-like n=9 and Unc+Dpy n=7 animals. Black horizontal lines, average ± SEM. Student’s t-test, *** p

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Expressing, Infection, Quantitation Assay

    EFF-1 expression in BWMs produces Unc+Dpy worms with multinucleated cells (A-H) Images of fluorescent Z-stack projections and respective DIC of animals with extrachromosomal myo-3p ::EFF-1, myo-3p ::mCherry. (G) White arrowhead, bridge formed between 2 BWMs from opposing quadrants. Yellow arrows, myo-3p ::mCherry accumulations also in DIC (H). Asterisks, clustered nuclei within one BWM. Scale bars, 100 µm. (I) Number of nuclei per myo-3p ::mCherry (+) BWM cell in L2s. wt-like (n=12); Unc+Dpy (n=15). Each dot represents the average number of nuclei/BWM cell/worm. Total average ± SEM for each phenotype. Two tailed Student’s t-test p

    Journal: bioRxiv

    Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: EFF-1 expression in BWMs produces Unc+Dpy worms with multinucleated cells (A-H) Images of fluorescent Z-stack projections and respective DIC of animals with extrachromosomal myo-3p ::EFF-1, myo-3p ::mCherry. (G) White arrowhead, bridge formed between 2 BWMs from opposing quadrants. Yellow arrows, myo-3p ::mCherry accumulations also in DIC (H). Asterisks, clustered nuclei within one BWM. Scale bars, 100 µm. (I) Number of nuclei per myo-3p ::mCherry (+) BWM cell in L2s. wt-like (n=12); Unc+Dpy (n=15). Each dot represents the average number of nuclei/BWM cell/worm. Total average ± SEM for each phenotype. Two tailed Student’s t-test p

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Expressing, Two Tailed Test

    VSVΔG-AFF-1 does not infect PVD and other sensory neurons ectopically expressing AFF-1/EFF-1 (A-C) SDC microscope Z-stack projections of animals infected with 82-103 IU VSVΔG-AFF-1 (red pins). (For genotypes and quantitation see Table S2). Scale bar, 100 µm. (A) Young adult expressing mCherry in PVD. (B) eff-1(ts) adult expressing AFF-1 in PVD. (C) eff-1(ts) adult expressing EFF-1 and dsRed in 12 sensory neurons. See also Table S2 and Movie S2.

    Journal: bioRxiv

    Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: VSVΔG-AFF-1 does not infect PVD and other sensory neurons ectopically expressing AFF-1/EFF-1 (A-C) SDC microscope Z-stack projections of animals infected with 82-103 IU VSVΔG-AFF-1 (red pins). (For genotypes and quantitation see Table S2). Scale bar, 100 µm. (A) Young adult expressing mCherry in PVD. (B) eff-1(ts) adult expressing AFF-1 in PVD. (C) eff-1(ts) adult expressing EFF-1 and dsRed in 12 sensory neurons. See also Table S2 and Movie S2.

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Expressing, Microscopy, Infection, Quantitation Assay

    EFF-1 expression in BWMs induces their fusion ( A-C ) Confocal images of wt-like adult worms with membrane bound ( MB ) myo-3p::MB::YFP (cyan) and extrachromosomal array containing myo-3p:: EFF-1, myo-3p:: mCherry (magenta). ( D-F ) Confocal images of Unc+Dpy [ myo-3p::MB::YFP (cyan); myo-3p::EFF-1, myo-3p::mCherry] . Arrows, unfused BWMs with MB (cyan). Note only two unfused BWMs, all the others appear fused with no MB separating them. Insets correspond to white-dotted area. Scale bars 100 µm. See also Movie S1.

    Journal: bioRxiv

    Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: EFF-1 expression in BWMs induces their fusion ( A-C ) Confocal images of wt-like adult worms with membrane bound ( MB ) myo-3p::MB::YFP (cyan) and extrachromosomal array containing myo-3p:: EFF-1, myo-3p:: mCherry (magenta). ( D-F ) Confocal images of Unc+Dpy [ myo-3p::MB::YFP (cyan); myo-3p::EFF-1, myo-3p::mCherry] . Arrows, unfused BWMs with MB (cyan). Note only two unfused BWMs, all the others appear fused with no MB separating them. Insets correspond to white-dotted area. Scale bars 100 µm. See also Movie S1.

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Expressing

    Retargeting of VSVΔG-AFF-1 to body wall muscle cells Wild-type worms and animals with extrachromosomal array containing myo-3p:: EFF-1 and myo-3p:: mCherry were injected with VSVΔG-AFF-1 (35-63 IU, red pins; n=39 wt, n=50 wt-like and n=27 Unc+Dpy worms) or VSVΔG-G (3*10 5 IU, white pins; n=30 wt-like and 14 Unc+Dpy) respectively. Wt worms injected with VSVΔG-G (2300-4700 IU, n=56) were taken from figure 2I. Animals were analysed by SDC microscopy. Data represents average percentage of worms with GFP(+) BWMs ± SEM. Student’s t-test: *p

    Journal: bioRxiv

    Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: Retargeting of VSVΔG-AFF-1 to body wall muscle cells Wild-type worms and animals with extrachromosomal array containing myo-3p:: EFF-1 and myo-3p:: mCherry were injected with VSVΔG-AFF-1 (35-63 IU, red pins; n=39 wt, n=50 wt-like and n=27 Unc+Dpy worms) or VSVΔG-G (3*10 5 IU, white pins; n=30 wt-like and 14 Unc+Dpy) respectively. Wt worms injected with VSVΔG-G (2300-4700 IU, n=56) were taken from figure 2I. Animals were analysed by SDC microscopy. Data represents average percentage of worms with GFP(+) BWMs ± SEM. Student’s t-test: *p

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Injection, Microscopy

    Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).

    Journal: bioRxiv

    Article Title: gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

    doi: 10.1101/2021.02.11.430729

    Figure Lengend Snippet: Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).

    Article Snippet: The amplicon was purified and digested using 30 units of SacI-HF and SalI-HF (NEB, #R3156S and R3138S respectively) at 37 °C for 45 min. pBAD33 was similarly treated with SacI-HF and SalI-HF, and after 15 min incubation at 37 °C, the plasmid digestion was supplemented with 1.5 units of recombinant shrimp alkaline phosphatase (rSAP; NEB #M0371S).

    Techniques: Molecular Cloning, Expressing, Clone Assay, Sequencing, Staining, Acrylamide Gel Assay, Western Blot, Produced, Molecular Weight, Plasmid Preparation

    Picolinic acid does not inhibit infection by non-enveloped viruses and bacteriophages. HeLa c ells were either pre-treated for 3hr with 2mM PA, infected with 10 MOI CVB3 in presence of the drug and collected 3hr later (−3hr); or treated during infection (T0), or virus and drug were incubated together for 1hr and used for infection (1hr virus+PA). (A) shows quantification of virus infection by western blot using VP1 antibody. (B) HeLa cells were pre-treated with increasing concentrations of PA as indicated and infected with 0.1 MOI CVB3 in the presence of drugs. 48hr p.i, cell culture supernatants were used to quantify infectious virus by plaque assay. (C) HEK293T cells pre-treated with 2mM PA were infected with RRV, fixed with 4% formalin after 12hr and immunolabelled with VP6 antibody and DAPI to label the virus particles and nuclei respectively. (D) Percentage positive cells were quantified using ImageJ/Fiji. (E) HEK293T cells pre-treated with 2mM PA were infected with AAV6-GFP particles in presence of the drug at different volumes as indicated. 48hr p.i, cells were harvested and analyzed for GFP positive cells by flow cytometry. (F) HEK293 cells pre-treated with 2mM PA were infected with 10MOI Adenovirus 5 expressing eGFP in the presence of the drug and harvested 24hr later for quantification of GFP positive cells by flow cytometry. (G) M.smegmatis cells in a 48 well plate were treated with increasing concentrations of PA as indicated and OD600 measurements were taken up to 24hr. (H) log-phase secondary bacterial cultures were treated with 1mM PA at regular time intervals as indicated and infected with 10 MOI TM4 mycobacteriophage. OD600 measurements were taken up to 60hr p.i. **p

    Journal: bioRxiv

    Article Title: A natural broad-spectrum inhibitor of enveloped virus entry, effective against SARS-CoV-2 and Influenza A Virus in preclinical animal models

    doi: 10.1101/2022.02.16.480801

    Figure Lengend Snippet: Picolinic acid does not inhibit infection by non-enveloped viruses and bacteriophages. HeLa c ells were either pre-treated for 3hr with 2mM PA, infected with 10 MOI CVB3 in presence of the drug and collected 3hr later (−3hr); or treated during infection (T0), or virus and drug were incubated together for 1hr and used for infection (1hr virus+PA). (A) shows quantification of virus infection by western blot using VP1 antibody. (B) HeLa cells were pre-treated with increasing concentrations of PA as indicated and infected with 0.1 MOI CVB3 in the presence of drugs. 48hr p.i, cell culture supernatants were used to quantify infectious virus by plaque assay. (C) HEK293T cells pre-treated with 2mM PA were infected with RRV, fixed with 4% formalin after 12hr and immunolabelled with VP6 antibody and DAPI to label the virus particles and nuclei respectively. (D) Percentage positive cells were quantified using ImageJ/Fiji. (E) HEK293T cells pre-treated with 2mM PA were infected with AAV6-GFP particles in presence of the drug at different volumes as indicated. 48hr p.i, cells were harvested and analyzed for GFP positive cells by flow cytometry. (F) HEK293 cells pre-treated with 2mM PA were infected with 10MOI Adenovirus 5 expressing eGFP in the presence of the drug and harvested 24hr later for quantification of GFP positive cells by flow cytometry. (G) M.smegmatis cells in a 48 well plate were treated with increasing concentrations of PA as indicated and OD600 measurements were taken up to 24hr. (H) log-phase secondary bacterial cultures were treated with 1mM PA at regular time intervals as indicated and infected with 10 MOI TM4 mycobacteriophage. OD600 measurements were taken up to 60hr p.i. **p

    Article Snippet: Briefly, pCB3/T7 DNA was linearized using SalI-HF enzyme (R3138S, NEB) and CVB3 RNA was produced by in vitro transcription reaction.

    Techniques: Infection, Incubation, Western Blot, Cell Culture, Plaque Assay, Flow Cytometry, Expressing

    EFF-1 in BWM enables VSVΔG-AFF-1 and VSVΔG-G spreading along fused muscles (A-L) Z-stack projections of wt-like (A-F) and Unc+Dpy animals (G-L) expressing myo-3p:: EFF-1 and myo-3p:: mCherry infected with VSVΔG-AFF-1 (35-63 IU red pins) or VSVΔG-G (3*10 5 IU; white pins). Insets and their corresponding images (yellow frames). Arrows, individual infected (cyan) BWMs. Dashed lines outline grouped BWMs that express myo-3p ::mCherry and myo-3p:: EFF-1 (magenta) and infected with virus (cyan) showing spreading of GFP. Scale bars, 100 µm. (M-N) Number of BWM cells/worm expressing EFF-1 (magenta cell), infected (cyan cell) or expressing EFF-1 and infected (magenta and cyan). wt-like (circles) and Unc+Dpy (triangles). Each point represents a single worm. (O-P) Quantitation of multinucleation of infected BWMs. Each dot represents an average number of nuclei/ GFP(+) BWM, calculated from 1-6 multinucleated BWMs of a single worm. (M and O) wt-like n=6 and Unc+Dpy n=10 animals. (N and P) wt-like n=9 and Unc+Dpy n=7 animals. Black horizontal lines, average ± SEM. Student’s t-test, *** p

    Journal: bioRxiv

    Article Title: Tissue-specific delivery system via AFF-1-coated pseudotyped Vesicular Stomatitis Virus in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: EFF-1 in BWM enables VSVΔG-AFF-1 and VSVΔG-G spreading along fused muscles (A-L) Z-stack projections of wt-like (A-F) and Unc+Dpy animals (G-L) expressing myo-3p:: EFF-1 and myo-3p:: mCherry infected with VSVΔG-AFF-1 (35-63 IU red pins) or VSVΔG-G (3*10 5 IU; white pins). Insets and their corresponding images (yellow frames). Arrows, individual infected (cyan) BWMs. Dashed lines outline grouped BWMs that express myo-3p ::mCherry and myo-3p:: EFF-1 (magenta) and infected with virus (cyan) showing spreading of GFP. Scale bars, 100 µm. (M-N) Number of BWM cells/worm expressing EFF-1 (magenta cell), infected (cyan cell) or expressing EFF-1 and infected (magenta and cyan). wt-like (circles) and Unc+Dpy (triangles). Each point represents a single worm. (O-P) Quantitation of multinucleation of infected BWMs. Each dot represents an average number of nuclei/ GFP(+) BWM, calculated from 1-6 multinucleated BWMs of a single worm. (M and O) wt-like n=6 and Unc+Dpy n=10 animals. (N and P) wt-like n=9 and Unc+Dpy n=7 animals. Black horizontal lines, average ± SEM. Student’s t-test, *** p

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Expressing, Infection, Quantitation Assay

    EFF-1 ectopic expression in BWMs results in Uncoordinated and Dumpy (Unc+Dpy) phenotypes Mixed population of worms with extrachromosomal pmyo-3::mCherry and pmyo-3::EFF-1 . White arrowhead point to mCherry(+) worm that is Unc+Dpy. White arrow point to mCherry(+) worm that is wt-like. Yellow arrowhead points to a wt-like mCherry(-) worm, which left the frame within seconds. Elapsed time (seconds) indicated in top left corner.

    Journal: bioRxiv

    Article Title: Tissue-specific delivery system via AFF-1-coated pseudotyped Vesicular Stomatitis Virus in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: EFF-1 ectopic expression in BWMs results in Uncoordinated and Dumpy (Unc+Dpy) phenotypes Mixed population of worms with extrachromosomal pmyo-3::mCherry and pmyo-3::EFF-1 . White arrowhead point to mCherry(+) worm that is Unc+Dpy. White arrow point to mCherry(+) worm that is wt-like. Yellow arrowhead points to a wt-like mCherry(-) worm, which left the frame within seconds. Elapsed time (seconds) indicated in top left corner.

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Expressing

    EFF-1 expression in BWMs produces Unc+Dpy worms with multinucleated cells (A-H) Images of fluorescent Z-stack projections and respective DIC of animals with extrachromosomal myo-3p ::EFF-1, myo-3p ::mCherry. (G) White arrowhead, bridge formed between 2 BWMs from opposing quadrants. Yellow arrows, myo-3p ::mCherry accumulations also in DIC (H). Asterisks, clustered nuclei within one BWM. Scale bars, 100 µm. (I) Number of nuclei per myo-3p ::mCherry (+) BWM cell in L2s. wt-like (n=12); Unc+Dpy (n=15). Each dot represents the average number of nuclei/BWM cell/worm. Total average ± SEM for each phenotype. Two tailed Student’s t-test p

    Journal: bioRxiv

    Article Title: Tissue-specific delivery system via AFF-1-coated pseudotyped Vesicular Stomatitis Virus in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: EFF-1 expression in BWMs produces Unc+Dpy worms with multinucleated cells (A-H) Images of fluorescent Z-stack projections and respective DIC of animals with extrachromosomal myo-3p ::EFF-1, myo-3p ::mCherry. (G) White arrowhead, bridge formed between 2 BWMs from opposing quadrants. Yellow arrows, myo-3p ::mCherry accumulations also in DIC (H). Asterisks, clustered nuclei within one BWM. Scale bars, 100 µm. (I) Number of nuclei per myo-3p ::mCherry (+) BWM cell in L2s. wt-like (n=12); Unc+Dpy (n=15). Each dot represents the average number of nuclei/BWM cell/worm. Total average ± SEM for each phenotype. Two tailed Student’s t-test p

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Expressing, Two Tailed Test

    VSVΔG-AFF-1 does not infect PVD and other sensory neurons ectopically expressing AFF-1/EFF-1 (A-C) SDC microscope Z-stack projections of animals infected with 82-103 IU VSVΔG-AFF-1 (red pins). (For genotypes and quantitation see Table S2 ). Scale bar, 100 µm. (A) Young adult expressing mCherry in PVD. (B) eff-1(ts) adult expressing AFF-1 in PVD. (C) eff-1(ts) adult expressing EFF-1 and dsRed in 12 sensory neurons. See also Table S2 and Movie S2 .

    Journal: bioRxiv

    Article Title: Tissue-specific delivery system via AFF-1-coated pseudotyped Vesicular Stomatitis Virus in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: VSVΔG-AFF-1 does not infect PVD and other sensory neurons ectopically expressing AFF-1/EFF-1 (A-C) SDC microscope Z-stack projections of animals infected with 82-103 IU VSVΔG-AFF-1 (red pins). (For genotypes and quantitation see Table S2 ). Scale bar, 100 µm. (A) Young adult expressing mCherry in PVD. (B) eff-1(ts) adult expressing AFF-1 in PVD. (C) eff-1(ts) adult expressing EFF-1 and dsRed in 12 sensory neurons. See also Table S2 and Movie S2 .

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Expressing, Microscopy, Infection, Quantitation Assay

    EFF-1 expression in BWMs induces their fusion ( A-C ) Confocal images of wt-like adult worms with membrane bound ( MB ) myo-3p::MB::YFP (cyan) and extrachromosomal array containing myo-3p:: EFF-1, myo-3p:: mCherry (magenta). ( D-F ) Confocal images of Unc+Dpy [ myo-3p::MB::YFP (cyan); myo-3p::EFF-1, myo-3p::mCherry] . Arrows, unfused BWMs with MB (cyan). Note only two unfused BWMs, all the others appear fused with no MB separating them. Insets correspond to white-dotted area. Scale bars 100 µm. See also Movie S1.

    Journal: bioRxiv

    Article Title: Tissue-specific delivery system via AFF-1-coated pseudotyped Vesicular Stomatitis Virus in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: EFF-1 expression in BWMs induces their fusion ( A-C ) Confocal images of wt-like adult worms with membrane bound ( MB ) myo-3p::MB::YFP (cyan) and extrachromosomal array containing myo-3p:: EFF-1, myo-3p:: mCherry (magenta). ( D-F ) Confocal images of Unc+Dpy [ myo-3p::MB::YFP (cyan); myo-3p::EFF-1, myo-3p::mCherry] . Arrows, unfused BWMs with MB (cyan). Note only two unfused BWMs, all the others appear fused with no MB separating them. Insets correspond to white-dotted area. Scale bars 100 µm. See also Movie S1.

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Expressing

    Retargeting of VSVΔG-AFF-1 to body wall muscle cells Wild-type worms and animals with extrachromosomal array containing myo-3p:: EFF-1 and myo-3p:: mCherry were injected with VSVΔG-AFF-1 (35-63 IU, red pins; n=39 wt, n=50 wt-like and n=27 Unc+Dpy worms) or VSVΔG-G (3*10 5 IU, white pins; n=30 wt-like and 14 Unc+Dpy) respectively. Wt worms injected with VSVΔG-G (2300-4700 IU, n=56) were taken from figure 2I . Animals were analysed by SDC microscopy. Data represents average percentage of worms with GFP(+) BWMs ± SEM. Student’s t-test: *p

    Journal: bioRxiv

    Article Title: Tissue-specific delivery system via AFF-1-coated pseudotyped Vesicular Stomatitis Virus in C. elegans

    doi: 10.1101/2020.05.17.099622

    Figure Lengend Snippet: Retargeting of VSVΔG-AFF-1 to body wall muscle cells Wild-type worms and animals with extrachromosomal array containing myo-3p:: EFF-1 and myo-3p:: mCherry were injected with VSVΔG-AFF-1 (35-63 IU, red pins; n=39 wt, n=50 wt-like and n=27 Unc+Dpy worms) or VSVΔG-G (3*10 5 IU, white pins; n=30 wt-like and 14 Unc+Dpy) respectively. Wt worms injected with VSVΔG-G (2300-4700 IU, n=56) were taken from figure 2I . Animals were analysed by SDC microscopy. Data represents average percentage of worms with GFP(+) BWMs ± SEM. Student’s t-test: *p

    Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

    Techniques: Injection, Microscopy