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clonal genes  (Twist Bioscience)


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    Structured Review

    Twist Bioscience clonal genes
    Clonal Genes, supplied by Twist Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/vector+construct/Clonal+Genes+Twist+Expression+Vector+per+Construct/pmc12887479-131-11-18
    Average 94 stars, based on 1 article reviews
    clonal genes - by Bioz Stars, 2026-11
    94/100 stars

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    Related Articles

    Synthesized:

    Article Title: Systematic Analysis of the EXO70 Gene Family in Kiwifruit Species: Evolutionary Selection and Potential Functions in Plant Immunity
    Article Snippet: .. Selected AccEXO70 clades were synthesized and cloned into the pTwist Gateway entry vector (Twist Bioscience) and recombined into the prey vector pDEST22. ..

    Article Title: A Novel Patient-Derived Mutation in Glucocorticoid Receptor Reveals an Essential Role for DNA binding in Development and Endocrine Regulation
    Article Snippet: .. DNA containing the T444I mutation and flanking homology arms was synthesized by Twist Biosciences and cloned into the pGMC00018 vector backbone using Gibson assembly , yielding the construct pMG0658. .. This plasmid was then used as a template for long ssDNA production with the Guide-it Long ssDNA Production System v2 (Takara).

    Article Title: Activation of Arp2/3 complex by a SPIN90 dimer in linear actin-filament nucleation.
    Article Snippet: .. Additionally, a truncated SPIN90 (326–722) construct, the W401A mutant and the triple mutant (Q397A, Q398A and W401A) genes were synthesized (Twist Bioscience) and cloned into a pET-28a(+) vector. ..

    Article Title: Predicting TCR antigen specificity at proteome-scale with synthetic immune cells and machine learning
    Article Snippet: Assembly reactions were performed using a 2:1 molar ratio of insert to vector (120 fmol vector), with 2 μL of NEB Golden Gate Enzyme Mix. .. TCR constructs were synthesized (Twist Bioscience) and cloned into the mammalian expression vector pTwist_BetaGlobin_CMV_WPRE (Twist Bioscience). .. Assembled products were amplified by PCR using primer pair KH241/KH240 (see Supplementary Table 6), and purified using the DNA Clean & Concentrator-25 kit (Zymo Research, #D4005) to generate the final HDR templates.

    Article Title: Selective MENA binding peptides
    Article Snippet: .. All sequences of constructed plasmids were confirmed by Sanger sequencing. mRuby2-Pcare and Mito-mRuby2-Pcare inserts were synthesized (Twist Bioscience) and subcloned into a SFFV-promoter lentiviral expression vector. ..

    Clone Assay:

    Article Title: Systematic Analysis of the EXO70 Gene Family in Kiwifruit Species: Evolutionary Selection and Potential Functions in Plant Immunity
    Article Snippet: .. Selected AccEXO70 clades were synthesized and cloned into the pTwist Gateway entry vector (Twist Bioscience) and recombined into the prey vector pDEST22. ..

    Article Title: A Novel Patient-Derived Mutation in Glucocorticoid Receptor Reveals an Essential Role for DNA binding in Development and Endocrine Regulation
    Article Snippet: .. DNA containing the T444I mutation and flanking homology arms was synthesized by Twist Biosciences and cloned into the pGMC00018 vector backbone using Gibson assembly , yielding the construct pMG0658. .. This plasmid was then used as a template for long ssDNA production with the Guide-it Long ssDNA Production System v2 (Takara).

    Article Title: Activation of Arp2/3 complex by a SPIN90 dimer in linear actin-filament nucleation.
    Article Snippet: .. Additionally, a truncated SPIN90 (326–722) construct, the W401A mutant and the triple mutant (Q397A, Q398A and W401A) genes were synthesized (Twist Bioscience) and cloned into a pET-28a(+) vector. ..

    Article Title: Predicting TCR antigen specificity at proteome-scale with synthetic immune cells and machine learning
    Article Snippet: Assembly reactions were performed using a 2:1 molar ratio of insert to vector (120 fmol vector), with 2 μL of NEB Golden Gate Enzyme Mix. .. TCR constructs were synthesized (Twist Bioscience) and cloned into the mammalian expression vector pTwist_BetaGlobin_CMV_WPRE (Twist Bioscience). .. Assembled products were amplified by PCR using primer pair KH241/KH240 (see Supplementary Table 6), and purified using the DNA Clean & Concentrator-25 kit (Zymo Research, #D4005) to generate the final HDR templates.

    Plasmid Preparation:

    Article Title: Systematic Analysis of the EXO70 Gene Family in Kiwifruit Species: Evolutionary Selection and Potential Functions in Plant Immunity
    Article Snippet: .. Selected AccEXO70 clades were synthesized and cloned into the pTwist Gateway entry vector (Twist Bioscience) and recombined into the prey vector pDEST22. ..

    Article Title: Shared PRAME epitopes are T-cell targets in NUT carcinoma
    Article Snippet: .. One primary fusion construct and two auxiliary constructs were generated as clonal genes in mammalian expression vector plasmids (Twist Bioscience). .. One primary fusion construct and two auxiliary constructs were generated as clonal genes in mammalian expression vector plasmids (Twist Bioscience).

    Article Title: A Novel Patient-Derived Mutation in Glucocorticoid Receptor Reveals an Essential Role for DNA binding in Development and Endocrine Regulation
    Article Snippet: .. DNA containing the T444I mutation and flanking homology arms was synthesized by Twist Biosciences and cloned into the pGMC00018 vector backbone using Gibson assembly , yielding the construct pMG0658. .. This plasmid was then used as a template for long ssDNA production with the Guide-it Long ssDNA Production System v2 (Takara).

    Article Title: Predicting TCR antigen specificity at proteome-scale with synthetic immune cells and machine learning
    Article Snippet: Assembly reactions were performed using a 2:1 molar ratio of insert to vector (120 fmol vector), with 2 μL of NEB Golden Gate Enzyme Mix. .. TCR constructs were synthesized (Twist Bioscience) and cloned into the mammalian expression vector pTwist_BetaGlobin_CMV_WPRE (Twist Bioscience). .. Assembled products were amplified by PCR using primer pair KH241/KH240 (see Supplementary Table 6), and purified using the DNA Clean & Concentrator-25 kit (Zymo Research, #D4005) to generate the final HDR templates.

    Construct:

    Article Title: Shared PRAME epitopes are T-cell targets in NUT carcinoma
    Article Snippet: .. One primary fusion construct and two auxiliary constructs were generated as clonal genes in mammalian expression vector plasmids (Twist Bioscience). .. One primary fusion construct and two auxiliary constructs were generated as clonal genes in mammalian expression vector plasmids (Twist Bioscience).

    Article Title: A Novel Patient-Derived Mutation in Glucocorticoid Receptor Reveals an Essential Role for DNA binding in Development and Endocrine Regulation
    Article Snippet: .. DNA containing the T444I mutation and flanking homology arms was synthesized by Twist Biosciences and cloned into the pGMC00018 vector backbone using Gibson assembly , yielding the construct pMG0658. .. This plasmid was then used as a template for long ssDNA production with the Guide-it Long ssDNA Production System v2 (Takara).

    Article Title: Activation of Arp2/3 complex by a SPIN90 dimer in linear actin-filament nucleation.
    Article Snippet: .. Additionally, a truncated SPIN90 (326–722) construct, the W401A mutant and the triple mutant (Q397A, Q398A and W401A) genes were synthesized (Twist Bioscience) and cloned into a pET-28a(+) vector. ..

    Article Title: Predicting TCR antigen specificity at proteome-scale with synthetic immune cells and machine learning
    Article Snippet: Assembly reactions were performed using a 2:1 molar ratio of insert to vector (120 fmol vector), with 2 μL of NEB Golden Gate Enzyme Mix. .. TCR constructs were synthesized (Twist Bioscience) and cloned into the mammalian expression vector pTwist_BetaGlobin_CMV_WPRE (Twist Bioscience). .. Assembled products were amplified by PCR using primer pair KH241/KH240 (see Supplementary Table 6), and purified using the DNA Clean & Concentrator-25 kit (Zymo Research, #D4005) to generate the final HDR templates.

    Article Title: Selective MENA binding peptides
    Article Snippet: .. All sequences of constructed plasmids were confirmed by Sanger sequencing. mRuby2-Pcare and Mito-mRuby2-Pcare inserts were synthesized (Twist Bioscience) and subcloned into a SFFV-promoter lentiviral expression vector. ..

    Generated:

    Article Title: Shared PRAME epitopes are T-cell targets in NUT carcinoma
    Article Snippet: .. One primary fusion construct and two auxiliary constructs were generated as clonal genes in mammalian expression vector plasmids (Twist Bioscience). .. One primary fusion construct and two auxiliary constructs were generated as clonal genes in mammalian expression vector plasmids (Twist Bioscience).

    Expressing:

    Article Title: Shared PRAME epitopes are T-cell targets in NUT carcinoma
    Article Snippet: .. One primary fusion construct and two auxiliary constructs were generated as clonal genes in mammalian expression vector plasmids (Twist Bioscience). .. One primary fusion construct and two auxiliary constructs were generated as clonal genes in mammalian expression vector plasmids (Twist Bioscience).

    Article Title: Predicting TCR antigen specificity at proteome-scale with synthetic immune cells and machine learning
    Article Snippet: Assembly reactions were performed using a 2:1 molar ratio of insert to vector (120 fmol vector), with 2 μL of NEB Golden Gate Enzyme Mix. .. TCR constructs were synthesized (Twist Bioscience) and cloned into the mammalian expression vector pTwist_BetaGlobin_CMV_WPRE (Twist Bioscience). .. Assembled products were amplified by PCR using primer pair KH241/KH240 (see Supplementary Table 6), and purified using the DNA Clean & Concentrator-25 kit (Zymo Research, #D4005) to generate the final HDR templates.

    Article Title: Selective MENA binding peptides
    Article Snippet: .. All sequences of constructed plasmids were confirmed by Sanger sequencing. mRuby2-Pcare and Mito-mRuby2-Pcare inserts were synthesized (Twist Bioscience) and subcloned into a SFFV-promoter lentiviral expression vector. ..

    Mutagenesis:

    Article Title: A Novel Patient-Derived Mutation in Glucocorticoid Receptor Reveals an Essential Role for DNA binding in Development and Endocrine Regulation
    Article Snippet: .. DNA containing the T444I mutation and flanking homology arms was synthesized by Twist Biosciences and cloned into the pGMC00018 vector backbone using Gibson assembly , yielding the construct pMG0658. .. This plasmid was then used as a template for long ssDNA production with the Guide-it Long ssDNA Production System v2 (Takara).

    Article Title: Activation of Arp2/3 complex by a SPIN90 dimer in linear actin-filament nucleation.
    Article Snippet: .. Additionally, a truncated SPIN90 (326–722) construct, the W401A mutant and the triple mutant (Q397A, Q398A and W401A) genes were synthesized (Twist Bioscience) and cloned into a pET-28a(+) vector. ..

    other:

    Article Title: Vibrio MARTX toxin binding of biantennary N-glycans at host cell surfaces
    Article Snippet: The construct encoding Aknot-HA (residues 244 to 571 of the MARTX toxin from V. vulnificus CMCP6), with a 6xHis tag and a C-terminal HA tag or with an N-terminal Avidin (Avi) tag (Avi-Aknot), was cloned into the pET28A+ vector by Twist Bioscience (South San Francisco, CA).

    Article Title: Identification and Characterization of the Two Glycosyltransferases Required for the Polymerization of the HS:1 Serotype Capsular Polysaccharide of Campylobacter jejuni G1.
    Article Snippet: The HS1.09286−703 construct was chemically synthesized by Twist Biosciences and cloned into a pET-29b(+) vector with a polyhistidine purification tag at the C-terminus.

    Sequencing:

    Article Title: Selective MENA binding peptides
    Article Snippet: .. All sequences of constructed plasmids were confirmed by Sanger sequencing. mRuby2-Pcare and Mito-mRuby2-Pcare inserts were synthesized (Twist Bioscience) and subcloned into a SFFV-promoter lentiviral expression vector. ..



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    Image Search Results


    RT induces LUAD cell radio-resistance by evoking MTHFD2 overexpression. A Baseline MTHFD2 expression: left: immunoblots of MTHFD2 protein levels across LUAD cell lines. Right: densitometric quantification normalized to β-tubulin. B Left: Representative colony formation images post-X-ray irradiation (0–4 Gy). Right: cell survival curves. ( C - D ) Radiation-induced MTHFD2 regulation: ( C ) time-course immunoblot (0–48 h post-6 Gy) demonstrating temporal coordination of MTHFD2 and γ-H2AX expression. ( D ) dose-dependent upregulation (0–10 Gy) of MTHFD2 and DNA damage markers. E Nuclear/cytoplasmic fractionation showing radiation-triggered (6 Gy) MTHFD2 nuclear accumulation over time (lamin a/c nuclear marker, GAPDH cytoplasmic control). F Schematic of iterative fractionated irradiation (2 Gy × 30 fractions) for generating radioresistant clones. G Left: immunoblots of MTHFD2 and γ-H2AX in parental vs. radioresistant lines. Middle and right: quantitative comparison showing MTHFD2 elevation and γ-H2AX reduction in resistant clones. H DNA repair capacity assessment: left: neutral comet assay images post-6 Gy irradiation. Right: olive tail moment quantification demonstrating impaired DNA repair in radioresistant cells (scale bar: 50 μm). Data represent mean ± SD from three biologically independent experiments. Statistical significance determined by two-tailed Student’s t-test ( n = 3, * p < 0.05, ** p< 0.001,*** p< 0.001, **** p < 0.0001)

    Journal: Journal of Translational Medicine

    Article Title: MTHFD2 is required for DNA repair and implicated in LUAD radiotherapy resistance

    doi: 10.1186/s12967-026-07680-7

    Figure Lengend Snippet: RT induces LUAD cell radio-resistance by evoking MTHFD2 overexpression. A Baseline MTHFD2 expression: left: immunoblots of MTHFD2 protein levels across LUAD cell lines. Right: densitometric quantification normalized to β-tubulin. B Left: Representative colony formation images post-X-ray irradiation (0–4 Gy). Right: cell survival curves. ( C - D ) Radiation-induced MTHFD2 regulation: ( C ) time-course immunoblot (0–48 h post-6 Gy) demonstrating temporal coordination of MTHFD2 and γ-H2AX expression. ( D ) dose-dependent upregulation (0–10 Gy) of MTHFD2 and DNA damage markers. E Nuclear/cytoplasmic fractionation showing radiation-triggered (6 Gy) MTHFD2 nuclear accumulation over time (lamin a/c nuclear marker, GAPDH cytoplasmic control). F Schematic of iterative fractionated irradiation (2 Gy × 30 fractions) for generating radioresistant clones. G Left: immunoblots of MTHFD2 and γ-H2AX in parental vs. radioresistant lines. Middle and right: quantitative comparison showing MTHFD2 elevation and γ-H2AX reduction in resistant clones. H DNA repair capacity assessment: left: neutral comet assay images post-6 Gy irradiation. Right: olive tail moment quantification demonstrating impaired DNA repair in radioresistant cells (scale bar: 50 μm). Data represent mean ± SD from three biologically independent experiments. Statistical significance determined by two-tailed Student’s t-test ( n = 3, * p < 0.05, ** p< 0.001,*** p< 0.001, **** p < 0.0001)

    Article Snippet: Recombinant lentiviral vectors carrying MTHFD2/XRCC6 overexpression constructs (LV-MTHFD2/LV-XRCC6) and short hairpin RNA (shRNA) sequences targeting MTHFD2/XRCC6 (LV-shMTHFD2/LV-shXRCC6) were commercially purchased from GeneChem Co., Ltd (Shanghai, China).

    Techniques: Over Expression, Expressing, Western Blot, Irradiation, Fractionation, Marker, Control, Clone Assay, Comparison, Neutral Comet Assay, Two Tailed Test

    MTHFD2 interacts with XRCC6 and controls XRCC6 expression. A Venn diagram illustrating the overlap between MTHFD2-associated proteins identified by LC-MS/MS analysis ( n = 1,247) and curated DNA repair factors (HR/NHEJ pathways). Six candidate interactors are highlighted. B Co-IP validation of MTHFD2-binding partners in H1299 cells using anti-flag antibody. C Co-immunoprecipitation assay detection of MTHFD2, SMC6 and XRCC6 with endogenous MTHFD2 in whole-cell lysates. D Co-immunoprecipitation assay detection of MTHFD2, SMC6 and XRCC6 with endogenous SMC6 and XRCC6 in whole-cell lysates. E Correlation between MTHFD2 and XRCC6 expression levels in radiotherapy-sensitive (case 1) versus radiotherapy-resistant (case 2) LUAD cohorts. Scale bars: 100 μm/25 μm(inset). F XRCC6 protein levels were positively regulated in cells with MTHFD2 deletion or overexpression. G XRCC6 perturbation (knockdown or overexpression) failed to alter MTHFD2 protein abundance. H Co-IP analysis of FLAG-MTHFD2/XRCC6 interaction dynamics in MTHFD2-manipulated LUAD cells (β-tubulin as loading control). I Co-IP confirming interaction specificity: XRCC6 antibody pulls down exogenous MTHFD2 in overexpression models. J in vivo functional correlation: MTHFD2 depletion (shRNA) impairs XRCC6 expression in xenograft tumours post-ionizing radiation treatment, as shown by dual immunofluorescence test. Scale bars: 100 μm/25 μm(inset). Statistical significance determined by two-tailed Student’s t-test ( n = 3, ** p < 0.01)

    Journal: Journal of Translational Medicine

    Article Title: MTHFD2 is required for DNA repair and implicated in LUAD radiotherapy resistance

    doi: 10.1186/s12967-026-07680-7

    Figure Lengend Snippet: MTHFD2 interacts with XRCC6 and controls XRCC6 expression. A Venn diagram illustrating the overlap between MTHFD2-associated proteins identified by LC-MS/MS analysis ( n = 1,247) and curated DNA repair factors (HR/NHEJ pathways). Six candidate interactors are highlighted. B Co-IP validation of MTHFD2-binding partners in H1299 cells using anti-flag antibody. C Co-immunoprecipitation assay detection of MTHFD2, SMC6 and XRCC6 with endogenous MTHFD2 in whole-cell lysates. D Co-immunoprecipitation assay detection of MTHFD2, SMC6 and XRCC6 with endogenous SMC6 and XRCC6 in whole-cell lysates. E Correlation between MTHFD2 and XRCC6 expression levels in radiotherapy-sensitive (case 1) versus radiotherapy-resistant (case 2) LUAD cohorts. Scale bars: 100 μm/25 μm(inset). F XRCC6 protein levels were positively regulated in cells with MTHFD2 deletion or overexpression. G XRCC6 perturbation (knockdown or overexpression) failed to alter MTHFD2 protein abundance. H Co-IP analysis of FLAG-MTHFD2/XRCC6 interaction dynamics in MTHFD2-manipulated LUAD cells (β-tubulin as loading control). I Co-IP confirming interaction specificity: XRCC6 antibody pulls down exogenous MTHFD2 in overexpression models. J in vivo functional correlation: MTHFD2 depletion (shRNA) impairs XRCC6 expression in xenograft tumours post-ionizing radiation treatment, as shown by dual immunofluorescence test. Scale bars: 100 μm/25 μm(inset). Statistical significance determined by two-tailed Student’s t-test ( n = 3, ** p < 0.01)

    Article Snippet: Recombinant lentiviral vectors carrying MTHFD2/XRCC6 overexpression constructs (LV-MTHFD2/LV-XRCC6) and short hairpin RNA (shRNA) sequences targeting MTHFD2/XRCC6 (LV-shMTHFD2/LV-shXRCC6) were commercially purchased from GeneChem Co., Ltd (Shanghai, China).

    Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Biomarker Discovery, Binding Assay, Over Expression, Knockdown, Quantitative Proteomics, Control, In Vivo, Functional Assay, shRNA, Immunofluorescence, Two Tailed Test

    XRCC6 overexpression partially rescues MTHFD2 knockdown-induced functional impairment. A Immunoblot analysis of MTHFD2 and XRCC6 expression in LUAD cells transfected with specific shRNA or overexpression constructs. B Cell viability rescue: Cell Counting Kit-8 (CCK-8) assays demonstrated that XRCC6 overexpression restored cellular viability in MTHFD2-silenced cells. C Clonogenic recovery: quantitative colony formation assays confirmed partial reversal of proliferation defects by XRCC6 reconstitution. D DNA damage mitigation: γ-H2AX immunoblotting post-6 Gy irradiation showed XRCC6 overexpression reduced DNA damage signaling. E Comet assay validation: neutral comet quantification revealed XRCC6 reconstitution shortened olive tail moments in MTHFD2-deficient cells (scale bar: 50 μm). Data represent mean ± SD from three biologically independent experiments. Statistical significance determined by two-tailed Student’s t-test ( n = 3, * p < 0.05, ** p < 0.01)

    Journal: Journal of Translational Medicine

    Article Title: MTHFD2 is required for DNA repair and implicated in LUAD radiotherapy resistance

    doi: 10.1186/s12967-026-07680-7

    Figure Lengend Snippet: XRCC6 overexpression partially rescues MTHFD2 knockdown-induced functional impairment. A Immunoblot analysis of MTHFD2 and XRCC6 expression in LUAD cells transfected with specific shRNA or overexpression constructs. B Cell viability rescue: Cell Counting Kit-8 (CCK-8) assays demonstrated that XRCC6 overexpression restored cellular viability in MTHFD2-silenced cells. C Clonogenic recovery: quantitative colony formation assays confirmed partial reversal of proliferation defects by XRCC6 reconstitution. D DNA damage mitigation: γ-H2AX immunoblotting post-6 Gy irradiation showed XRCC6 overexpression reduced DNA damage signaling. E Comet assay validation: neutral comet quantification revealed XRCC6 reconstitution shortened olive tail moments in MTHFD2-deficient cells (scale bar: 50 μm). Data represent mean ± SD from three biologically independent experiments. Statistical significance determined by two-tailed Student’s t-test ( n = 3, * p < 0.05, ** p < 0.01)

    Article Snippet: Recombinant lentiviral vectors carrying MTHFD2/XRCC6 overexpression constructs (LV-MTHFD2/LV-XRCC6) and short hairpin RNA (shRNA) sequences targeting MTHFD2/XRCC6 (LV-shMTHFD2/LV-shXRCC6) were commercially purchased from GeneChem Co., Ltd (Shanghai, China).

    Techniques: Over Expression, Knockdown, Functional Assay, Western Blot, Expressing, Transfection, shRNA, Construct, Cell Counting, CCK-8 Assay, Irradiation, Single Cell Gel Electrophoresis, Biomarker Discovery, Two Tailed Test

    MTHFD2 recruits the LIG4/XRCC4/XLF complex to activate DNA damage repair pathways. A Immunoblot analysis of key NHEJ components (MTHFD2, XRCC6, XLF, XRCC4, LIG4) in LUAD cells transfected with specific shRNA. B Immunofluorescence revealed that MTHFD2 depletion markedly attenuated nuclear foci formation of LIG4/XRCC4/XLF complexes (Scale bar: 20 μm). Data represent mean ± SD from three biologically independent experiments. Statistical significance determined by two-tailed Student’s t-test ( n = 3, * p < 0.05, ** p < 0.01)

    Journal: Journal of Translational Medicine

    Article Title: MTHFD2 is required for DNA repair and implicated in LUAD radiotherapy resistance

    doi: 10.1186/s12967-026-07680-7

    Figure Lengend Snippet: MTHFD2 recruits the LIG4/XRCC4/XLF complex to activate DNA damage repair pathways. A Immunoblot analysis of key NHEJ components (MTHFD2, XRCC6, XLF, XRCC4, LIG4) in LUAD cells transfected with specific shRNA. B Immunofluorescence revealed that MTHFD2 depletion markedly attenuated nuclear foci formation of LIG4/XRCC4/XLF complexes (Scale bar: 20 μm). Data represent mean ± SD from three biologically independent experiments. Statistical significance determined by two-tailed Student’s t-test ( n = 3, * p < 0.05, ** p < 0.01)

    Article Snippet: Recombinant lentiviral vectors carrying MTHFD2/XRCC6 overexpression constructs (LV-MTHFD2/LV-XRCC6) and short hairpin RNA (shRNA) sequences targeting MTHFD2/XRCC6 (LV-shMTHFD2/LV-shXRCC6) were commercially purchased from GeneChem Co., Ltd (Shanghai, China).

    Techniques: Western Blot, Transfection, shRNA, Immunofluorescence, Two Tailed Test

    A Schematic representation of the Lox-Stop-Lox-dCas9-DNMT3A-P2A-GFP (LSL-dC9-D) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of DNMT3A, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-DNMT3A-GFP mice and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-DNMT3A-GFP and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, GFP, dCas9 colocalization in mice injected contralaterally with either AAV9-mCherry or AAV9-mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-DNMT3A induction efficiency in mCherry and mCherry-Cre labeled cells. ( n = 3 mice per group, two-sided t test, P = 0.000010).

    Journal: Nature Communications

    Article Title: Editing DNA methylation in vivo

    doi: 10.1038/s41467-025-67222-5

    Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-DNMT3A-P2A-GFP (LSL-dC9-D) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of DNMT3A, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-DNMT3A-GFP mice and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-DNMT3A-GFP and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, GFP, dCas9 colocalization in mice injected contralaterally with either AAV9-mCherry or AAV9-mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-DNMT3A induction efficiency in mCherry and mCherry-Cre labeled cells. ( n = 3 mice per group, two-sided t test, P = 0.000010).

    Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

    Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling

    A Schematic representation of the Lox-Stop-Lox-dCas9-TET1-P2A-GFP (LSL-dC-T) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of GFP, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-TET1-GFP mice and LSL-dCas9-TET1-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-TET1-GFP and LSL-dCas9-TET1-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, eGFP, dCas9 colocalization in mice injected contralaterally with either mCherry or mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-TET1 induction efficiency in mCherry-Cre and mCherry-labeled cells. ( n = 3 mice per group, two-sided t test, P = 1.2 × 10 −7 ). F Quantification of the percentage of NeuN+ cells in mCherry− and mCherry+ populations. ( n = 8 mice per group, two-sided t test, P = 2.81 × 10 −8 ).

    Journal: Nature Communications

    Article Title: Editing DNA methylation in vivo

    doi: 10.1038/s41467-025-67222-5

    Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-TET1-P2A-GFP (LSL-dC-T) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of GFP, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-TET1-GFP mice and LSL-dCas9-TET1-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-TET1-GFP and LSL-dCas9-TET1-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, eGFP, dCas9 colocalization in mice injected contralaterally with either mCherry or mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-TET1 induction efficiency in mCherry-Cre and mCherry-labeled cells. ( n = 3 mice per group, two-sided t test, P = 1.2 × 10 −7 ). F Quantification of the percentage of NeuN+ cells in mCherry− and mCherry+ populations. ( n = 8 mice per group, two-sided t test, P = 2.81 × 10 −8 ).

    Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

    Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling