Journal: Nucleic Acids Research

Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

doi: 10.1093/nar/gkx1249

Figure Lengend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions . Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

Article Snippet: Ten micrograms of genomic DNA and 200 ng of 2.2-kb p15A-cm linear vector (1 μg of 8-kb linear BAC vector) were assembled in 20 μl reactions consisting of 2 μl of 10 × NEBuffer 2.1 and 0.13 μl of 3 U μl −1 T4pol (NEB, cat. no. M0203).

Techniques: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

doi: 10.1093/nar/gkx1249

Figure Lengend Snippet: ExoCET mechanism. ( A ) Juxtaposition of the 80-bp homology arms between the p15A-cm (chloramphenicol) vector and the 14-kb lux genomic segment is illustrated: (a) both homology arms were located at the termini; (b and c) one homology arm was located at a terminus and the other 1 kb from the other end; (d) both homology arms were 1 kb from each end. ( B ) Number of colonies obtained from ETgA, T4pol or ExoCET using the homology arm combinations (a–d) as indicated. Reaction conditions were the same as for Figure . ( C ) Protein combinations as indicated expressed from pSC101 plasmids in GB2005 were tested for direct cloning of the 14-kb lux gene cluster using terminal homology arms and ExoCET conditions except for the omission of RecA (ETg); RecA and RecT (Eg), RecA and RecE (Tg) and all (pSC101-tet). Error bars, s.d.; n = 3. Corresponding DNA analyses are shown in .

Article Snippet: Ten micrograms of genomic DNA and 200 ng of 2.2-kb p15A-cm linear vector (1 μg of 8-kb linear BAC vector) were assembled in 20 μl reactions consisting of 2 μl of 10 × NEBuffer 2.1 and 0.13 μl of 3 U μl −1 T4pol (NEB, cat. no. M0203).

Techniques: Plasmid Preparation, Clone Assay

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Schematic of experimental design. (B) Flow cytometry contour plots demonstrating the loss of the endogenous splenic CD4 + Foxp3 -GFP + Treg population in Foxp3 GFP-DTR mice following administration of diphtheria toxin (DTx). (C) Lung and spleen Foxp3 -GFP + Treg cell quantification in Foxp3 GFP-DTR mice that received influenza A virus but no DTx (No DTx + flu, n = 3) or influenza and DTx (DTx + flu, n = 4) at 13 DPI compared with mice that received DTx but no influenza (DTx No flu, n = 3). (D) Mass over time of Foxp3 GFP-DTR mice receiving no DTx or DTx until pre-specified timepoints (DTx withdrawal) following intra-tracheal inoculation of 5 plaque-forming units (PFU) of influenza A/WSN/33 H1N1 virus (n = 5 per group). (E) Representative lung histopathology (H&E staining) at 60 DPI of control (No DTx) and DTx withdrawal mice following influenza A virus infection. Original magnification x10, scale bar = 1 mm. (F) Metagene analysis of DNA methylation across Treg-SE of Tregs recovered from the mice described in 1D compared with splenic nTregs and iTregs from culture. Data presented as mean and SD. * q < 0.05 according to one-way ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (C).* q < 0.05 according to mixed-effects model (REML) with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (D).

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Flow Cytometry, Virus, Histopathology, Staining, Control, Infection, DNA Methylation Assay

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Flow cytometry contour plots for phenotyping of the CD4 + Foxp3 -GFP + tdTomato + iTreg population following culture in the presence of αCD3/αCD28, IL-2, TGF-β, and tamoxifen. (B) Mass over time of Foxp3 GFP-DTR mice receiving adoptive transfer of nTreg (n = 27), PBS (n = 21), Tconv (n = 25), or iTreg (n = 18) cells 5 days following intra-tracheal inoculation of 6.5 PFU of influenza A/WSN/33 H1N1 virus. Negative controls included mice that received influenza but no DTx (no DTx + flu, n = 9) and DTx but no influenza (DTx no flu, n = 3). (C) Total number of lung CD45 + cells at 24 DPI in mice that received No DTx (n = 3), nTreg (n = 15), PBS (n = 11), or Tconv (n = 10). (D-H) Total number of lung CD326 + CD31 − CD45 − cells (D), CD326 + MHCII + T1A − cells (E), KRT5 + CD326 + cells (F), Ki-67 + CD326 + MHCII + cells (G), and CD326 − CD31 + cells (H) at 24 DPI in mice that received No DTx (n = 3), nTreg (n = 15), PBS (n = 11), or Tconv (n = 10) cells. * q < 0.05 according to mixed-effects model (REML) with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (B). Data presented as mean and SD (C, F-H) with * q < 0.05 according to multiple Mann-Whitney tests and correcting for multiple comparisons using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (C). UHRF1 is dispensable for iTreg FOXP3 induction and stability but is required to maintain transcriptional and epigenetic programs in vitro .

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Flow Cytometry, Adoptive Transfer Assay, Virus, MANN-WHITNEY, In Vitro

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Schematic of experimental design. (B) Frequency of Foxp3 -GFP + and Foxp3 -GFP − tdTomato + (ex-FOXP3) cells in culture of iTregs with UHRF1 deleted concurrent with (early) or 5 days after (delayed) FOXP3 induction compared with Uhrf1 +/+ controls on days 5 and 12 of culture (n = 3 per group). (C) Division index of CD4 + CTV + Foxp3 -GFP − splenic responder T cells co-cultured for 72 hours with varying ratios of Uhrf1 +/+ CD4 + Foxp3 -GFP + iTregs, Uhrf1 fl/fl CD4 + Foxp3 -GFP + iTregs, or Uhrf1 +/+ nTregs (n = 3 per group). (D) Principal component analysis of 6,978 differentially expressed genes from sorted cells described in A, identified from ANOVA-like testing with FDR q < 0.05. Ellipses represent normal contour lines with 1 standard deviation probability. (E) K-means clustering of 127 genes with an FDR q < 0.05 comparing the Uhrf1 fl/fl and Uhrf1 +/+ iTreg populations on day 12 in which UHRF1 was deleted after FOXP3 induction (delayed) with k = 2. (F) MA plot comparing gene expression of delayed Uhrf1 +/+ and Uhrf1 fl/fl iTregs on day 12 of culture. Genes of interest are annotated. (G) Enrichment plot of the GSE14415_INDUCED_TREG_VS_TCONV_UP, GSE14415_INDUCED_TREG_VS_TCONV_DN, GSE14415_INDUCED_TREG_VS_FAILED_INDUCED_TREG_UP, GSE37605_TREG_VS_TCONV_C57BL6_FOXP3_FUSION_GFP_UP, GSE13306_TREG_VS_TCONV_UP gene sets ( p < 0.05, FDR q < 0.25) generated through GSEA preranked testing of the expressed genes of delayed Uhrf1 +/+ iTregs (control) and delayed Uhrf1 fl/fl iTregs on day 12 of culture. (H) PCA of 81,179 differentially methylated cytosines identified from ANOVA-like testing with FDR q < 0.05. Ellipses represent normal contour lines with 1 standard deviation probability. (I) Cumulative distribution function plot of differentially methylated cytosines expressed as β scores, with 0 representing unmethylated and 1 representing fully methylated; a shift in the cumulative distribution function up and to the left represents relative hypomethylation. * q < 0.05 according to two-way ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Cell Culture, Standard Deviation, Gene Expression, Generated, Control, Methylation

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) MA plot comparing gene expression of Uhrf1 +/+ iTregs (control) with CD4 + Foxp3 -GFP + nTregs on day 5 of culture. Genes of interest are annotated. (B) Enrichment plots of the HALLMARK_MYC_TARGETS_V1, HALLMARK_E2F_TARGETS, HALLMARK_TGF_BETA_SIGNALING, HALLMARK_WNT_BETA_CATENIN_SIGNALING, HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_MTORC1_SIGNALING, HALLMARK_IL2_STAT5_SIGNALING, and HALLMARK_KRAS_SIGNALING_UP gene sets generated through GSEA preranked testing of the expressed genes of delayed Uhrf1 +/+ iTregs (control) and delayed Uhrf1 fl/fl iTregs on day 12 of culture.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Gene Expression, Control, Generated

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A-F) Total numbers of CD45 + (A), CD3 + (B), CD4 + CD8 − (C), CD4 − CD8 + (D), CD11b − CD64 + CD11c + SiglecF + (E), and CD11b + Ly6G + (F) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 8), Uhrf1 +/+ iTregs (n = 16), or U hrf1 fl/fl iTregs. (n = 16) (G) Total number of CD326 + CD31 − CD45 − (epithelial) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 7), Uhrf1 +/+ iTregs (n = 15), or U hrf1 fl/fl iTregs (n = 16). (H) Total number of KRT5 + CD326 + epithelial cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 7), Tconv (n = 7), PBS (n = 7), Uhrf1 +/+ iTregs (n = 13), or U hrf1 fl/fl iTregs (n = 14). (I) Total number of CD326 − CD31 + (endothelial) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 8), Uhrf1 +/+ iTregs (n = 14), or U hrf1 fl/fl iTregs (n = 15). Data presented as mean and SD. ns = not significant by multiple Mann-Whitney tests and correcting for multiple comparisons using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% test.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: MANN-WHITNEY