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previous mouse ko sgrna pooled library  (Addgene inc)


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    Addgene inc previous mouse ko sgrna pooled library
    Overview of the Experimental KO Screening Strategy (A) In our culture system, naive, ex vivo T cells are differentiated into Th2 cells by IL4. Potential alternative T cell fates that may be open to genetically perturbed cells are indicated. In vivo , T cells develop into different subtypes dependent on stimuli. (B) The retrovirus is based on murine stem cell virus (MSCV), encoding one <t>sgRNA</t> per virus, and allows for BFP and puro selection. For the screening we have used a pool of plasmids, encoding over 86,000 sgRNAs, from all of which we produced viruses. The library is subcloned from a previous mouse sgRNA library ( <xref ref-type=Tzelepis et al., 2016 ). (C) For genome-wide screens, we pool cells from up to 30 mice. After infection and puromycin selection, the cells are sorted based on fluorescence for the investigated gene. sgRNAs affecting gene expression are identified by genomic PCR. Differential sgRNA expression analysis then allows us to find genes affecting either viability (drop-out screen) or differentiation. (D) The top enriched and depleted genes (“hits”) were analyzed based on their dynamics measured by RNA-seq, ATAC-seq, and ChIP-seq. (E) Particularly interesting genes were further validated by individual KO and RNA-seq. (F) By using all this data and curating the literature, we provide a Th2 gene regulatory network. " width="250" height="auto" />
    Previous Mouse Ko Sgrna Pooled Library, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94/100 stars

    Images

    1) Product Images from "Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation"

    Article Title: Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation

    Journal: Cell

    doi: 10.1016/j.cell.2018.11.044

    Overview of the Experimental KO Screening Strategy (A) In our culture system, naive, ex vivo T cells are differentiated into Th2 cells by IL4. Potential alternative T cell fates that may be open to genetically perturbed cells are indicated. In vivo , T cells develop into different subtypes dependent on stimuli. (B) The retrovirus is based on murine stem cell virus (MSCV), encoding one sgRNA per virus, and allows for BFP and puro selection. For the screening we have used a pool of plasmids, encoding over 86,000 sgRNAs, from all of which we produced viruses. The library is subcloned from a previous mouse sgRNA library ( <xref ref-type=Tzelepis et al., 2016 ). (C) For genome-wide screens, we pool cells from up to 30 mice. After infection and puromycin selection, the cells are sorted based on fluorescence for the investigated gene. sgRNAs affecting gene expression are identified by genomic PCR. Differential sgRNA expression analysis then allows us to find genes affecting either viability (drop-out screen) or differentiation. (D) The top enriched and depleted genes (“hits”) were analyzed based on their dynamics measured by RNA-seq, ATAC-seq, and ChIP-seq. (E) Particularly interesting genes were further validated by individual KO and RNA-seq. (F) By using all this data and curating the literature, we provide a Th2 gene regulatory network. " title="... on murine stem cell virus (MSCV), encoding one sgRNA per virus, and allows for BFP and puro ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Overview of the Experimental KO Screening Strategy (A) In our culture system, naive, ex vivo T cells are differentiated into Th2 cells by IL4. Potential alternative T cell fates that may be open to genetically perturbed cells are indicated. In vivo , T cells develop into different subtypes dependent on stimuli. (B) The retrovirus is based on murine stem cell virus (MSCV), encoding one sgRNA per virus, and allows for BFP and puro selection. For the screening we have used a pool of plasmids, encoding over 86,000 sgRNAs, from all of which we produced viruses. The library is subcloned from a previous mouse sgRNA library ( Tzelepis et al., 2016 ). (C) For genome-wide screens, we pool cells from up to 30 mice. After infection and puromycin selection, the cells are sorted based on fluorescence for the investigated gene. sgRNAs affecting gene expression are identified by genomic PCR. Differential sgRNA expression analysis then allows us to find genes affecting either viability (drop-out screen) or differentiation. (D) The top enriched and depleted genes (“hits”) were analyzed based on their dynamics measured by RNA-seq, ATAC-seq, and ChIP-seq. (E) Particularly interesting genes were further validated by individual KO and RNA-seq. (F) By using all this data and curating the literature, we provide a Th2 gene regulatory network.

    Techniques Used: Ex Vivo, In Vivo, Virus, Selection, Produced, Genome Wide, Infection, Fluorescence, Gene Expression, Expressing, RNA Sequencing, ChIP-sequencing



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    Overview of the Experimental KO Screening Strategy (A) In our culture system, naive, ex vivo T cells are differentiated into Th2 cells by IL4. Potential alternative T cell fates that may be open to genetically perturbed cells are indicated. In vivo , T cells develop into different subtypes dependent on stimuli. (B) The retrovirus is based on murine stem cell virus (MSCV), encoding one sgRNA per virus, and allows for BFP and puro selection. For the screening we have used a pool of plasmids, encoding over 86,000 sgRNAs, from all of which we produced viruses. The library is subcloned from a previous mouse sgRNA library ( <xref ref-type=Tzelepis et al., 2016 ). (C) For genome-wide screens, we pool cells from up to 30 mice. After infection and puromycin selection, the cells are sorted based on fluorescence for the investigated gene. sgRNAs affecting gene expression are identified by genomic PCR. Differential sgRNA expression analysis then allows us to find genes affecting either viability (drop-out screen) or differentiation. (D) The top enriched and depleted genes (“hits”) were analyzed based on their dynamics measured by RNA-seq, ATAC-seq, and ChIP-seq. (E) Particularly interesting genes were further validated by individual KO and RNA-seq. (F) By using all this data and curating the literature, we provide a Th2 gene regulatory network. " width="100%" height="100%">

    Journal: Cell

    Article Title: Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation

    doi: 10.1016/j.cell.2018.11.044

    Figure Lengend Snippet: Overview of the Experimental KO Screening Strategy (A) In our culture system, naive, ex vivo T cells are differentiated into Th2 cells by IL4. Potential alternative T cell fates that may be open to genetically perturbed cells are indicated. In vivo , T cells develop into different subtypes dependent on stimuli. (B) The retrovirus is based on murine stem cell virus (MSCV), encoding one sgRNA per virus, and allows for BFP and puro selection. For the screening we have used a pool of plasmids, encoding over 86,000 sgRNAs, from all of which we produced viruses. The library is subcloned from a previous mouse sgRNA library ( Tzelepis et al., 2016 ). (C) For genome-wide screens, we pool cells from up to 30 mice. After infection and puromycin selection, the cells are sorted based on fluorescence for the investigated gene. sgRNAs affecting gene expression are identified by genomic PCR. Differential sgRNA expression analysis then allows us to find genes affecting either viability (drop-out screen) or differentiation. (D) The top enriched and depleted genes (“hits”) were analyzed based on their dynamics measured by RNA-seq, ATAC-seq, and ChIP-seq. (E) Particularly interesting genes were further validated by individual KO and RNA-seq. (F) By using all this data and curating the literature, we provide a Th2 gene regulatory network.

    Article Snippet: To produce the pooled library pMSCV-U6gRNA(lib)-PGKpuroT2ABFP (Addgene: #104861) the sgRNA part of a previous mouse KO sgRNA pooled library ( ) (Addgene: #67988) was PCR-amplified using the primers gib_sgRNAlib_fwd/rev.

    Techniques: Ex Vivo, In Vivo, Virus, Selection, Produced, Genome Wide, Infection, Fluorescence, Gene Expression, Expressing, RNA Sequencing, ChIP-sequencing