Journal: Microbiology Spectrum

Article Title: Efficient and rapid one-step method to generate gene deletions in Streptococcus pyogenes

doi: 10.1128/spectrum.01185-24

Figure Lengend Snippet: Efficiency of mutant generation using the pKOSpy strategy in different genomic contexts and different M-types/strains ^a

Article Snippet: Both empty pSWITCH and pKOSpy- sagB plasmids are available from Addgene (222402 and 222345, respectively).

Techniques: Mutagenesis

Journal: Microbiology Spectrum

Article Title: Efficient and rapid one-step method to generate gene deletions in Streptococcus pyogenes

doi: 10.1128/spectrum.01185-24

Figure Lengend Snippet: ( A ) Construction strategy for sagB allelic exchange with the pKOSpy vector. Upstream (FR1) and downstream (FR2) flanking regions of the sagB gene and the kanamycin resistance gene was inserted using Golden gate assembly with Bsa I enzyme in the pKOSpy backbone consisting of the colE1 ori and bla promoter (P bla ) controlling the expression of the pheS** mutant allele and the aad9 spectinomycin resistance gene. Primers used to amplify the three PCR fragments as well as origin of all fragments are depicted. ( B ) Map of the pKOSpy- sagB plasmid. Colored as in A. gDNA, GAS genomic DNA; bp, base pairs.

Article Snippet: Both empty pSWITCH and pKOSpy- sagB plasmids are available from Addgene (222402 and 222345, respectively).

Techniques: Plasmid Preparation, Expressing, Mutagenesis

Journal: Microbiology Spectrum

Article Title: Efficient and rapid one-step method to generate gene deletions in Streptococcus pyogenes

doi: 10.1128/spectrum.01185-24

Figure Lengend Snippet: ( A ) Strategy used to generate GAS KO mutants in 3 working days. Day 1: the suicide vector is transformed in GAS cells and GAS cells are plated on THY supplemented with Kanamycin. Day 2: Kanamycin-resistant colonies are streaked on plates containing either kanamycin or spectinomycin. Day 3: Spectinomycin-sensitive and kanamycin-resistant colonies are screened by PCR using primers annealing outside the cloned FR1 and FR2 and in the aphA3 gene. Double recombinants (Kanamycin R and Spectinomycin S ) present two bands at around 1,000 bp. Simple recombinants (Kanamycin R and Spectinomycin R ) present only one PCR band (either from FR1 or FR2). Red arrows indicate primer annealing. gDNA, genomic DNA; pDNA, plasmidic DNA; bp, base pairs. ( B ) PCR screening of the sagB mutant candidates by PCR. Clones 1 and 2 are spectinomycin R and kanamycin R (single recombinants, taken as an example), and clone 3 is spectinomycin S and kanamycin R (double recombinant). LO1, the wild-type strain; FR, flanking region.

Article Snippet: Both empty pSWITCH and pKOSpy- sagB plasmids are available from Addgene (222402 and 222345, respectively).

Techniques: Plasmid Preparation, Transformation Assay, Clone Assay, Mutagenesis, Recombinant

Journal: Microbiology Spectrum

Article Title: Efficient and rapid one-step method to generate gene deletions in Streptococcus pyogenes

doi: 10.1128/spectrum.01185-24

Figure Lengend Snippet: ( A ) Schematic representation of the sag operon responsible for Streptolysin S production in GAS. A rho-independent terminator sequence is schematized between the sagA and sagB gene and at the end of the sag operon. ( B ) Quantification by RT-qPCR of the fold changes of sagB and sagC RNA transcripts between the sagB mutant ( DsagB ) compared with the WT strain. ( C ) Phenotypic validation of the sagB mutant (D sagB ) and its complemented derivative (D sagB + ) compared with the parent LO1 strain (WT) on 5% sheep blood agar plates. Bacteria with functional hemolysin show a clear halo around the colonies. ( D ) Quantitative analysis of SLS activity of the sagB mutant (D sagB ) and its complemented derivative (D sagB + ) compared with the parent LO1 strain (WT). Release of hemoglobin was measured after incubation of bacterial supernatant with red blood cells for 3 h at 37°C. The percentage of hemolysis has been calculated compared with the WT strain, considered as 100%. A one-way ANOVA test was used for statistical analysis.

Article Snippet: Both empty pSWITCH and pKOSpy- sagB plasmids are available from Addgene (222402 and 222345, respectively).

Techniques: Sequencing, Quantitative RT-PCR, Mutagenesis, Biomarker Discovery, Bacteria, Functional Assay, Activity Assay, Incubation

Journal: Nature Communications

Article Title: Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

doi: 10.1038/s41467-021-23320-8

Figure Lengend Snippet: a FACS analysis of the percentages of OCT4-Tdtomato+ H1-EPS cells with indicated factor treatment after 2 passages. n = 2 biologically independent samples. None, LCDM medium. b Analysis of cell numbers of feeder-free cultured hEPS cells under the treatments of vitamin C, catalase, and their combination. VC, vitamin C. n = 3 biologically independent samples. ES1-EPS cells were used. c Analysis of cell numbers of feeder-free cultured hEPS cells in different basal culturing media. n = 4 biologically independent samples. ES1-EPS cells were used. d – f Analysis of the effects of different matrix proteins on feeder-free cultured hEPS cells. The percentages of adherent hEPS cells at 1.5 h after seeding was shown in d . Survival of dissociated hEPS cells 24 h after seeding was shown in e . Proliferation of hEPS cells at 72 h after seeding was shown in f . For e , f , index represents the cell number at a specific time point divided by the number of seeding cells. For d – f , n = 3 biologically independent samples. ES1-EPS cells were used. g Representative morphologies of xeno-free hEPS cells with different genetic backgrounds. Similar images were obtained in at least 5 independent experiments. Error bars, mean ± SD. All differences between means with P < 0.01 are indicated. ** P < 0.01; *** P < 0.001; **** P < 0.0001. Statistical significance was analyzed using one-way ANOVA with Tukey multiple comparison test in b – f . Scale bar, 100 μm. Experiments in a – g were all independently repeated at least three times with similar results.

Article Snippet: To evaluate human OCT4 DE transcriptional regulation in primed hPSCs and hEPS cells cultured in different conditions, OCT4 -DE luciferase plasmid (Addgene, 52414) was transfected into the cells by nucleofection (4D-NucleofectorTM System, Lonza).

Techniques: Cell Culture, Comparison

Journal: Nature Communications

Article Title: Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

doi: 10.1038/s41467-021-23320-8

Figure Lengend Snippet: a Representative FACS analysis of the percentages of OCT4 + or NANOG + cells in xeno-free hEPS cells. b Hierarchical clustering of global gene expression of EPS cells cultured in XF-LCDM and F-LCDM. Distance is calculated by 1-Spearman correlation coefficient. c GSEA analysis of enrichment of module C and module D in xeno-free and feeder-cultured hEPS cells. Bulk RNA-seq data for xeno-free and feeder-cultured hEPS cells were used. Gene signatures for module C and module D were defined in our previous study (Yang et al.) . As the control, dataset for pre-implantation human embryos (GSE36552, Yan et al.) were chosen. d GSEA analysis of enrichment of gene signatures for E5/E6/E7 epiblasts or naive pluripotent stem cells in xeno-free and feeder-cultured hEPS cells. Bulk RNA-seq data for xeno-free and feeder-cultured hEPS cells were used. Gene signatures for E5/E6/E7 epiblasts were defined in the study performed by Petropoulos et al. , and naive signatures were defined in the study performed by Liu et al. . As the control, dataset for pre-implantation human embryos (GSE36552, Yan et al.) , and naive or primed hPSCs (E-MTAB-4461, Guo et al.; E-MTAB-5674, Guo et al.) , were chosen. e Mean FPKM values at TE marker genes in XF-LCDM, F-LCDM and mTeSR1 cultured primed cells. The signal density of given region (2 kb up- and down- streams of TSS) and randomly selected genome regions of the same length were analyzed. TE marker genes were defined in the study performed by Petropoulos et al. .

Article Snippet: To evaluate human OCT4 DE transcriptional regulation in primed hPSCs and hEPS cells cultured in different conditions, OCT4 -DE luciferase plasmid (Addgene, 52414) was transfected into the cells by nucleofection (4D-NucleofectorTM System, Lonza).

Techniques: Gene Expression, Cell Culture, RNA Sequencing, Control, Marker

Journal: Nature Communications

Article Title: Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

doi: 10.1038/s41467-021-23320-8

Figure Lengend Snippet: a Representative images showing histological analysis of teratomas generated from xeno-free hEPS cells. Scale bars, 100 µm. Similar images were obtained in at least 3 independent experiments. b Phase contrast images of primed and XF-EPS cells. To derive hTSC-like cells, primed hPSCs and XF-EPS cells were cultured in hTSC medium. Primed H1 and XF H1-EPS cells were used. Scale bars, 100 µm. Similar images were obtained in at least three independent experiments. c Representative immune-staining images showing the marker expression of XF-EPS cells and hTSC-like cells. XF H1-EPS cells were used. Scale bars, 100 µm. Similar images were obtained in at least three independent experiments. d Representative images showing chimerism of xeno-free hEPS cells in E4.5 mouse blastocysts. GFP, anti-GFP antibody. White and yellow arrows indicate GFP + cells with OCT4 and CDX2 expression respectively. Scale bar, 100 µm. Similar images were obtained in at least 5 independent experiments. e Chimerism of XF-EPS cells, naive hPSCs and primed hPSCs in E6.5 mouse conceptuses. Representative images of chimerism were shown. A summary of the chimeric experiment was also shown. Scale bar, 500 µm. f Immunofluorescent staining of E6.5 chimeric embryos showing lineage contributions of xeno-free hEPS derivatives to epiblast or trophoblast lineages. mclover+ ES1-EPS cells were used. Embryos were stained for OCT4 (epiblast), CK18 (trophoblast compartment) and mclover (GFP). White and yellow arrows indicate GFP + cells with OCT4 and CK18 expression respectively. Scale bars, 50 µm. Similar images were obtained in at least 5 independent experiments. Experiments in a – f were all independently repeated at least three times with similar results.

Article Snippet: To evaluate human OCT4 DE transcriptional regulation in primed hPSCs and hEPS cells cultured in different conditions, OCT4 -DE luciferase plasmid (Addgene, 52414) was transfected into the cells by nucleofection (4D-NucleofectorTM System, Lonza).

Techniques: Generated, Cell Culture, Staining, Marker, Expressing