293fectin lipid transfection  (Thermo Fisher)


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    Name:
    293fectin Transfection Reagent
    Description:
    293fectin Transfection Reagent is a proprietary cationic lipid based formulation for transfecting DNA into eukaryotic cells This reagent is optimized for transfecting suspension 293 human embryonic kidney cells e g FreeStyle 293 F cells Cat No R790 07 in defined serum free FreeStyle 293 Expression Medium Cat No 12338 018 and is intended for use with the FreeStyle 293 Expression System Cat No K9000 01 293fectin Transfection Reagent provides the following advantages • Demonstrates high transfection efficiency in suspension 293 cells and is also suitable for transfecting adherent 293 cells• Suspension FreeStyle 293 F cells may be transfected in FreeStyle 293 Expression Medium no medium change is required• Add 293fectin reagent complexes directly to cells in culture medium• No need to remove complexes or change or add medium following transfection
    Catalog Number:
    12347019
    Price:
    None
    Applications:
    Bioproduction|Cell Culture|Mammalian Expression|Mammalian Protein Production|Plasmid Transfection|Protein Biology|Protein Expression|Transient Protein Production & High-Throughput Screening|Antibody & Cell Culture Production|Transfection
    Category:
    Cell Culture Transfection Reagents
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    Structured Review

    Thermo Fisher 293fectin lipid transfection
    293fectin Transfection Reagent is a proprietary cationic lipid based formulation for transfecting DNA into eukaryotic cells This reagent is optimized for transfecting suspension 293 human embryonic kidney cells e g FreeStyle 293 F cells Cat No R790 07 in defined serum free FreeStyle 293 Expression Medium Cat No 12338 018 and is intended for use with the FreeStyle 293 Expression System Cat No K9000 01 293fectin Transfection Reagent provides the following advantages • Demonstrates high transfection efficiency in suspension 293 cells and is also suitable for transfecting adherent 293 cells• Suspension FreeStyle 293 F cells may be transfected in FreeStyle 293 Expression Medium no medium change is required• Add 293fectin reagent complexes directly to cells in culture medium• No need to remove complexes or change or add medium following transfection
    https://www.bioz.com/result/293fectin lipid transfection/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    293fectin lipid transfection - by Bioz Stars, 2020-09
    99/100 stars

    Related Products / Commonly Used Together

    antibody expression antibodies
    hek-293 cells

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    Functional Assay:

    Article Title: Tumor suppressive microRNA-1285 regulates novel molecular targets: Aberrant expression and functional significance in renal cell carcinoma
    Article Snippet: .. Effect of miR-1285 restoration on cell proliferation, migration and invasion in RCC cell lines To investigate the functional significance of miR-1285 , we performed gain-of-function studies using transient transfection with mature miR-1285 . .. We utilized two sources of mature miR-1285 (Ambion and Thermo) to ensure reproducibility of the data.

    Transfection:

    Article Title: Mitochondria-associated protein LRPPRC exerts cardioprotective effects against doxorubicin-induced toxicity, potentially via inhibition of ROS accumulation
    Article Snippet: .. Overexpression of LRPPRC was achieved via transient transfection as aforementioned. ..

    Article Title: Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism
    Article Snippet: .. Transient transfection of Nsp1 Approximately 2×105 cells were seeded without antibiotics on 24-well plates. .. The following day, 0.2 μg of FH-Nsp1 or Nsp1-HF was transfected into cells using Lipofectamine 2000 (Thermo-Fisher) according to the manufacturer’s protocol.

    Article Title: PROTEIN THERAPEUTICS FOR JUNCTIONAL EPIDERMOLYSIS BULLOSA: INCORPORATION OF RECOMBINANT ?3 CHAIN INTO LAMININ 332 IN ?3-/- KERATINOCYTES IN VITRO
    Article Snippet: .. Optimization of DNA transfection into 293F cells was carried out using 293Fectin™ transfection reagent (Invitrogen, Carlsbad, CA) and Amaxa nucleofection reaction (Amaxa Inc, Köln, Germany). .. Two and 4 μg of the pMaxGFP plasmid encoding green fluorescent protein (Amaxa Inc, Köln, Germany) were used for transfection and nucleofection, respectively.

    Article Title: Tumor suppressive microRNA-1285 regulates novel molecular targets: Aberrant expression and functional significance in renal cell carcinoma
    Article Snippet: .. Effect of miR-1285 restoration on cell proliferation, migration and invasion in RCC cell lines To investigate the functional significance of miR-1285 , we performed gain-of-function studies using transient transfection with mature miR-1285 . .. We utilized two sources of mature miR-1285 (Ambion and Thermo) to ensure reproducibility of the data.

    Article Title: Activating NK- receptors, homing selectins and inhibitory Siglecs recognize EBOLA-GP and HPV-L1NK
    Article Snippet: .. P24 antigen capture assay Lentivirus particles based on the human immunodeficiency virus-1 (HIV-1) were produced in HEK-293 cells through transient transfection of 2 plasmids encoding components of the virus envelope. .. Cell culture medium containing viral particles produced by packaging cells was harvested after 72 h. HEK-293 cells stably transfected with the plasmid pcDNA6/V5 HisB-EBOV-GP) and control HEK-293 cells stably transfected with pcDNA6/V5 HisB were used to produce lentiviral particles displaying EBOV-GP (lenti-EBOV-GP) and control particles devoid of EBOV-GP.

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities
    Article Snippet: .. Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes. .. Briefly, 150,000 4T1 cells were seeded onto 24 wells plates or 225,000 mES cells were seeded onto gelatin coated 24 well plates, followed by transfection of 100 ng transposase plasmid and 400 ng transponon plasmid prepared via mixing with Lipofectamine 2000.

    Article Title: hnRNP K Supports High-Amplitude D Site-Binding Protein mRNA (Dbp mRNA) Oscillation To Sustain Circadian Rhythms
    Article Snippet: .. Transient transfection and RNAi. .. Lipofectamine 2000 (Invitrogen), Metafectene (Bionex), and the Neon transfection system (Invitrogen) were used for transient transfection as recommended by the manufacturers.

    Produced:

    Article Title: Activating NK- receptors, homing selectins and inhibitory Siglecs recognize EBOLA-GP and HPV-L1NK
    Article Snippet: .. P24 antigen capture assay Lentivirus particles based on the human immunodeficiency virus-1 (HIV-1) were produced in HEK-293 cells through transient transfection of 2 plasmids encoding components of the virus envelope. .. Cell culture medium containing viral particles produced by packaging cells was harvested after 72 h. HEK-293 cells stably transfected with the plasmid pcDNA6/V5 HisB-EBOV-GP) and control HEK-293 cells stably transfected with pcDNA6/V5 HisB were used to produce lentiviral particles displaying EBOV-GP (lenti-EBOV-GP) and control particles devoid of EBOV-GP.

    Generated:

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities
    Article Snippet: .. Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes. .. Briefly, 150,000 4T1 cells were seeded onto 24 wells plates or 225,000 mES cells were seeded onto gelatin coated 24 well plates, followed by transfection of 100 ng transposase plasmid and 400 ng transponon plasmid prepared via mixing with Lipofectamine 2000.

    Migration:

    Article Title: Tumor suppressive microRNA-1285 regulates novel molecular targets: Aberrant expression and functional significance in renal cell carcinoma
    Article Snippet: .. Effect of miR-1285 restoration on cell proliferation, migration and invasion in RCC cell lines To investigate the functional significance of miR-1285 , we performed gain-of-function studies using transient transfection with mature miR-1285 . .. We utilized two sources of mature miR-1285 (Ambion and Thermo) to ensure reproducibility of the data.

    Over Expression:

    Article Title: Mitochondria-associated protein LRPPRC exerts cardioprotective effects against doxorubicin-induced toxicity, potentially via inhibition of ROS accumulation
    Article Snippet: .. Overexpression of LRPPRC was achieved via transient transfection as aforementioned. ..

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    Thermo Fisher transient transfection
    Effect of silencing of TGM2 in two RCC cell lines (A) Cell proliferation determined with XTT assays of A498 and 768-O cell lines after 72 h <t>transfection</t> with 10nM si- TRM2 , miR-control or mock. Cell proliferation was significantly inhibited in the two si- TGM2 transfectants in comparison with the nontransfectants (mock) and the si-control transfectants. Thus, the percentage of cell viability for A498 was 60.6 ± 4.2%, 62.0 ± 5.9%, 100.0 ± 5.5%, and 92.3 ± 6.3%, respectively (P
    Transient Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Thermo Fisher
    Average 99 stars, based on 5255 article reviews
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    transient transfection - by Bioz Stars, 2020-09
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    Effect of silencing of TGM2 in two RCC cell lines (A) Cell proliferation determined with XTT assays of A498 and 768-O cell lines after 72 h transfection with 10nM si- TRM2 , miR-control or mock. Cell proliferation was significantly inhibited in the two si- TGM2 transfectants in comparison with the nontransfectants (mock) and the si-control transfectants. Thus, the percentage of cell viability for A498 was 60.6 ± 4.2%, 62.0 ± 5.9%, 100.0 ± 5.5%, and 92.3 ± 6.3%, respectively (P

    Journal: Oncotarget

    Article Title: Tumor suppressive microRNA-1285 regulates novel molecular targets: Aberrant expression and functional significance in renal cell carcinoma

    doi:

    Figure Lengend Snippet: Effect of silencing of TGM2 in two RCC cell lines (A) Cell proliferation determined with XTT assays of A498 and 768-O cell lines after 72 h transfection with 10nM si- TRM2 , miR-control or mock. Cell proliferation was significantly inhibited in the two si- TGM2 transfectants in comparison with the nontransfectants (mock) and the si-control transfectants. Thus, the percentage of cell viability for A498 was 60.6 ± 4.2%, 62.0 ± 5.9%, 100.0 ± 5.5%, and 92.3 ± 6.3%, respectively (P

    Article Snippet: Effect of miR-1285 restoration on cell proliferation, migration and invasion in RCC cell lines To investigate the functional significance of miR-1285 , we performed gain-of-function studies using transient transfection with mature miR-1285 .

    Techniques: Transfection

    Effect of mature miR-1285 transfection in RCC cell lines (A) Cell proliferation was determined with XTT assays of A498 and 768-O cell lines after 72 h transfection with 10 nM miR-1285 , miR-control and mock. Cell proliferation was significantly inhibited in miR-1285 transfectants in comparison with the mock cells. Thus, with the Ambion products, the percentage of cell viability relative to mock cells was 20.3 ± 1.7% for A498, and 20.1 ± 0.7% for 786-O (both P

    Journal: Oncotarget

    Article Title: Tumor suppressive microRNA-1285 regulates novel molecular targets: Aberrant expression and functional significance in renal cell carcinoma

    doi:

    Figure Lengend Snippet: Effect of mature miR-1285 transfection in RCC cell lines (A) Cell proliferation was determined with XTT assays of A498 and 768-O cell lines after 72 h transfection with 10 nM miR-1285 , miR-control and mock. Cell proliferation was significantly inhibited in miR-1285 transfectants in comparison with the mock cells. Thus, with the Ambion products, the percentage of cell viability relative to mock cells was 20.3 ± 1.7% for A498, and 20.1 ± 0.7% for 786-O (both P

    Article Snippet: Effect of miR-1285 restoration on cell proliferation, migration and invasion in RCC cell lines To investigate the functional significance of miR-1285 , we performed gain-of-function studies using transient transfection with mature miR-1285 .

    Techniques: Transfection

    miR-1285 directly regulates TGM2 in RCC cells (A) Expression level of TGM2 mRNA in clinical RCC cell specimens. Relative TGM2 mRNA expression levels are expressed in box plots. (B) The mRNA expression levels of TGM2 in RCC cell lines (A498 and 786-O) compared to normal kidney RNA. GUSB was used as an internal control. (C) TGM2 mRNA expression in RCC cell lines (A498 and 786-O). TGM2 mRNA expression 24 h after transfection with 10 nM miR-1285 . GUSB was used as an internal control. (D) TGM2 protein expression in RCC cell lines (A498 and 786-O). TGM2 protein expression 72 h after transfection with 10 nM miR-1285 . GAPDH was used as a loading control. E) miRNA-1285 binding sites in the 3'UTR of TGM2 mRNA. A luciferase assay using the vector encoding full-length 3'UTR of TGM2 mRNA. The Renilla luciferase values were normalized to firefly luciferase values. *P

    Journal: Oncotarget

    Article Title: Tumor suppressive microRNA-1285 regulates novel molecular targets: Aberrant expression and functional significance in renal cell carcinoma

    doi:

    Figure Lengend Snippet: miR-1285 directly regulates TGM2 in RCC cells (A) Expression level of TGM2 mRNA in clinical RCC cell specimens. Relative TGM2 mRNA expression levels are expressed in box plots. (B) The mRNA expression levels of TGM2 in RCC cell lines (A498 and 786-O) compared to normal kidney RNA. GUSB was used as an internal control. (C) TGM2 mRNA expression in RCC cell lines (A498 and 786-O). TGM2 mRNA expression 24 h after transfection with 10 nM miR-1285 . GUSB was used as an internal control. (D) TGM2 protein expression in RCC cell lines (A498 and 786-O). TGM2 protein expression 72 h after transfection with 10 nM miR-1285 . GAPDH was used as a loading control. E) miRNA-1285 binding sites in the 3'UTR of TGM2 mRNA. A luciferase assay using the vector encoding full-length 3'UTR of TGM2 mRNA. The Renilla luciferase values were normalized to firefly luciferase values. *P

    Article Snippet: Effect of miR-1285 restoration on cell proliferation, migration and invasion in RCC cell lines To investigate the functional significance of miR-1285 , we performed gain-of-function studies using transient transfection with mature miR-1285 .

    Techniques: Expressing, Transfection, Binding Assay, Luciferase, Plasmid Preparation

    Silencing of TGM2 in two RCC cell lines by si- TGM2 (A) TGM2 mRNA expression after 24 hr of transfection with 10 nM si- TGM2 in RCC cell lines (A498 and 786-O). GUSB was used as an internal control. (B) TGM2 protein expression after 72 hr transfection with si- TGM2 . GAPDH was used a loading control. *P

    Journal: Oncotarget

    Article Title: Tumor suppressive microRNA-1285 regulates novel molecular targets: Aberrant expression and functional significance in renal cell carcinoma

    doi:

    Figure Lengend Snippet: Silencing of TGM2 in two RCC cell lines by si- TGM2 (A) TGM2 mRNA expression after 24 hr of transfection with 10 nM si- TGM2 in RCC cell lines (A498 and 786-O). GUSB was used as an internal control. (B) TGM2 protein expression after 72 hr transfection with si- TGM2 . GAPDH was used a loading control. *P

    Article Snippet: Effect of miR-1285 restoration on cell proliferation, migration and invasion in RCC cell lines To investigate the functional significance of miR-1285 , we performed gain-of-function studies using transient transfection with mature miR-1285 .

    Techniques: Expressing, Transfection

    Screening of tumor suppressive microRNAs in RCC (A-D) Effect of cell proliferation determined by XTT assays using mature miRNAs ( miR-141 , miR-200c , miR-187 , miR-509-5p , miR-135a, miR-508-3p, miR-1285, miR-206, miR-218, miR-133b, miR-1291, let-7g *, miR-204, miR-429, miR-370, miR-363, miR-335, miR-1, miR-1255b , and miR-362-3p ) after 72 h transfection of RCC cell lines (A498, 786-O, ACHN and caki2). *P

    Journal: Oncotarget

    Article Title: Tumor suppressive microRNA-1285 regulates novel molecular targets: Aberrant expression and functional significance in renal cell carcinoma

    doi:

    Figure Lengend Snippet: Screening of tumor suppressive microRNAs in RCC (A-D) Effect of cell proliferation determined by XTT assays using mature miRNAs ( miR-141 , miR-200c , miR-187 , miR-509-5p , miR-135a, miR-508-3p, miR-1285, miR-206, miR-218, miR-133b, miR-1291, let-7g *, miR-204, miR-429, miR-370, miR-363, miR-335, miR-1, miR-1255b , and miR-362-3p ) after 72 h transfection of RCC cell lines (A498, 786-O, ACHN and caki2). *P

    Article Snippet: Effect of miR-1285 restoration on cell proliferation, migration and invasion in RCC cell lines To investigate the functional significance of miR-1285 , we performed gain-of-function studies using transient transfection with mature miR-1285 .

    Techniques: Transfection

    Related to Figure 6: Functionality of individual arms of the ramified cascade of pGuides A. Schema depicting single pGuide activation that results in mutagenesis of APT1A1 and resistance to ouabain. B. A previously reported sgRNA sequence (REF)(Agudelo et al, Nat. Meth. 2017) was used to generate ouabain-resistance mutations in Cas9-expressing cells. The same target sequence was used for a pGuide, which was activated in cells with co-expression of an aGuide. The ability of sgRNA or pGuide to mediate ouabain resistance was compared by transfecting HEK293 cells with guide RNA and Cas9 expression plasmids. Cells were by treated with ouabain five days after transfection and allowed to grow for six days before measuring viability. Viability was determined by staining cells with Calcein AM and dividing the number of Calcein AM positive cells by the total number of starting cells for each condition. Data represent mean +/−standard deviation of n = 3 biological replicates. C. Representative images from the Celigo detecting all cells: brightfield and Hoeschst, and live cells: Calcein AM in conditions containing pGuides only (-aGuide) or mGuides (+ aGuide). D. For the traffic light reporter gene to be converted to encode for a functional red fluorescent protein (RFP), a +2bp insertion or a - 1bp deletion is required to shift the frame and enable the RFP to be translated in frame. (Top). Three sgRNA (TLR#1-#3) were assessed for the frequency by which they generated RFP-positive cells after transfection into AAVS::TLR cells. Note that TLR#3 is identical to GFP5 ( Fig. S1C ). (bottom) RFP positive cells were detected using Celigo-based imaging. Brightfield was used as the measure for total cell number and the subsequent denominator to determine percent RFP positive cells. Data are displayed as the mean +/−standard deviation of n = 3 biological replicates E. Representative images from the Celigo instrument used for counting total cells and RFP-positive cells and used for comparison of sgRNA in panel A. F. Schematic depicting activation of a single pGuide to target the TLR and induce expression of RFP. G. Induction of RFP resulting from sgRNA or a tetraloop pGuide was measured at 48 and 96 hours post transfection of sgRNA and Cas9 expression plasmids. Data represent mean +/−standard deviation of 3 biological replicates.

    Journal: bioRxiv

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

    doi: 10.1101/2020.06.20.162982

    Figure Lengend Snippet: Related to Figure 6: Functionality of individual arms of the ramified cascade of pGuides A. Schema depicting single pGuide activation that results in mutagenesis of APT1A1 and resistance to ouabain. B. A previously reported sgRNA sequence (REF)(Agudelo et al, Nat. Meth. 2017) was used to generate ouabain-resistance mutations in Cas9-expressing cells. The same target sequence was used for a pGuide, which was activated in cells with co-expression of an aGuide. The ability of sgRNA or pGuide to mediate ouabain resistance was compared by transfecting HEK293 cells with guide RNA and Cas9 expression plasmids. Cells were by treated with ouabain five days after transfection and allowed to grow for six days before measuring viability. Viability was determined by staining cells with Calcein AM and dividing the number of Calcein AM positive cells by the total number of starting cells for each condition. Data represent mean +/−standard deviation of n = 3 biological replicates. C. Representative images from the Celigo detecting all cells: brightfield and Hoeschst, and live cells: Calcein AM in conditions containing pGuides only (-aGuide) or mGuides (+ aGuide). D. For the traffic light reporter gene to be converted to encode for a functional red fluorescent protein (RFP), a +2bp insertion or a - 1bp deletion is required to shift the frame and enable the RFP to be translated in frame. (Top). Three sgRNA (TLR#1-#3) were assessed for the frequency by which they generated RFP-positive cells after transfection into AAVS::TLR cells. Note that TLR#3 is identical to GFP5 ( Fig. S1C ). (bottom) RFP positive cells were detected using Celigo-based imaging. Brightfield was used as the measure for total cell number and the subsequent denominator to determine percent RFP positive cells. Data are displayed as the mean +/−standard deviation of n = 3 biological replicates E. Representative images from the Celigo instrument used for counting total cells and RFP-positive cells and used for comparison of sgRNA in panel A. F. Schematic depicting activation of a single pGuide to target the TLR and induce expression of RFP. G. Induction of RFP resulting from sgRNA or a tetraloop pGuide was measured at 48 and 96 hours post transfection of sgRNA and Cas9 expression plasmids. Data represent mean +/−standard deviation of 3 biological replicates.

    Article Snippet: Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes.

    Techniques: Activation Assay, Mutagenesis, Sequencing, Expressing, Transfection, Staining, Standard Deviation, Functional Assay, Generated, Imaging

    Conversion of pGuide to an active mGuide state by removal of ribozyme encoding DNA. A, A’. The overall experimental approach is illustrated in Fig. 2E . The activity of mGuides was measured by loss of EGFP expression from Rex1::GFPd2 cells harboring genomically integrated pGuide DNA at indicated times after transient transfection of aGuide expression plasmid. EGFP disruption was assessed by loss of GFP fluorescence detected by flow cytometry. A: EGFP disruption values over 125 hours for tetraloop pGuides +/−aGuide, hairpin 1 pGuides +/−aGuides, and the positive control sgRNA targeting EGFP. A’: Rate of EGFP disruption as a function of time for the data in (A). Data are displayed as dots representing n = 3 biological replicates, lines being the average of all three dots for each condition, and gray shading representing the standard deviation. B. Schematic depicting the process of ribozyme excision via genome editing. Identical target cut sites flank the ribozyme within sequences called “stems.” The example shown depicts excision of the ribozyme from the tetraloop variant; the same design was applied to the hairpin variant. Excision of the ribozyme via spCas9 cutting at each stem and repair by end joining results in a deletion of 85bp. Note that the stems are designed to favor this large excision to be the predominant repair event in cells. C. Stem sequences stimulate deletion of entire ribozyme from a genomically integrated pGuide. A tetraloop variant pGuide (diagrammed in A) was integrated into the genome of mESC::Rex1 cells. These cells were transiently transfected with plasmids expressing spCas9 and an aGuide, and genomic DNA was isolated 40 hours after transfection. The pGuide/mGuide was amplified via PCR and subjected to deep sequencing. The predominant mutation detected was an 85 bp deletion (middle graph, blue bars). Data represent distribution of 29,241 DNA sequencing reads from one of two replicates. D. Non-converting indel mutations within the stems sequences occur via indel mutation at each stem sequence without deletion between the two stems (Left). Deep sequencing showed that double indels was a rare event, occurring in less than 1.5% of the 29,241 sequencing reads. Data is representative from one of two replicates.

    Journal: bioRxiv

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

    doi: 10.1101/2020.06.20.162982

    Figure Lengend Snippet: Conversion of pGuide to an active mGuide state by removal of ribozyme encoding DNA. A, A’. The overall experimental approach is illustrated in Fig. 2E . The activity of mGuides was measured by loss of EGFP expression from Rex1::GFPd2 cells harboring genomically integrated pGuide DNA at indicated times after transient transfection of aGuide expression plasmid. EGFP disruption was assessed by loss of GFP fluorescence detected by flow cytometry. A: EGFP disruption values over 125 hours for tetraloop pGuides +/−aGuide, hairpin 1 pGuides +/−aGuides, and the positive control sgRNA targeting EGFP. A’: Rate of EGFP disruption as a function of time for the data in (A). Data are displayed as dots representing n = 3 biological replicates, lines being the average of all three dots for each condition, and gray shading representing the standard deviation. B. Schematic depicting the process of ribozyme excision via genome editing. Identical target cut sites flank the ribozyme within sequences called “stems.” The example shown depicts excision of the ribozyme from the tetraloop variant; the same design was applied to the hairpin variant. Excision of the ribozyme via spCas9 cutting at each stem and repair by end joining results in a deletion of 85bp. Note that the stems are designed to favor this large excision to be the predominant repair event in cells. C. Stem sequences stimulate deletion of entire ribozyme from a genomically integrated pGuide. A tetraloop variant pGuide (diagrammed in A) was integrated into the genome of mESC::Rex1 cells. These cells were transiently transfected with plasmids expressing spCas9 and an aGuide, and genomic DNA was isolated 40 hours after transfection. The pGuide/mGuide was amplified via PCR and subjected to deep sequencing. The predominant mutation detected was an 85 bp deletion (middle graph, blue bars). Data represent distribution of 29,241 DNA sequencing reads from one of two replicates. D. Non-converting indel mutations within the stems sequences occur via indel mutation at each stem sequence without deletion between the two stems (Left). Deep sequencing showed that double indels was a rare event, occurring in less than 1.5% of the 29,241 sequencing reads. Data is representative from one of two replicates.

    Article Snippet: Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes.

    Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Fluorescence, Flow Cytometry, Positive Control, Standard Deviation, Variant Assay, Isolation, Amplification, Polymerase Chain Reaction, Sequencing, Mutagenesis, DNA Sequencing

    Related to Figure 1 and 2: pGuide inactivity is RNA-based on cis -cleaving ribozymes. A. Several modalities for inactivating sgRNA were tested via insertion of elements into the sgRNA-encoding gene. Initial testing of ribozymes used insertions of a new sequence at the end of hairpin 1. Shown here are the structures of three ribozymes that were inserted and tested in vitro and in cells. The red arrow denotes the cis-cleavage site for each ribozyme. B. pGuides self-destruct during in vitro transcription. Each pGuide variant containing a unique ribozyme at hairpin 1 was cloned into plasmid in which a T7 promoter directed in vitro transcription of the pGuide. Transcription reaction products were analyzed via EtBr staining on a TBE-urea denaturing gel. The sgRNA gene (no ribozyme insertion) generated a single 100nt band, corresponding to intact sgRNA. All three pGuide gene variants result in high levels of cleavage of the transcribed RNA. Low levels of full-length, uncleaved pGuide were at detected at varying amounts depending on the ribozyme. The HDV ribozyme appeared to be the most efficient in vitro . Uncleaved ribozymes may result from an insufficient levels of Mg 2+ in the in vitro reaction; Mg 2+ is used both by T7 RNA polymerase for transcription and by ribozymes as a cofactor required for catalysis. C. Schematic showing locations of target sites utilized for assessing genome editing by pGuides. The DNA sequences for sites GFP1-6 are listed in Table S1. Base pair numbering begins with +1 at the first coding nucleotide for EGFP. D. Mouse 4T1 cells harboring a single genomic insertion of a heterologous EGFP gene were used to assess spCas9 activity. In separate transient transfections, sgRNA targeting six different sites (sgGFP1-6) throughout the EGFP coding sequence were tested. For each target site, the normal sgRNA without a ribozyme insertion and pGuides with either a Hammerhead, HDV, or Twister ribozyme were generated and tested. Mutagenesis of the EGFP gene is inferred from loss of EGFP fluorescence measured by flow cytometry performed five days after transfections. Data represent mean +/−range of n = 2 biological replicates.

    Journal: bioRxiv

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

    doi: 10.1101/2020.06.20.162982

    Figure Lengend Snippet: Related to Figure 1 and 2: pGuide inactivity is RNA-based on cis -cleaving ribozymes. A. Several modalities for inactivating sgRNA were tested via insertion of elements into the sgRNA-encoding gene. Initial testing of ribozymes used insertions of a new sequence at the end of hairpin 1. Shown here are the structures of three ribozymes that were inserted and tested in vitro and in cells. The red arrow denotes the cis-cleavage site for each ribozyme. B. pGuides self-destruct during in vitro transcription. Each pGuide variant containing a unique ribozyme at hairpin 1 was cloned into plasmid in which a T7 promoter directed in vitro transcription of the pGuide. Transcription reaction products were analyzed via EtBr staining on a TBE-urea denaturing gel. The sgRNA gene (no ribozyme insertion) generated a single 100nt band, corresponding to intact sgRNA. All three pGuide gene variants result in high levels of cleavage of the transcribed RNA. Low levels of full-length, uncleaved pGuide were at detected at varying amounts depending on the ribozyme. The HDV ribozyme appeared to be the most efficient in vitro . Uncleaved ribozymes may result from an insufficient levels of Mg 2+ in the in vitro reaction; Mg 2+ is used both by T7 RNA polymerase for transcription and by ribozymes as a cofactor required for catalysis. C. Schematic showing locations of target sites utilized for assessing genome editing by pGuides. The DNA sequences for sites GFP1-6 are listed in Table S1. Base pair numbering begins with +1 at the first coding nucleotide for EGFP. D. Mouse 4T1 cells harboring a single genomic insertion of a heterologous EGFP gene were used to assess spCas9 activity. In separate transient transfections, sgRNA targeting six different sites (sgGFP1-6) throughout the EGFP coding sequence were tested. For each target site, the normal sgRNA without a ribozyme insertion and pGuides with either a Hammerhead, HDV, or Twister ribozyme were generated and tested. Mutagenesis of the EGFP gene is inferred from loss of EGFP fluorescence measured by flow cytometry performed five days after transfections. Data represent mean +/−range of n = 2 biological replicates.

    Article Snippet: Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes.

    Techniques: Sequencing, In Vitro, Variant Assay, Clone Assay, Plasmid Preparation, Staining, Generated, Activity Assay, Transfection, Mutagenesis, Fluorescence, Flow Cytometry

    Related to Figure 6: A. Data represents raw values prior to normalization corresponding to Fig. 6B . Plasmid DNAs encoding Cas9Cdt1 and pGuides targeted to ATP1A1 and the TLR were transfected into HEK293 harboring a single genomic TLR cassette (see Fig. 6A for sequential targeting strategy). Two and four days after transfections, cells were plated into ouabain followed by immediate measurement of RFP positive cells using a Celigo imager. After 6 days of continuous culturing in ouabain, viable cells were measured via Calcein AM staining and divided by the initial cell number measured upon plating into ouabain. Data represent mean +/−standard deviation of 3 biological replicates. B. Data represents raw values prior to normalization corresponding to Fig. 6D . To compare the efficiency and kinetics of the pGuide cascade to sgRNA in generating double positive ouabain resistant, RFP expressing cells, viable cells that were all Calcein AM positive cells were counted followed by assessment of RFP positivity within the viable subpopulation. p-values are presented to compare the sgRNA condition to the pGuide condition to address differences in kinetics, revealing that the pGuide cascade exhibits delayed kinetics but similar efficiencies as sgRNAs. p-values are located above or below the lines drawn between two conditions and were calculated using Welch’s two sided t-test. Data represent mean +/−standard deviation of n=3 biological replicates.

    Journal: bioRxiv

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

    doi: 10.1101/2020.06.20.162982

    Figure Lengend Snippet: Related to Figure 6: A. Data represents raw values prior to normalization corresponding to Fig. 6B . Plasmid DNAs encoding Cas9Cdt1 and pGuides targeted to ATP1A1 and the TLR were transfected into HEK293 harboring a single genomic TLR cassette (see Fig. 6A for sequential targeting strategy). Two and four days after transfections, cells were plated into ouabain followed by immediate measurement of RFP positive cells using a Celigo imager. After 6 days of continuous culturing in ouabain, viable cells were measured via Calcein AM staining and divided by the initial cell number measured upon plating into ouabain. Data represent mean +/−standard deviation of 3 biological replicates. B. Data represents raw values prior to normalization corresponding to Fig. 6D . To compare the efficiency and kinetics of the pGuide cascade to sgRNA in generating double positive ouabain resistant, RFP expressing cells, viable cells that were all Calcein AM positive cells were counted followed by assessment of RFP positivity within the viable subpopulation. p-values are presented to compare the sgRNA condition to the pGuide condition to address differences in kinetics, revealing that the pGuide cascade exhibits delayed kinetics but similar efficiencies as sgRNAs. p-values are located above or below the lines drawn between two conditions and were calculated using Welch’s two sided t-test. Data represent mean +/−standard deviation of n=3 biological replicates.

    Article Snippet: Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes.

    Techniques: Plasmid Preparation, Transfection, Staining, Standard Deviation, Expressing

    Utility of plasmid based pGuides. A. Schema highlighting steps in the conversion of an episomal pGuide to an mGuide via an episomal aGuide. Elements of the conversion are provided via transient transfection of plasmid DNA, which do not require genomic integration for their activity. Multiple copies of a pGuide plasmid causes cells to express both latent pGuide (OFF) and activated mGuide (ON) RNA following activity of the aGuide. The presence of the mGuide RNA targets spCas9 to the genome. B. The relative effectiveness of a genomically integrated pGuide (Genomic) and an episomal pGuide (Plasmids) was assessed in Rex1::GFPd2 cells by loss of GFP fluorescence following transient transfection of plasmid DNA. As a positive control, an sgRNA targeting the same sequence in EGFP was transfected without pGuides. Exclusion of the aGuide plasmid from transfections was used to assess activity that was dependent upon conversion of the pGuide. Data are displayed as the mean +/− standard deviation of n = 3 biological replicates. C. The effect of the mGuide target site on its genome editing activity was assessed for three different target site sequences (GFP1, GFP2, GFP5; see also Fig. S1C and Table S1). spCas9-, pGuide-, and aGuide-expression plasmids were transiently transfected into Rex1::GFPd2 cells, and GFP disruption was measured three days after transfection. The sgRNA and –aGuide controls are as described in (B). Data are displayed as the mean +/−range of n = 2 biological replicates. D. The effects of elements of the stem sequences on conversion of pGuides was assessed for six different stem sequences containing different spCas9 target sites (see Fig. 1G, H and Table S1 for nucleotide sequences). Conversion was measured by targeted each pGuide to EGFP and assessing disruption frequency as in B and C. Relative EGFP disruption frequencies were calculated by dividing EGF disruption from the pGuide conditions by EGFP disruption frequencies mediated by the sgRNA positive control. Data are displayed as mean +/−the range for n = 2 biological replicates.

    Journal: bioRxiv

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

    doi: 10.1101/2020.06.20.162982

    Figure Lengend Snippet: Utility of plasmid based pGuides. A. Schema highlighting steps in the conversion of an episomal pGuide to an mGuide via an episomal aGuide. Elements of the conversion are provided via transient transfection of plasmid DNA, which do not require genomic integration for their activity. Multiple copies of a pGuide plasmid causes cells to express both latent pGuide (OFF) and activated mGuide (ON) RNA following activity of the aGuide. The presence of the mGuide RNA targets spCas9 to the genome. B. The relative effectiveness of a genomically integrated pGuide (Genomic) and an episomal pGuide (Plasmids) was assessed in Rex1::GFPd2 cells by loss of GFP fluorescence following transient transfection of plasmid DNA. As a positive control, an sgRNA targeting the same sequence in EGFP was transfected without pGuides. Exclusion of the aGuide plasmid from transfections was used to assess activity that was dependent upon conversion of the pGuide. Data are displayed as the mean +/− standard deviation of n = 3 biological replicates. C. The effect of the mGuide target site on its genome editing activity was assessed for three different target site sequences (GFP1, GFP2, GFP5; see also Fig. S1C and Table S1). spCas9-, pGuide-, and aGuide-expression plasmids were transiently transfected into Rex1::GFPd2 cells, and GFP disruption was measured three days after transfection. The sgRNA and –aGuide controls are as described in (B). Data are displayed as the mean +/−range of n = 2 biological replicates. D. The effects of elements of the stem sequences on conversion of pGuides was assessed for six different stem sequences containing different spCas9 target sites (see Fig. 1G, H and Table S1 for nucleotide sequences). Conversion was measured by targeted each pGuide to EGFP and assessing disruption frequency as in B and C. Relative EGFP disruption frequencies were calculated by dividing EGF disruption from the pGuide conditions by EGFP disruption frequencies mediated by the sgRNA positive control. Data are displayed as mean +/−the range for n = 2 biological replicates.

    Article Snippet: Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes.

    Techniques: Plasmid Preparation, Transfection, Activity Assay, Fluorescence, Positive Control, Sequencing, Standard Deviation, Expressing

    Related to Figure 5: Four event linear cascade of pGuides in cells. Transient transfection of guide RNA and spCas9 expression plasmids into Rex1::GFPd2 cells followed by analysis of EGFP levels was performed to assess the efficiency of cascade completion. Experiment and analyses are identical to those for Fig 5F , with the exception that spCas9 was used instead of Cas9Cdt1. Error bars represent +/−s.d. for n = 3 biological replicates. p-values were calculated using Welch’s two sided t-test.

    Journal: bioRxiv

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

    doi: 10.1101/2020.06.20.162982

    Figure Lengend Snippet: Related to Figure 5: Four event linear cascade of pGuides in cells. Transient transfection of guide RNA and spCas9 expression plasmids into Rex1::GFPd2 cells followed by analysis of EGFP levels was performed to assess the efficiency of cascade completion. Experiment and analyses are identical to those for Fig 5F , with the exception that spCas9 was used instead of Cas9Cdt1. Error bars represent +/−s.d. for n = 3 biological replicates. p-values were calculated using Welch’s two sided t-test.

    Article Snippet: Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes.

    Techniques: Transfection, Expressing

    Programming temporal ordering of spCas9 activities via a genome-independent, plasmid-based cascades of pGuides. A. Schematic of a ramified five-step pGuide cascade that targets the genome at two sites (TLR and ATP1A1) through execution of these five spCas9-mediated activieis: (left side) 1) activation of pGuide 1.1 by aGuide, 2) activation of pGuide 2 by mGuide 1.1, 3) editing of TLR gene by mGuide 2 to induce RFP expression, and (right side) 4) activation of pGuide 1.2 by aGuide, and 5) editing of ATP1A1 gene by mGuide 1.2 to provide ouabain resistance. See Fig S5B-C for pGuide editing the ATP1A1 gene and Fig. S65F-G for pGuide editing the TLR gene. B. Left: Plasmid DNA constituting the ramified cascade and Cas9Cdt1 shown in (A) were transiently transfected into HEK293 cells harboring a genomic copy of TLR. Progression to the two genomic endpoints was assessed by resistance to 0.5uM ouabain for the ATP1A1 gene and RFP fluorescence for the TLR gene. Resistance to ouabain was determined by viable cell staining with Calcein AM after six days of selection in ouabain-containing media. Right: Percent viability was determined by dividing the number Calcein AM positive cells after six days of ouabain treatment by the number of starting cells plated at two days or four days after transfection. The percent RFP positive was determined by measuring individual cell fluorescence with a Celigo instrument within an hour of plating cells (two and four days). Data are normalized to levels of viable cells and RFP positive cells generated by transfection of sgRNA targeting ATP1A1 and TLR, respectively, from concurrent transfections. See Fig. S6 for data prior to normalization. Data represent means +/−standard deviation of n = 3 biological replicates. p-values are located above or below the lines drawn between two conditions and were calculated using Welch’s two sided t-test. C. Representative images of cells that underwent six days of ouabain treatment initiated two days after transfection of plasmid DNA. Fluorescent images of entire well from a 96 well plate using a Celigo instrument. Images are representative of three biological replicates. D. Percentage of cells that were resistant to ouabain starting at two days and four days and have progressed to become RFP positive by the end of ouabain treatment at eight days and ten days after transfection. Viability and RFP positivity were measured as described in B. Data are normalized to levels of viable cells and RFP positive cells generated by transfection of sgRNA targeting ATP1A1 and TLR, respectively, from concurrent transfections. See Fig. S6 for data prior to normalization. p-values were calculated using Welch’s two sided t-test.

    Journal: bioRxiv

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

    doi: 10.1101/2020.06.20.162982

    Figure Lengend Snippet: Programming temporal ordering of spCas9 activities via a genome-independent, plasmid-based cascades of pGuides. A. Schematic of a ramified five-step pGuide cascade that targets the genome at two sites (TLR and ATP1A1) through execution of these five spCas9-mediated activieis: (left side) 1) activation of pGuide 1.1 by aGuide, 2) activation of pGuide 2 by mGuide 1.1, 3) editing of TLR gene by mGuide 2 to induce RFP expression, and (right side) 4) activation of pGuide 1.2 by aGuide, and 5) editing of ATP1A1 gene by mGuide 1.2 to provide ouabain resistance. See Fig S5B-C for pGuide editing the ATP1A1 gene and Fig. S65F-G for pGuide editing the TLR gene. B. Left: Plasmid DNA constituting the ramified cascade and Cas9Cdt1 shown in (A) were transiently transfected into HEK293 cells harboring a genomic copy of TLR. Progression to the two genomic endpoints was assessed by resistance to 0.5uM ouabain for the ATP1A1 gene and RFP fluorescence for the TLR gene. Resistance to ouabain was determined by viable cell staining with Calcein AM after six days of selection in ouabain-containing media. Right: Percent viability was determined by dividing the number Calcein AM positive cells after six days of ouabain treatment by the number of starting cells plated at two days or four days after transfection. The percent RFP positive was determined by measuring individual cell fluorescence with a Celigo instrument within an hour of plating cells (two and four days). Data are normalized to levels of viable cells and RFP positive cells generated by transfection of sgRNA targeting ATP1A1 and TLR, respectively, from concurrent transfections. See Fig. S6 for data prior to normalization. Data represent means +/−standard deviation of n = 3 biological replicates. p-values are located above or below the lines drawn between two conditions and were calculated using Welch’s two sided t-test. C. Representative images of cells that underwent six days of ouabain treatment initiated two days after transfection of plasmid DNA. Fluorescent images of entire well from a 96 well plate using a Celigo instrument. Images are representative of three biological replicates. D. Percentage of cells that were resistant to ouabain starting at two days and four days and have progressed to become RFP positive by the end of ouabain treatment at eight days and ten days after transfection. Viability and RFP positivity were measured as described in B. Data are normalized to levels of viable cells and RFP positive cells generated by transfection of sgRNA targeting ATP1A1 and TLR, respectively, from concurrent transfections. See Fig. S6 for data prior to normalization. p-values were calculated using Welch’s two sided t-test.

    Article Snippet: Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes.

    Techniques: Plasmid Preparation, Activation Assay, Expressing, Transfection, Fluorescence, Staining, Selection, Generated, Standard Deviation

    Related to Figures 4-6: Cell cycle regulation of spCas9 modulates mGuide mutagenesis kinetics. A. Data show raw values prior to normalization corresponding to Fig. 4D . The effect of the stem sequence (Cas9 target sites flanking the ribozyme) on pGuide to mGuide conversion was examined through generating 6 different tetraloop pGuide variants harboring different stem sequences, followed by an EGFP disruption assay to measure Cas9 activity. mESC::Rex1 cells were transfected with guide RNA and Cas9 expression plasmids, followed by flow cytometry analysis of EGFP levels after 48 hours. Data are displayed as mean +/−range of n = 2 biological replicates. Stem nucleotide sequences are located in Table S1. B. Data represents raw values prior to normalization corresponding to Fig. 5B . A linear cascade of two pGuides executing three sequential genome edits was assessed. Guide RNA plasmids were transfected into cells and EGFP levels were measured 48 hours after via flow cytometry. Data are displayed as standard deviation of n = 3 biological replicates. C. Expression of Cas9-degron fusions. Protein levels of the spCas9 and variants were measured by western blot 24 hours after transiently transfecting Rex1::GFPd2 cells with each plasmid. Cas9Gem and Cas9Cdt1 exhibit ~50% less protein levels than spCas9 and migrate at a higher molecular weight due to fusion of the degron to Cas9. D. Activation of episomal pGuides with Cas9-degron fusions reveals different kinetics associated with cell cycle regulation of Cas9. Data are displayed as mean +/−standard deviation of n = 3 biological replicates. E. Next generation sequencing confirms that modulation of pGuide activation via Cas9Cdt1 and Cas9Gem is due to increases or decreases in mutation frequencies, not changes in mutational outcomes. We examined the possibility that EGFP disruption frequencies were different as a result from cell cycle timed DSBs altering the frequency of in frame mutations relative to frameshift indels. Allele frequencies were determined by deep sequencing of DNA harvested from Rex1::GFPd2 cells 48 hours after transfection with tetraloop pGuides and the spCas9 or variant plasmids from experiment shown in (D). Analysis reveals three informative results: 1) the mutational profiles generated by spCas9 and the two variants are highly concordant, 2) mutation frequencies are increased or decreased for Cas9Cdt1 and Cas9Gem, respectively, and 3) the top two mutant alleles are in frame deletions (12 bp), potentially leading to an observed EGFP loss values that are below actual mutation frequencies. Allele maps are representative of one of two replicates.

    Journal: bioRxiv

    Article Title: Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

    doi: 10.1101/2020.06.20.162982

    Figure Lengend Snippet: Related to Figures 4-6: Cell cycle regulation of spCas9 modulates mGuide mutagenesis kinetics. A. Data show raw values prior to normalization corresponding to Fig. 4D . The effect of the stem sequence (Cas9 target sites flanking the ribozyme) on pGuide to mGuide conversion was examined through generating 6 different tetraloop pGuide variants harboring different stem sequences, followed by an EGFP disruption assay to measure Cas9 activity. mESC::Rex1 cells were transfected with guide RNA and Cas9 expression plasmids, followed by flow cytometry analysis of EGFP levels after 48 hours. Data are displayed as mean +/−range of n = 2 biological replicates. Stem nucleotide sequences are located in Table S1. B. Data represents raw values prior to normalization corresponding to Fig. 5B . A linear cascade of two pGuides executing three sequential genome edits was assessed. Guide RNA plasmids were transfected into cells and EGFP levels were measured 48 hours after via flow cytometry. Data are displayed as standard deviation of n = 3 biological replicates. C. Expression of Cas9-degron fusions. Protein levels of the spCas9 and variants were measured by western blot 24 hours after transiently transfecting Rex1::GFPd2 cells with each plasmid. Cas9Gem and Cas9Cdt1 exhibit ~50% less protein levels than spCas9 and migrate at a higher molecular weight due to fusion of the degron to Cas9. D. Activation of episomal pGuides with Cas9-degron fusions reveals different kinetics associated with cell cycle regulation of Cas9. Data are displayed as mean +/−standard deviation of n = 3 biological replicates. E. Next generation sequencing confirms that modulation of pGuide activation via Cas9Cdt1 and Cas9Gem is due to increases or decreases in mutation frequencies, not changes in mutational outcomes. We examined the possibility that EGFP disruption frequencies were different as a result from cell cycle timed DSBs altering the frequency of in frame mutations relative to frameshift indels. Allele frequencies were determined by deep sequencing of DNA harvested from Rex1::GFPd2 cells 48 hours after transfection with tetraloop pGuides and the spCas9 or variant plasmids from experiment shown in (D). Analysis reveals three informative results: 1) the mutational profiles generated by spCas9 and the two variants are highly concordant, 2) mutation frequencies are increased or decreased for Cas9Cdt1 and Cas9Gem, respectively, and 3) the top two mutant alleles are in frame deletions (12 bp), potentially leading to an observed EGFP loss values that are below actual mutation frequencies. Allele maps are representative of one of two replicates.

    Article Snippet: Generation of cell linesCell lines were generated through transient transfection of piggbac transposase plasmids (HyperPiggybac) and respective pGuide transposon plasmids harboring puromycin resistance cassettes.

    Techniques: Mutagenesis, Sequencing, Activity Assay, Transfection, Expressing, Flow Cytometry, Standard Deviation, Western Blot, Plasmid Preparation, Molecular Weight, Activation Assay, Next-Generation Sequencing, Variant Assay, Generated

    . (B) Motif analysis showed that the CCC motif was frequently found along with a CCT motif. (C) Coomassie blue staining showed that one thick band was bound onto the forward poly(C) motif oligonucleotide, which was identified as hnRNP K by mass spectrometry. In contrast, there were no prominent proteins detected at the complementary poly(C) motif oligonucleotide. (D) Purification of GST and GST-hnRNP K was also carried out for pulldown assay. GST-hnRNP K was detected at the wild-type oligonucleotide but not at the mutant oligonucleotide, which demonstrates that hnRNP K is directly bound to the poly(C) motif. (E) To identify the effect of knockdown of hnRNP K on WT(Short) and the poly(C) mutant, a promoter assay was conducted with siRNA transfection. It showed a significant decrease only for WT(Short) promoter activity ( n = 3; n.s., not significant; *, P

    Journal: Molecular and Cellular Biology

    Article Title: hnRNP K Supports High-Amplitude D Site-Binding Protein mRNA (Dbp mRNA) Oscillation To Sustain Circadian Rhythms

    doi: 10.1128/MCB.00537-19

    Figure Lengend Snippet: . (B) Motif analysis showed that the CCC motif was frequently found along with a CCT motif. (C) Coomassie blue staining showed that one thick band was bound onto the forward poly(C) motif oligonucleotide, which was identified as hnRNP K by mass spectrometry. In contrast, there were no prominent proteins detected at the complementary poly(C) motif oligonucleotide. (D) Purification of GST and GST-hnRNP K was also carried out for pulldown assay. GST-hnRNP K was detected at the wild-type oligonucleotide but not at the mutant oligonucleotide, which demonstrates that hnRNP K is directly bound to the poly(C) motif. (E) To identify the effect of knockdown of hnRNP K on WT(Short) and the poly(C) mutant, a promoter assay was conducted with siRNA transfection. It showed a significant decrease only for WT(Short) promoter activity ( n = 3; n.s., not significant; *, P

    Article Snippet: Transient transfection and RNAi.

    Techniques: Countercurrent Chromatography, Staining, Mass Spectrometry, Purification, Mutagenesis, Promoter Assay, Transfection, Activity Assay

    hnRNP K controls the Dbp mRNA expression but not stability. (A) Knockdown of hnRNP K was mediated by siRNA transfection in NIH 3T3 cells. The group with hnRNP K siRNA pool transfection showed a significant decrease of the hnRNP K protein level ( n = 5; *, P

    Journal: Molecular and Cellular Biology

    Article Title: hnRNP K Supports High-Amplitude D Site-Binding Protein mRNA (Dbp mRNA) Oscillation To Sustain Circadian Rhythms

    doi: 10.1128/MCB.00537-19

    Figure Lengend Snippet: hnRNP K controls the Dbp mRNA expression but not stability. (A) Knockdown of hnRNP K was mediated by siRNA transfection in NIH 3T3 cells. The group with hnRNP K siRNA pool transfection showed a significant decrease of the hnRNP K protein level ( n = 5; *, P

    Article Snippet: Transient transfection and RNAi.

    Techniques: Expressing, Transfection

    LRPPRC overexpression exerts protective effects against DOX-induced cell injury. (A) A total of 48 h post-transfection with siLRPPRC and the coding sequence of LRPPRC, the protein levels of LRPPRC were detected via western blotting. (B) Following LRPPRC overexpression, the cytotoxicity of DOX in H9C2 cells was measured. (C) Cell Counting Kit-8 assay was performed to detect the effect of LRPRC overexpression on cell proliferation under DOX treatment. (D) DOX at IC 30 was utilized for cell treatment for 24 h, followed by PI staining and flow cytometric analysis to detect the cell cycle phases. (E) DOX at IC 50 was employed for cell treatment for 24 h, followed by Annexin V-FITC/PI double staining and flow cytometric analysis to detect apoptotic and non-apoptotic cell death. * P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Mitochondria-associated protein LRPPRC exerts cardioprotective effects against doxorubicin-induced toxicity, potentially via inhibition of ROS accumulation

    doi: 10.3892/etm.2020.9111

    Figure Lengend Snippet: LRPPRC overexpression exerts protective effects against DOX-induced cell injury. (A) A total of 48 h post-transfection with siLRPPRC and the coding sequence of LRPPRC, the protein levels of LRPPRC were detected via western blotting. (B) Following LRPPRC overexpression, the cytotoxicity of DOX in H9C2 cells was measured. (C) Cell Counting Kit-8 assay was performed to detect the effect of LRPRC overexpression on cell proliferation under DOX treatment. (D) DOX at IC 30 was utilized for cell treatment for 24 h, followed by PI staining and flow cytometric analysis to detect the cell cycle phases. (E) DOX at IC 50 was employed for cell treatment for 24 h, followed by Annexin V-FITC/PI double staining and flow cytometric analysis to detect apoptotic and non-apoptotic cell death. * P

    Article Snippet: Overexpression of LRPPRC was achieved via transient transfection as aforementioned.

    Techniques: Over Expression, Transfection, Sequencing, Western Blot, Cell Counting, Staining, Double Staining