Journal: PLOS ONE

Article Title: A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer

doi: 10.1371/journal.pone.0311120

Figure Lengend Snippet: (A) Mutant mESC lines were generated by recombinase-mediated cassette exchange (RMCE). Heterotypic Flp recognition target (FRT) sites and an mCherry expression reporter were inserted around Klf4 enhancer E2 in mESCs. In the presence of an exchange plasmid, which contains the enhancer E2 flanked by the heterotypic FRT sites, Flp recombinase facilitates integration of the desired enhancer E2 sequence. Single cells were selected after RMCE, isolated for clonal outgrowth, and confirmed by PCR and sequencing. (B-F) Analysis of gene expression in the mutant mESC lines. For each enhancer E2 sequence, multiple clonal populations were grown under naïve-state conditions, and total mRNA was harvested from each and reverse transcribed into cDNA. Quantitative PCR was performed on the cDNA to measure levels of Klf4 (B), Rad23b (C), Pou5f1 (D), Sox2 (E), Nanog (F), Esrrb (G), Tbx3 (H), Klf2 (I), and Klf5 (J). Data was analyzed using the ΔΔCT method. Each data point represents the average from 2–3 technical replicates for each isolated clone, and error bars represent mean of clones ± 95% confidence interval. Asterisks represent statistically significant differences between averages (*p<0.05; **p<0.01, unpaired t-test), with brackets to indicate samples compared. If not shown, averages were not statistically different. Outliers, as determined by the Grubb’s test, are denoted by unfilled shapes and were not utilized in statistical tests.

Article Snippet: The PGK promoter was inserted at the KpnI/XhoI cut sites; the optimized Flp recombinase expression cassette was amplified from pDIRE (Addgene #26745) and inserted at EcoRI/NotI; and a cassette with an internal ribosome entry sequence (IRES) sequence followed by the puromycin resistance gene was inserted at NotI/SacI.

Techniques: Mutagenesis, Generated, Expressing, Plasmid Preparation, Sequencing, Isolation, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Clone Assay

Journal: Current Research in Biotechnology

Article Title: Functional study of residual iCre activity relevant for split-Cre applications

doi: 10.1016/j.crbiot.2024.100263

Figure Lengend Snippet: Fig. 1. A 3D ribbon model of both homo-tetramer and monomer iCre recombinase generated with SWISS-MODEL web server https://swissmodel.expasy.org (Waterhouse et al., 2018). The model illustrates the protein’s interaction with DNA. The active site tyrosine is marked with a black star in the C-terminal catalytic domain. B Amino acid sequence of iCre recombinase with the different domains reported. In grey the nuclear localization sequence (NLS). The red arrow indicates the split location used in (Hirrlinger et al., 2009). α-Helices are highlighted as a thicker cylinder (A-N), β-strands are labelled 1–5 and polylinker between E and F α-helices connects the N-terminal to the C-terminal of the recombinase, which contains the catalytic domain. Active-site residues are boxed; asterisks show amino acids that contact DNA in the subunit that cleaves lox site; in red boxes are highlighted the methionine residues and the position of the progressively ATG-deleted iCre isoforms, starting from the iCre sequence deleted of its first methionine (namely iCreΔ1) to that one deleted of its 6th methionine (namely iCreΔ6). (For inter pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: PFU DNA polymerase (Promega, Madison, WI) was used in presence of the pDIRE vector harboring iCre gene (Addgene #26745) used as a template.

Techniques: Generated, Sequencing

Journal: Current Research in Biotechnology

Article Title: Functional study of residual iCre activity relevant for split-Cre applications

doi: 10.1016/j.crbiot.2024.100263

Figure Lengend Snippet: Fig. 3. Comparison of ribbon models (both homo-tetramer and monomer) for each N-terminal deleted iCre isoform generated using the resource https://swissmodel. expasy.org/ (Waterhouse et al., 2018).

Article Snippet: PFU DNA polymerase (Promega, Madison, WI) was used in presence of the pDIRE vector harboring iCre gene (Addgene #26745) used as a template.

Techniques: Comparison, Generated

Journal: Current Research in Biotechnology

Article Title: Functional study of residual iCre activity relevant for split-Cre applications

doi: 10.1016/j.crbiot.2024.100263

Figure Lengend Snippet: Fig. 4. A Schematic representation of the different isoforms of progressively N-terminal deleted iCre gene isoforms. Methionine residues present in the iCre cDNA, which serve as potential translation start sites, are highlighted as red bars. B Electrophoresis analysis of each isoform of iCre amplified by PCR highlighting the progressively amplicon size decrease. This confirms the successful generation of these truncated isoforms of iCre sequence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: PFU DNA polymerase (Promega, Madison, WI) was used in presence of the pDIRE vector harboring iCre gene (Addgene #26745) used as a template.

Techniques: Electrophoresis, Amplification, Sequencing

Journal: Current Research in Biotechnology

Article Title: Functional study of residual iCre activity relevant for split-Cre applications

doi: 10.1016/j.crbiot.2024.100263

Figure Lengend Snippet: Fig. 5. In vitro functional iCreΔ isoforms assay. Images of transfected HEK293T cells. The Cre-mediated recombination directly allows the expression of red fluorescent protein. Beside each line, schematic versions of the transfected constructs are shown. pIRES-eGFP was used as control of cell viability and transfection efficiency. pDIO-RFP was transfected alone to verify absence of aspecific RFP fluorescent signal. Scale bar: 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: PFU DNA polymerase (Promega, Madison, WI) was used in presence of the pDIRE vector harboring iCre gene (Addgene #26745) used as a template.

Techniques: In Vitro, Functional Assay, Transfection, Expressing, Construct, Control

Journal: Current Research in Biotechnology

Article Title: Functional study of residual iCre activity relevant for split-Cre applications

doi: 10.1016/j.crbiot.2024.100263

Figure Lengend Snippet: Fig. 6. High magnification confocal images of direct functional iCre assay. Images of co-transfected cells with bicistronic vector simultaneously expressing GFP and iCreΔ isoform, and a Cre-dependent RFP reporter plasmid. iCreΔ2-4 show residual recombinase activity and allow the co-expression of green and red fluorescent protein. DAPI was used to label nuclei Scale bar: 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: PFU DNA polymerase (Promega, Madison, WI) was used in presence of the pDIRE vector harboring iCre gene (Addgene #26745) used as a template.

Techniques: Functional Assay, Transfection, Plasmid Preparation, Expressing, Activity Assay