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novel polyclonal anti human per2 antibodies  (Cocalico Inc)

 
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    Cocalico Inc novel polyclonal anti human per2 antibodies
    Efficient targeting of Bmal1 and <t>Per2</t> genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.
    Novel Polyclonal Anti Human Per2 Antibodies, supplied by Cocalico Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/novel+polyclonal+anti+human+per2+antibodies/novel+polyclonal+anti+human+per2+antibodies/pmc06342919-223-1-19
    Average 90 stars, based on 1 article reviews
    novel polyclonal anti human per2 antibodies - by Bioz Stars, 2026-10
    90/100 stars

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    1) Product Images from "Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9"

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36736-y

    Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.
    Figure Legend Snippet: Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.

    Techniques Used: CRISPR, Plasmid Preparation, Software, Sequencing, Expressing, Transfection, Clone Assay

    Efficient generation of Bmal1 and Per2 knockout clones in U2OS cells by the all-in-one vector. ( A ) Function of Bmal1 is essential for the clock in U2OS cells. Bmal1 promoter driven Luciferase rhythms are completely disrupted by overexpressing a mutant BMAL1 lacking the DNA-binding domain. Transduction efficiency by the adenovirus was ~100% (see Supplementary Fig. 5) as shown in the merged image between bright field and GFP. Expression levels of the transgene were ~10-fold higher than those of endogenous Bmal1 (right panel). *Indicates a nonspecific band. Note that the mutant BMAL1 is smaller than the endogenous one. The scale bar represents 50 μm. ( B ) Circadian rhythms are still observed when Bmal1 exon 6 and 7 are targeted by transfection of the all-in-one CRISPR-Cas9 vector, and positive cells were selected by FACS and subjected to bioluminescence assay. Amplitude was reduced in both cases. ( C ) Arrhythmic clones are easily selected by single cell isolation and expansion from the FACS sorted U2OS cells. Four arrhythmic clones from Bmal1 exon 6 targeted cells are shown along with two control traces. Raw bioluminescence rhythms are shown. Efficiency of knockouts (number of arrhythmic clones/total number) for E6 = 23/37 (62%); for E8 = 9/17 (53%); for E9 = 5/16 (31%). ( D ) Arrhythmicity is not due to off-target effect of the all-in-one CRISPR-Cas9. All arrhythmic clones tested were rescued by expressing wt BMAL1 protein using the adenoviral vector. Two representative cases are shown. Blue: arrhythmic clone; Green: the arrhythmic clone plus AV- wtBmal1 ; Black: control cell. ( E ) Knockouts were confirmed by immunoblots and sequencing (Supplementary Fig. 2). Representative arrhythmic clones from three different experiments ( Bmal1 exon 6, 8 and 9) are shown. Note that the BMAL1-rescued sample was under-loaded to show the difference in size clearly between endogenous and transgenic BMAL1 due to 3XFlag tag. ( F,G ) Knockout clones for Per2 were efficiently generated by the all-in-one vector targeting exon 5. Three knockout clones are shown, which was confirmed by immunoblots ( G ) and sequencing (Supplementary Fig. 4). Note that all knockout clones (red) show damped rhythms compared to control cells (black). The same control traces and the same scale for Y-axis were used in all three graphs. The graphs were de-trended to compare amplitudes between control and knockout samples. ( G ) PER2 exhibits robust rhythms in U2OS (left panel). The samples were harvested at the indicated times after 2-hr serum shock. The majority of clones with damped rhythms show absence of PER2 (middle panel). Note that serially diluted control samples (1, 1/2 and 1/3) were loaded next to the candidate samples to show the detection of PER2 at those levels and the corresponding absence in knockout clones. Note that clones 5–13 and 5–62 retain PER2 expression. *Indicates a nonspecific band. ( H ) Knockout was independently confirmed by a novel monoclonal antibody. Probing of time-course samples with the antibody showed similarly robust oscillations (Supplementary Fig. 7).
    Figure Legend Snippet: Efficient generation of Bmal1 and Per2 knockout clones in U2OS cells by the all-in-one vector. ( A ) Function of Bmal1 is essential for the clock in U2OS cells. Bmal1 promoter driven Luciferase rhythms are completely disrupted by overexpressing a mutant BMAL1 lacking the DNA-binding domain. Transduction efficiency by the adenovirus was ~100% (see Supplementary Fig. 5) as shown in the merged image between bright field and GFP. Expression levels of the transgene were ~10-fold higher than those of endogenous Bmal1 (right panel). *Indicates a nonspecific band. Note that the mutant BMAL1 is smaller than the endogenous one. The scale bar represents 50 μm. ( B ) Circadian rhythms are still observed when Bmal1 exon 6 and 7 are targeted by transfection of the all-in-one CRISPR-Cas9 vector, and positive cells were selected by FACS and subjected to bioluminescence assay. Amplitude was reduced in both cases. ( C ) Arrhythmic clones are easily selected by single cell isolation and expansion from the FACS sorted U2OS cells. Four arrhythmic clones from Bmal1 exon 6 targeted cells are shown along with two control traces. Raw bioluminescence rhythms are shown. Efficiency of knockouts (number of arrhythmic clones/total number) for E6 = 23/37 (62%); for E8 = 9/17 (53%); for E9 = 5/16 (31%). ( D ) Arrhythmicity is not due to off-target effect of the all-in-one CRISPR-Cas9. All arrhythmic clones tested were rescued by expressing wt BMAL1 protein using the adenoviral vector. Two representative cases are shown. Blue: arrhythmic clone; Green: the arrhythmic clone plus AV- wtBmal1 ; Black: control cell. ( E ) Knockouts were confirmed by immunoblots and sequencing (Supplementary Fig. 2). Representative arrhythmic clones from three different experiments ( Bmal1 exon 6, 8 and 9) are shown. Note that the BMAL1-rescued sample was under-loaded to show the difference in size clearly between endogenous and transgenic BMAL1 due to 3XFlag tag. ( F,G ) Knockout clones for Per2 were efficiently generated by the all-in-one vector targeting exon 5. Three knockout clones are shown, which was confirmed by immunoblots ( G ) and sequencing (Supplementary Fig. 4). Note that all knockout clones (red) show damped rhythms compared to control cells (black). The same control traces and the same scale for Y-axis were used in all three graphs. The graphs were de-trended to compare amplitudes between control and knockout samples. ( G ) PER2 exhibits robust rhythms in U2OS (left panel). The samples were harvested at the indicated times after 2-hr serum shock. The majority of clones with damped rhythms show absence of PER2 (middle panel). Note that serially diluted control samples (1, 1/2 and 1/3) were loaded next to the candidate samples to show the detection of PER2 at those levels and the corresponding absence in knockout clones. Note that clones 5–13 and 5–62 retain PER2 expression. *Indicates a nonspecific band. ( H ) Knockout was independently confirmed by a novel monoclonal antibody. Probing of time-course samples with the antibody showed similarly robust oscillations (Supplementary Fig. 7).

    Techniques Used: Knock-Out, Clone Assay, Plasmid Preparation, Luciferase, Mutagenesis, Binding Assay, Transduction, Expressing, Transfection, CRISPR, ATP Bioluminescent Assay, Single-cell Isolation, Western Blot, Sequencing, Transgenic Assay, Generated

    PCR primers for T7E1 assay.
    Figure Legend Snippet: PCR primers for T7E1 assay.

    Techniques Used:

    Related Articles

    Generated:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    CRISPR:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Plasmid Preparation:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Software:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Sequencing:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Expressing:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Transfection:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Clone Assay:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Knock-Out:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Luciferase:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Mutagenesis:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Binding Assay:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Transduction:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    ATP Bioluminescent Assay:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Single-cell Isolation:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Western Blot:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.

    Transgenic Assay:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 7 and Per2 exon 15.



    Similar Products

    novel polyclonal anti human per2 antibodies  (Cocalico Inc)
    90
    Cocalico Inc novel polyclonal anti human per2 antibodies
    Efficient targeting of Bmal1 and <t>Per2</t> genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.
    Novel Polyclonal Anti Human Per2 Antibodies, supplied by Cocalico Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/novel+polyclonal+anti+human+per2+antibodies/novel+polyclonal+anti+human+per2+antibodies/pmc06342919-223-1-19
    Average 90 stars, based on 1 article reviews
    novel polyclonal anti human per2 antibodies - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier

    Image Search Results


    Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.

    Journal: Scientific Reports

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

    doi: 10.1038/s41598-018-36736-y

    Figure Lengend Snippet: Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.

    Article Snippet: For human PER2, novel polyclonal anti human PER2 antibodies were generated in guinea pigs using aa 1–200 peptide by Cocalico Biologicals, Inc (449 Stevens Rd, Reamstown, PA). hP2-GP49 was used in the current study.

    Techniques: CRISPR, Plasmid Preparation, Software, Sequencing, Expressing, Transfection, Clone Assay

    Efficient generation of Bmal1 and Per2 knockout clones in U2OS cells by the all-in-one vector. ( A ) Function of Bmal1 is essential for the clock in U2OS cells. Bmal1 promoter driven Luciferase rhythms are completely disrupted by overexpressing a mutant BMAL1 lacking the DNA-binding domain. Transduction efficiency by the adenovirus was ~100% (see Supplementary Fig. 5) as shown in the merged image between bright field and GFP. Expression levels of the transgene were ~10-fold higher than those of endogenous Bmal1 (right panel). *Indicates a nonspecific band. Note that the mutant BMAL1 is smaller than the endogenous one. The scale bar represents 50 μm. ( B ) Circadian rhythms are still observed when Bmal1 exon 6 and 7 are targeted by transfection of the all-in-one CRISPR-Cas9 vector, and positive cells were selected by FACS and subjected to bioluminescence assay. Amplitude was reduced in both cases. ( C ) Arrhythmic clones are easily selected by single cell isolation and expansion from the FACS sorted U2OS cells. Four arrhythmic clones from Bmal1 exon 6 targeted cells are shown along with two control traces. Raw bioluminescence rhythms are shown. Efficiency of knockouts (number of arrhythmic clones/total number) for E6 = 23/37 (62%); for E8 = 9/17 (53%); for E9 = 5/16 (31%). ( D ) Arrhythmicity is not due to off-target effect of the all-in-one CRISPR-Cas9. All arrhythmic clones tested were rescued by expressing wt BMAL1 protein using the adenoviral vector. Two representative cases are shown. Blue: arrhythmic clone; Green: the arrhythmic clone plus AV- wtBmal1 ; Black: control cell. ( E ) Knockouts were confirmed by immunoblots and sequencing (Supplementary Fig. 2). Representative arrhythmic clones from three different experiments ( Bmal1 exon 6, 8 and 9) are shown. Note that the BMAL1-rescued sample was under-loaded to show the difference in size clearly between endogenous and transgenic BMAL1 due to 3XFlag tag. ( F,G ) Knockout clones for Per2 were efficiently generated by the all-in-one vector targeting exon 5. Three knockout clones are shown, which was confirmed by immunoblots ( G ) and sequencing (Supplementary Fig. 4). Note that all knockout clones (red) show damped rhythms compared to control cells (black). The same control traces and the same scale for Y-axis were used in all three graphs. The graphs were de-trended to compare amplitudes between control and knockout samples. ( G ) PER2 exhibits robust rhythms in U2OS (left panel). The samples were harvested at the indicated times after 2-hr serum shock. The majority of clones with damped rhythms show absence of PER2 (middle panel). Note that serially diluted control samples (1, 1/2 and 1/3) were loaded next to the candidate samples to show the detection of PER2 at those levels and the corresponding absence in knockout clones. Note that clones 5–13 and 5–62 retain PER2 expression. *Indicates a nonspecific band. ( H ) Knockout was independently confirmed by a novel monoclonal antibody. Probing of time-course samples with the antibody showed similarly robust oscillations (Supplementary Fig. 7).

    Journal: Scientific Reports

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

    doi: 10.1038/s41598-018-36736-y

    Figure Lengend Snippet: Efficient generation of Bmal1 and Per2 knockout clones in U2OS cells by the all-in-one vector. ( A ) Function of Bmal1 is essential for the clock in U2OS cells. Bmal1 promoter driven Luciferase rhythms are completely disrupted by overexpressing a mutant BMAL1 lacking the DNA-binding domain. Transduction efficiency by the adenovirus was ~100% (see Supplementary Fig. 5) as shown in the merged image between bright field and GFP. Expression levels of the transgene were ~10-fold higher than those of endogenous Bmal1 (right panel). *Indicates a nonspecific band. Note that the mutant BMAL1 is smaller than the endogenous one. The scale bar represents 50 μm. ( B ) Circadian rhythms are still observed when Bmal1 exon 6 and 7 are targeted by transfection of the all-in-one CRISPR-Cas9 vector, and positive cells were selected by FACS and subjected to bioluminescence assay. Amplitude was reduced in both cases. ( C ) Arrhythmic clones are easily selected by single cell isolation and expansion from the FACS sorted U2OS cells. Four arrhythmic clones from Bmal1 exon 6 targeted cells are shown along with two control traces. Raw bioluminescence rhythms are shown. Efficiency of knockouts (number of arrhythmic clones/total number) for E6 = 23/37 (62%); for E8 = 9/17 (53%); for E9 = 5/16 (31%). ( D ) Arrhythmicity is not due to off-target effect of the all-in-one CRISPR-Cas9. All arrhythmic clones tested were rescued by expressing wt BMAL1 protein using the adenoviral vector. Two representative cases are shown. Blue: arrhythmic clone; Green: the arrhythmic clone plus AV- wtBmal1 ; Black: control cell. ( E ) Knockouts were confirmed by immunoblots and sequencing (Supplementary Fig. 2). Representative arrhythmic clones from three different experiments ( Bmal1 exon 6, 8 and 9) are shown. Note that the BMAL1-rescued sample was under-loaded to show the difference in size clearly between endogenous and transgenic BMAL1 due to 3XFlag tag. ( F,G ) Knockout clones for Per2 were efficiently generated by the all-in-one vector targeting exon 5. Three knockout clones are shown, which was confirmed by immunoblots ( G ) and sequencing (Supplementary Fig. 4). Note that all knockout clones (red) show damped rhythms compared to control cells (black). The same control traces and the same scale for Y-axis were used in all three graphs. The graphs were de-trended to compare amplitudes between control and knockout samples. ( G ) PER2 exhibits robust rhythms in U2OS (left panel). The samples were harvested at the indicated times after 2-hr serum shock. The majority of clones with damped rhythms show absence of PER2 (middle panel). Note that serially diluted control samples (1, 1/2 and 1/3) were loaded next to the candidate samples to show the detection of PER2 at those levels and the corresponding absence in knockout clones. Note that clones 5–13 and 5–62 retain PER2 expression. *Indicates a nonspecific band. ( H ) Knockout was independently confirmed by a novel monoclonal antibody. Probing of time-course samples with the antibody showed similarly robust oscillations (Supplementary Fig. 7).

    Article Snippet: For human PER2, novel polyclonal anti human PER2 antibodies were generated in guinea pigs using aa 1–200 peptide by Cocalico Biologicals, Inc (449 Stevens Rd, Reamstown, PA). hP2-GP49 was used in the current study.

    Techniques: Knock-Out, Clone Assay, Plasmid Preparation, Luciferase, Mutagenesis, Binding Assay, Transduction, Expressing, Transfection, CRISPR, ATP Bioluminescent Assay, Single-cell Isolation, Western Blot, Sequencing, Transgenic Assay, Generated

    PCR primers for T7E1 assay.

    Journal: Scientific Reports

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

    doi: 10.1038/s41598-018-36736-y

    Figure Lengend Snippet: PCR primers for T7E1 assay.

    Article Snippet: For human PER2, novel polyclonal anti human PER2 antibodies were generated in guinea pigs using aa 1–200 peptide by Cocalico Biologicals, Inc (449 Stevens Rd, Reamstown, PA). hP2-GP49 was used in the current study.

    Techniques: