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lentiviral crispr cas9 delivery vector  (Addgene inc)


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    Structured Review

    Addgene inc lentiviral crispr cas9 delivery vector
    Top: G9a IF in untreated OP6 cells. Bottom: Example of two nearby cells following transfection with the G9a-targeted <t>CRISPR-Cas9,</t> with the bottom cell having knocked out G9a and the top cell being un- transfected OP6. Scale bar = 5 mm. (B) Representative images showing G9a expression in scrambled (top panel) and G9a knockout (bottom panel) samples. Hashed boxes highlight a GFP+ cell in both the scrambled and knockout populations. Quantitation of average G9a IF intensity in untreated OP6 populations versus CRISPR-treated populations is shown in Supplementary Figure 2.
    Lentiviral Crispr Cas9 Delivery Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 97/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sgrna+lentiviral+delivery+vector/Cas9+sgRNA+vector+(Plasmid+%2368463)/bio_rxiv__2024__05__03__592460-265-13-34
    Average 97 stars, based on 430 article reviews
    lentiviral crispr cas9 delivery vector - by Bioz Stars, 2026-10
    97/100 stars

    Images

    1) Product Images from "Transformation of an olfactory placode-derived cell into one with stem cell characteristics by disrupting epigenetic barriers"

    Article Title: Transformation of an olfactory placode-derived cell into one with stem cell characteristics by disrupting epigenetic barriers

    Journal: bioRxiv

    doi: 10.1101/2024.05.03.592460

    Top: G9a IF in untreated OP6 cells. Bottom: Example of two nearby cells following transfection with the G9a-targeted CRISPR-Cas9, with the bottom cell having knocked out G9a and the top cell being un- transfected OP6. Scale bar = 5 mm. (B) Representative images showing G9a expression in scrambled (top panel) and G9a knockout (bottom panel) samples. Hashed boxes highlight a GFP+ cell in both the scrambled and knockout populations. Quantitation of average G9a IF intensity in untreated OP6 populations versus CRISPR-treated populations is shown in Supplementary Figure 2.
    Figure Legend Snippet: Top: G9a IF in untreated OP6 cells. Bottom: Example of two nearby cells following transfection with the G9a-targeted CRISPR-Cas9, with the bottom cell having knocked out G9a and the top cell being un- transfected OP6. Scale bar = 5 mm. (B) Representative images showing G9a expression in scrambled (top panel) and G9a knockout (bottom panel) samples. Hashed boxes highlight a GFP+ cell in both the scrambled and knockout populations. Quantitation of average G9a IF intensity in untreated OP6 populations versus CRISPR-treated populations is shown in Supplementary Figure 2.

    Techniques Used: Transfection, CRISPR, Expressing, Knock-Out, Quantitation Assay

    Example of colony maturation from G9a_ko populations over a 9-day period. (B) Colony formation incidence in three different immortalized cell lines (OP6, OP27, GD25) with both CRISPR treatment and using the BIX-01924 G9a inhibitor drug. (C) Left: Example of a colony formed in treated OP6 (top) and OP27 (bottom) populations upon G9a inhibition with BIX- 01924. Middle and Right: A pair of secondary colonies produced from passaging a primary OP6 colony derived from CRISPR treatment at Days 8, and 10 post-passage, shown in both brightfield (top panels) and GFP fluorescence (bottom panels). (D) Left: Immunofluorescence (IF) staining of G9a (red) in untreated OP6 cells (top) and within colonies (bottom). Nuclei are counterstained with DAPI (blue). Right: The distribution of ranked G9a IF intensities within colony cells (n=40 random cells from four representative colonies). Average IF intensity within scrambled cells (upper line) and G9a_kd populations (lower line) are indicated.
    Figure Legend Snippet: Example of colony maturation from G9a_ko populations over a 9-day period. (B) Colony formation incidence in three different immortalized cell lines (OP6, OP27, GD25) with both CRISPR treatment and using the BIX-01924 G9a inhibitor drug. (C) Left: Example of a colony formed in treated OP6 (top) and OP27 (bottom) populations upon G9a inhibition with BIX- 01924. Middle and Right: A pair of secondary colonies produced from passaging a primary OP6 colony derived from CRISPR treatment at Days 8, and 10 post-passage, shown in both brightfield (top panels) and GFP fluorescence (bottom panels). (D) Left: Immunofluorescence (IF) staining of G9a (red) in untreated OP6 cells (top) and within colonies (bottom). Nuclei are counterstained with DAPI (blue). Right: The distribution of ranked G9a IF intensities within colony cells (n=40 random cells from four representative colonies). Average IF intensity within scrambled cells (upper line) and G9a_kd populations (lower line) are indicated.

    Techniques Used: CRISPR, Inhibition, Produced, Passaging, Derivative Assay, Fluorescence, Immunofluorescence, Staining

    Immunofluorescence images of mouse embryonic stem cells (mESC) untreated OP6 cells, and BIX-treated OP6 colonies showing the expression of Sox2, Klf4, Nanog, cMyc, and Oct4 stem cell markers. Cell nuclei are visualized with DAPI staining (blue). Scale bars = 10 μm. Lower : Bar graph depicting the fold-increases of Sox2, Klf4, Nanog, cMyc, and Oct4 within BIX-treated OP6 cells (+) relative to untreated negative controls (-). (B) Up-regulation of stem cell markers Sox2, Klf4, Nanog, cMyc, and Oct4 in CRISPR-derived OP6 colonies compared to Scrambled Control and untransformed GFP+ cells. Cell nuclei are visualized with DAPI staining (blue). Scale bars = 10 μm. (C) Bar graph summarizing the normalized immunofluorescence (IF) intensities of stem cell markers from the analysis of several CRISPR-derived OP6 colonies (+) compared to Scrambled Control and untransformed GFP+ cells, as shown in (A). (D) Colonies stain with alkaline phosphatase.
    Figure Legend Snippet: Immunofluorescence images of mouse embryonic stem cells (mESC) untreated OP6 cells, and BIX-treated OP6 colonies showing the expression of Sox2, Klf4, Nanog, cMyc, and Oct4 stem cell markers. Cell nuclei are visualized with DAPI staining (blue). Scale bars = 10 μm. Lower : Bar graph depicting the fold-increases of Sox2, Klf4, Nanog, cMyc, and Oct4 within BIX-treated OP6 cells (+) relative to untreated negative controls (-). (B) Up-regulation of stem cell markers Sox2, Klf4, Nanog, cMyc, and Oct4 in CRISPR-derived OP6 colonies compared to Scrambled Control and untransformed GFP+ cells. Cell nuclei are visualized with DAPI staining (blue). Scale bars = 10 μm. (C) Bar graph summarizing the normalized immunofluorescence (IF) intensities of stem cell markers from the analysis of several CRISPR-derived OP6 colonies (+) compared to Scrambled Control and untransformed GFP+ cells, as shown in (A). (D) Colonies stain with alkaline phosphatase.

    Techniques Used: Immunofluorescence, Expressing, Staining, CRISPR, Derivative Assay

    Related Articles

    Cloning:

    Article Title: Pooled genetic perturbation screens with image-based phenotypes
    Article Snippet: HeLa cell line ( https://scicrunch.org/resolver/CVCL_0030 ; ATCC, cat. no. CCL-2) ! .. Custom sgRNA library cloning Custom oligonucleotide library, sequences designed in Steps 1–4 (Agilent, cat. no. G7222A or similar). sgRNA lentiviral delivery vector such as: CROPseq-Guide-Puro (Addgene, cat. no. 86708)CROPseq-Guide-Zeo (Addgene, cat. no. 127173)LentiGuide-BC-EF1a-Puro (Addgene, cat. no. 127170). .. PCR primer pairs to amplify oligo libraries for cloning (Integrated DNA Technologies, see Supplementary Table 2 for oligo sequences).

    Plasmid Preparation:

    Article Title: Pooled genetic perturbation screens with image-based phenotypes
    Article Snippet: HeLa cell line ( https://scicrunch.org/resolver/CVCL_0030 ; ATCC, cat. no. CCL-2) ! .. Custom sgRNA library cloning Custom oligonucleotide library, sequences designed in Steps 1–4 (Agilent, cat. no. G7222A or similar). sgRNA lentiviral delivery vector such as: CROPseq-Guide-Puro (Addgene, cat. no. 86708)CROPseq-Guide-Zeo (Addgene, cat. no. 127173)LentiGuide-BC-EF1a-Puro (Addgene, cat. no. 127170). .. PCR primer pairs to amplify oligo libraries for cloning (Integrated DNA Technologies, see Supplementary Table 2 for oligo sequences).

    Article Title: Pooled genetic perturbation screens with image-based phenotypes
    Article Snippet: .. Custom oligonucleotide library, sequences designed in Steps 1–4 (Agilent, cat. no. G7222A or similar). sgRNA lentiviral delivery vector such as: CROPseq-Guide-Puro (Addgene, cat. no. 86708)CROPseq-Guide-Zeo (Addgene, cat. no. 127173)LentiGuide-BC-EF1a-Puro (Addgene, cat. no. 127170). .. PCR primer pairs to amplify oligo libraries for cloning (Integrated DNA Technologies, see Supplementary Table 2 for oligo sequences).



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    Top: G9a IF in untreated OP6 cells. Bottom: Example of two nearby cells following transfection with the G9a-targeted <t>CRISPR-Cas9,</t> with the bottom cell having knocked out G9a and the top cell being un- transfected OP6. Scale bar = 5 mm. (B) Representative images showing G9a expression in scrambled (top panel) and G9a knockout (bottom panel) samples. Hashed boxes highlight a GFP+ cell in both the scrambled and knockout populations. Quantitation of average G9a IF intensity in untreated OP6 populations versus CRISPR-treated populations is shown in Supplementary Figure 2.
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    Top: G9a IF in untreated OP6 cells. Bottom: Example of two nearby cells following transfection with the G9a-targeted <t>CRISPR-Cas9,</t> with the bottom cell having knocked out G9a and the top cell being un- transfected OP6. Scale bar = 5 mm. (B) Representative images showing G9a expression in scrambled (top panel) and G9a knockout (bottom panel) samples. Hashed boxes highlight a GFP+ cell in both the scrambled and knockout populations. Quantitation of average G9a IF intensity in untreated OP6 populations versus CRISPR-treated populations is shown in Supplementary Figure 2.
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    Image Search Results


    Top: G9a IF in untreated OP6 cells. Bottom: Example of two nearby cells following transfection with the G9a-targeted CRISPR-Cas9, with the bottom cell having knocked out G9a and the top cell being un- transfected OP6. Scale bar = 5 mm. (B) Representative images showing G9a expression in scrambled (top panel) and G9a knockout (bottom panel) samples. Hashed boxes highlight a GFP+ cell in both the scrambled and knockout populations. Quantitation of average G9a IF intensity in untreated OP6 populations versus CRISPR-treated populations is shown in Supplementary Figure 2.

    Journal: bioRxiv

    Article Title: Transformation of an olfactory placode-derived cell into one with stem cell characteristics by disrupting epigenetic barriers

    doi: 10.1101/2024.05.03.592460

    Figure Lengend Snippet: Top: G9a IF in untreated OP6 cells. Bottom: Example of two nearby cells following transfection with the G9a-targeted CRISPR-Cas9, with the bottom cell having knocked out G9a and the top cell being un- transfected OP6. Scale bar = 5 mm. (B) Representative images showing G9a expression in scrambled (top panel) and G9a knockout (bottom panel) samples. Hashed boxes highlight a GFP+ cell in both the scrambled and knockout populations. Quantitation of average G9a IF intensity in untreated OP6 populations versus CRISPR-treated populations is shown in Supplementary Figure 2.

    Article Snippet: Complementary oligonucleotides 25 bp in length were synthesized, annealed, and cloned into the lentiviral CRISPR-Cas9 delivery vector containing spCas9-eGFP under control of the EFS promoter and the sgRNA under control of the U6 promoter (Addgene).

    Techniques: Transfection, CRISPR, Expressing, Knock-Out, Quantitation Assay

    Example of colony maturation from G9a_ko populations over a 9-day period. (B) Colony formation incidence in three different immortalized cell lines (OP6, OP27, GD25) with both CRISPR treatment and using the BIX-01924 G9a inhibitor drug. (C) Left: Example of a colony formed in treated OP6 (top) and OP27 (bottom) populations upon G9a inhibition with BIX- 01924. Middle and Right: A pair of secondary colonies produced from passaging a primary OP6 colony derived from CRISPR treatment at Days 8, and 10 post-passage, shown in both brightfield (top panels) and GFP fluorescence (bottom panels). (D) Left: Immunofluorescence (IF) staining of G9a (red) in untreated OP6 cells (top) and within colonies (bottom). Nuclei are counterstained with DAPI (blue). Right: The distribution of ranked G9a IF intensities within colony cells (n=40 random cells from four representative colonies). Average IF intensity within scrambled cells (upper line) and G9a_kd populations (lower line) are indicated.

    Journal: bioRxiv

    Article Title: Transformation of an olfactory placode-derived cell into one with stem cell characteristics by disrupting epigenetic barriers

    doi: 10.1101/2024.05.03.592460

    Figure Lengend Snippet: Example of colony maturation from G9a_ko populations over a 9-day period. (B) Colony formation incidence in three different immortalized cell lines (OP6, OP27, GD25) with both CRISPR treatment and using the BIX-01924 G9a inhibitor drug. (C) Left: Example of a colony formed in treated OP6 (top) and OP27 (bottom) populations upon G9a inhibition with BIX- 01924. Middle and Right: A pair of secondary colonies produced from passaging a primary OP6 colony derived from CRISPR treatment at Days 8, and 10 post-passage, shown in both brightfield (top panels) and GFP fluorescence (bottom panels). (D) Left: Immunofluorescence (IF) staining of G9a (red) in untreated OP6 cells (top) and within colonies (bottom). Nuclei are counterstained with DAPI (blue). Right: The distribution of ranked G9a IF intensities within colony cells (n=40 random cells from four representative colonies). Average IF intensity within scrambled cells (upper line) and G9a_kd populations (lower line) are indicated.

    Article Snippet: Complementary oligonucleotides 25 bp in length were synthesized, annealed, and cloned into the lentiviral CRISPR-Cas9 delivery vector containing spCas9-eGFP under control of the EFS promoter and the sgRNA under control of the U6 promoter (Addgene).

    Techniques: CRISPR, Inhibition, Produced, Passaging, Derivative Assay, Fluorescence, Immunofluorescence, Staining

    Immunofluorescence images of mouse embryonic stem cells (mESC) untreated OP6 cells, and BIX-treated OP6 colonies showing the expression of Sox2, Klf4, Nanog, cMyc, and Oct4 stem cell markers. Cell nuclei are visualized with DAPI staining (blue). Scale bars = 10 μm. Lower : Bar graph depicting the fold-increases of Sox2, Klf4, Nanog, cMyc, and Oct4 within BIX-treated OP6 cells (+) relative to untreated negative controls (-). (B) Up-regulation of stem cell markers Sox2, Klf4, Nanog, cMyc, and Oct4 in CRISPR-derived OP6 colonies compared to Scrambled Control and untransformed GFP+ cells. Cell nuclei are visualized with DAPI staining (blue). Scale bars = 10 μm. (C) Bar graph summarizing the normalized immunofluorescence (IF) intensities of stem cell markers from the analysis of several CRISPR-derived OP6 colonies (+) compared to Scrambled Control and untransformed GFP+ cells, as shown in (A). (D) Colonies stain with alkaline phosphatase.

    Journal: bioRxiv

    Article Title: Transformation of an olfactory placode-derived cell into one with stem cell characteristics by disrupting epigenetic barriers

    doi: 10.1101/2024.05.03.592460

    Figure Lengend Snippet: Immunofluorescence images of mouse embryonic stem cells (mESC) untreated OP6 cells, and BIX-treated OP6 colonies showing the expression of Sox2, Klf4, Nanog, cMyc, and Oct4 stem cell markers. Cell nuclei are visualized with DAPI staining (blue). Scale bars = 10 μm. Lower : Bar graph depicting the fold-increases of Sox2, Klf4, Nanog, cMyc, and Oct4 within BIX-treated OP6 cells (+) relative to untreated negative controls (-). (B) Up-regulation of stem cell markers Sox2, Klf4, Nanog, cMyc, and Oct4 in CRISPR-derived OP6 colonies compared to Scrambled Control and untransformed GFP+ cells. Cell nuclei are visualized with DAPI staining (blue). Scale bars = 10 μm. (C) Bar graph summarizing the normalized immunofluorescence (IF) intensities of stem cell markers from the analysis of several CRISPR-derived OP6 colonies (+) compared to Scrambled Control and untransformed GFP+ cells, as shown in (A). (D) Colonies stain with alkaline phosphatase.

    Article Snippet: Complementary oligonucleotides 25 bp in length were synthesized, annealed, and cloned into the lentiviral CRISPR-Cas9 delivery vector containing spCas9-eGFP under control of the EFS promoter and the sgRNA under control of the U6 promoter (Addgene).

    Techniques: Immunofluorescence, Expressing, Staining, CRISPR, Derivative Assay