Journal: Nature Communications

Article Title: Pluripotency-independent induction of human trophoblast stem cells from fibroblasts

doi: 10.1038/s41467-023-39104-1

Figure Lengend Snippet: a Schematic representation for the strategy to generate and reprogram knockout (KO) SOX2 fibroblasts into hiTSCs. b Schematic representation of the SOX2 gene locus, displaying the location of the gRNA used, as well as the primer pairs designed to examine indels . c DNA gel showing a WT band of 219 bp within the SOX2 coding region in WT fibroblasts and the same PCR reaction in seven independent hiTSC clones ( n = 7) from SOX2 KO fibroblasts. d Sequence alignment image of one indel event (i.e., Del A) within the SOX2 locus in hiTSC#3 SOX2-KO using Sequencher software (demo version). e DNA gel showing a WT band of 100 bp within the SOX2 coding region in WT fibroblasts and the same PCR reaction in seven independent hiTSC clones ( n = 7) from SOX2 KO fibroblasts. Note, 4 out of 7 colonies show a bi-allelic deletion within SOX2 coding region (SOX2del/del). f Bright field images of three SOX2 KO hiTSC colonies ( n = 3). g Schematic representation for the strategy to double knockout (DKO) NANOG and PRDM14 in fibroblasts and reprogramming into hiTSCs. h Graph depicting the number of hiTSC colonies that emerged either in DKO fibroblasts or in WT controls following reprogramming with GOKM or OSKM. Error bars indicate standard deviation between 3–4 experiments/replicates (for OSKM n = 3 and for GOKM n = 4). * indicates p value of 0.0383 for OSKM WT vs. GOKM WT and 0.0324 for OSKM WT vs. GOKM WT (95% confidence interval of 0.3851–9.282 and 1.081–17.42, respectively), using two-tailed unpaired t- test calculated by GraphPad Prism (8.3.0). Mean values (from left to right) are: 3.667, 1.000, 8.500, 17.75. i Sequences alignment image of various indel events within the NANOG and PRDM14 loci in seven independent hiTSC DKO clones ( n = 7) using Sequencher (Demo version). gRNA sequences and Sanger chromatograms are shown for 4 hiTSC DKO clones. Note that a significant enrichment for double KO events is evident in hiTSC clones that were derived from DKO fibroblasts. See also Supplementary Figs. – . Source data are provided as a Source Data file.

Article Snippet: gRNAs designed to target the first exon of SOX2, NANOG and PRDM14 (see Supplementary Table ) were cloned into pLentiCRISPR V2 vectors with puro (SOX2, NANOG) or hygromycin B (PRDM14) resistance (Addgene plasmids #52961 and #98291 respectively) using BsmBI restriction enzyme, as described in ref. .

Techniques: Knock-Out, Clone Assay, Sequencing, Software, Double Knockout, Standard Deviation, Two Tailed Test, Derivative Assay

Journal: Biotechnology for Biofuels and Bioproducts

Article Title: Thermotolerance improvement of engineered Saccharomyces cerevisiae ERG5 Delta ERG4 Delta ERG3 Delta , molecular mechanism, and its application in corn ethanol production

doi: 10.1186/s13068-023-02312-4

Figure Lengend Snippet: Screening of the putative transformants and molecular identification of S. cerevisiae ERG5ΔERG4ΔERG3Δ. A Construction strategy of four engineered S. cerevisiae strains; B Screening of four putative transformants on solid plates containing two antibiotics, while the wild-type strain could not grow on the screening plate; C Screening of vector loss of S. cerevisiae ERG5Δ transformants for further transformation of ERG4 . The cell proliferation of S. cerevisiae ERG5Δ transformant after transformation of ERG5-gRNA-trp-HyB and Cas9-NTC was carried out via the liquid fermentation approach. The proliferative colonies were screened and cultured on YPD plate a for 48 h at 30 °C. Then, the colonies on plate a were transferred to the corresponding positions on plates b and c. The media in a, b, and c were YPD medium, YPD medium containing 80 μg/mL of nourseothricin (NTC), and YPD medium containing 300 μg/mL of hygromycin B (HyB), respectively. The colonies (arrow) could not grow on both b and c, which meant that the corresponding colony on plate a had lost both ERG5-gRNA-trp-HyB and Cas9-NTC; D S. cerevisiae ERG5ΔERG4ΔERG3Δ identification by amplification of XYNA (lane 2, 1129 bp), CEL (lane 3, 880 bp), and MFC (lane 4, 628 bp). Lane M and 1 represented DNA Marker and the control, respectively

Article Snippet: The plasmid gRNA-trp-HyB carrying hygromycin B resistance gene for guide RNA synthesis and Cas9-NTC carrying nourseothricin resistance gene for S. cerevisiae genomic DNA digestion were from Addgene (Watertown, Massachusetts, USA).

Techniques: Plasmid Preparation, Transformation Assay, Cell Culture, Amplification, Marker