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    New England Biolabs engen spy cas9 nls
    CLEs have functional impacts. a Deletion of the b4galt2 CLE using <t>CRISPR/Cas9</t> rescues expression of downstream exons. Left: cut sites of the b4galt2 sgRNAs. CLE is indicated by capital letters. Center: PCR verification of Cas9 cleavage after injection of sgRNAs. Representative image; experiment performed five times. Right: RT-qPCR quantitation of the relative expression of the downstream b4galt2 exons in sfpq−/− embryos compared to siblings (±SD); n = 3 biologically independent replicates. Two-tailed unpaired t -test was performed. b In-situ hybridization of the epha4b CLE at 24 hpf, displaying strong expression in the midbrain and hindbrain of sfpq −/− embryos. c In-situ hybridization of rfng shows rhombomere boundary defects at 22ss after injection into WT embryos of the epha4b cryptic transcript or a mutated transcript with an early stop codon. Loss of boundaries seen in 8/10 embryos. d Left: in-situ hybridization of rfng shows rhombomere boundary defects of sfpq −/− embryos are rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Rhombomere boundaries are numbered. Right: quantification of staining in rhombomeres in three lateral view samples for each condition. Representative images; defect seen in 13/15 embryos. e Left: in-situ hybridization of DeltaA shows a loss of discrete neuronal clusters in sfpq −/− which is rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Right: quantification of number of DeltaA clusters in each condition. Each data point represents one embryo; embryos examined over two independent experiments. Two-tailed t -test was performed, *** p = 0.0005. c – e Upper: lateral view. Lower: dorsal view. Source data are provided as a Source Data file.
    Engen Spy Cas9 Nls, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs cas9 protein
    IRE1 facilitates migration of infected DCs in vivo . (A-B) IRE1 was depleted in bone marrow-derived DCs by <t>CRISPR/Cas9</t> and loss of IRE1 expression was assayed by RT- qPCR and immunoblot analyses. (C) WT or ire1 (-) DCs were infected for 18 h and the percentage of infection was determined by counting the number of parasites in 100 cells. (D) Infected WT and ire1 (-) cells were inoculated into mice by i.p. injection (10 6 infected cells). At the indicated hpi, the spleen of each mouse was harvested, and the number of parasites was determined by PCR. ***p
    Cas9 Protein, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs engen sgrna synthesis kit
    IRE1 facilitates migration of infected DCs in vivo . (A-B) IRE1 was depleted in bone marrow-derived DCs by <t>CRISPR/Cas9</t> and loss of IRE1 expression was assayed by RT- qPCR and immunoblot analyses. (C) WT or ire1 (-) DCs were infected for 18 h and the percentage of infection was determined by counting the number of parasites in 100 cells. (D) Infected WT and ire1 (-) cells were inoculated into mice by i.p. injection (10 6 infected cells). At the indicated hpi, the spleen of each mouse was harvested, and the number of parasites was determined by PCR. ***p
    Engen Sgrna Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CLEs have functional impacts. a Deletion of the b4galt2 CLE using CRISPR/Cas9 rescues expression of downstream exons. Left: cut sites of the b4galt2 sgRNAs. CLE is indicated by capital letters. Center: PCR verification of Cas9 cleavage after injection of sgRNAs. Representative image; experiment performed five times. Right: RT-qPCR quantitation of the relative expression of the downstream b4galt2 exons in sfpq−/− embryos compared to siblings (±SD); n = 3 biologically independent replicates. Two-tailed unpaired t -test was performed. b In-situ hybridization of the epha4b CLE at 24 hpf, displaying strong expression in the midbrain and hindbrain of sfpq −/− embryos. c In-situ hybridization of rfng shows rhombomere boundary defects at 22ss after injection into WT embryos of the epha4b cryptic transcript or a mutated transcript with an early stop codon. Loss of boundaries seen in 8/10 embryos. d Left: in-situ hybridization of rfng shows rhombomere boundary defects of sfpq −/− embryos are rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Rhombomere boundaries are numbered. Right: quantification of staining in rhombomeres in three lateral view samples for each condition. Representative images; defect seen in 13/15 embryos. e Left: in-situ hybridization of DeltaA shows a loss of discrete neuronal clusters in sfpq −/− which is rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Right: quantification of number of DeltaA clusters in each condition. Each data point represents one embryo; embryos examined over two independent experiments. Two-tailed t -test was performed, *** p = 0.0005. c – e Upper: lateral view. Lower: dorsal view. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A conserved role for the ALS-linked splicing factor SFPQ in repression of pathogenic cryptic last exons

    doi: 10.1038/s41467-021-22098-z

    Figure Lengend Snippet: CLEs have functional impacts. a Deletion of the b4galt2 CLE using CRISPR/Cas9 rescues expression of downstream exons. Left: cut sites of the b4galt2 sgRNAs. CLE is indicated by capital letters. Center: PCR verification of Cas9 cleavage after injection of sgRNAs. Representative image; experiment performed five times. Right: RT-qPCR quantitation of the relative expression of the downstream b4galt2 exons in sfpq−/− embryos compared to siblings (±SD); n = 3 biologically independent replicates. Two-tailed unpaired t -test was performed. b In-situ hybridization of the epha4b CLE at 24 hpf, displaying strong expression in the midbrain and hindbrain of sfpq −/− embryos. c In-situ hybridization of rfng shows rhombomere boundary defects at 22ss after injection into WT embryos of the epha4b cryptic transcript or a mutated transcript with an early stop codon. Loss of boundaries seen in 8/10 embryos. d Left: in-situ hybridization of rfng shows rhombomere boundary defects of sfpq −/− embryos are rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Rhombomere boundaries are numbered. Right: quantification of staining in rhombomeres in three lateral view samples for each condition. Representative images; defect seen in 13/15 embryos. e Left: in-situ hybridization of DeltaA shows a loss of discrete neuronal clusters in sfpq −/− which is rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Right: quantification of number of DeltaA clusters in each condition. Each data point represents one embryo; embryos examined over two independent experiments. Two-tailed t -test was performed, *** p = 0.0005. c – e Upper: lateral view. Lower: dorsal view. Source data are provided as a Source Data file.

    Article Snippet: To make gRNA:Cas9 RNP complexes, a mix was formed as follows: 1.5 μl each gRNA, 0.75 μl 2 M KCl, 1.25 μl EnGen Spy Cas9 NLS (NEB).

    Techniques: Functional Assay, CRISPR, Expressing, Polymerase Chain Reaction, Injection, Quantitative RT-PCR, Quantitation Assay, Two Tailed Test, In Situ Hybridization, Staining

    IRE1 facilitates migration of infected DCs in vivo . (A-B) IRE1 was depleted in bone marrow-derived DCs by CRISPR/Cas9 and loss of IRE1 expression was assayed by RT- qPCR and immunoblot analyses. (C) WT or ire1 (-) DCs were infected for 18 h and the percentage of infection was determined by counting the number of parasites in 100 cells. (D) Infected WT and ire1 (-) cells were inoculated into mice by i.p. injection (10 6 infected cells). At the indicated hpi, the spleen of each mouse was harvested, and the number of parasites was determined by PCR. ***p

    Journal: bioRxiv

    Article Title: Toxoplasma gondii co-opts the unfolded protein response to enhance migration and dissemination of infected host cells

    doi: 10.1101/2020.04.14.042069

    Figure Lengend Snippet: IRE1 facilitates migration of infected DCs in vivo . (A-B) IRE1 was depleted in bone marrow-derived DCs by CRISPR/Cas9 and loss of IRE1 expression was assayed by RT- qPCR and immunoblot analyses. (C) WT or ire1 (-) DCs were infected for 18 h and the percentage of infection was determined by counting the number of parasites in 100 cells. (D) Infected WT and ire1 (-) cells were inoculated into mice by i.p. injection (10 6 infected cells). At the indicated hpi, the spleen of each mouse was harvested, and the number of parasites was determined by PCR. ***p

    Article Snippet: Generation of IRE1 knockout cells Disruption of the IRE1 gene in MEF cells was carried out using the CRISPR/Cas9 method [ ].

    Techniques: Migration, Infection, In Vivo, Derivative Assay, CRISPR, Expressing, Quantitative RT-PCR, Mouse Assay, Injection, Polymerase Chain Reaction

    MEF cells were transfected with Cas9 bound to sgRNA targeted to IRE1 as described in the methods section; lowered IRE1 expression resulting from the CRISPR/Cas9 gene editing was evaluated by (A) RT-qPCR and (B) immunoblot analyses. GAPDH was included as a loading control for the immunoblot analyses. (C) WT and IRE1 -/- MEF cells were treated with 1 µM thapsigargin (TG), or no stress agent, for 6 h. Cells were then harvested and the XBP1s mRNA levels were measured by RT-qPCR. The values of XBP1s mRNAs were normalized to values of total XBP1 transcripts. (±SD, n=3) ***p

    Journal: bioRxiv

    Article Title: Toxoplasma gondii co-opts the unfolded protein response to enhance migration and dissemination of infected host cells

    doi: 10.1101/2020.04.14.042069

    Figure Lengend Snippet: MEF cells were transfected with Cas9 bound to sgRNA targeted to IRE1 as described in the methods section; lowered IRE1 expression resulting from the CRISPR/Cas9 gene editing was evaluated by (A) RT-qPCR and (B) immunoblot analyses. GAPDH was included as a loading control for the immunoblot analyses. (C) WT and IRE1 -/- MEF cells were treated with 1 µM thapsigargin (TG), or no stress agent, for 6 h. Cells were then harvested and the XBP1s mRNA levels were measured by RT-qPCR. The values of XBP1s mRNAs were normalized to values of total XBP1 transcripts. (±SD, n=3) ***p

    Article Snippet: Generation of IRE1 knockout cells Disruption of the IRE1 gene in MEF cells was carried out using the CRISPR/Cas9 method [ ].

    Techniques: Transfection, Expressing, CRISPR, Quantitative RT-PCR