Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: The illustration is based on a multiple sequence alignment using the Clustal W algorithm (see for accession numbers used in the alignment) of MCVSyn and all full length MCPyV sequences deposited in the NCBI Database as of August 2011. Aligned genomes were compared to the consensus sequence (which is identical to MCVSyn as well as the isolates 17b, 18b and 20b). Nucleotide substitutions/mismatches relative to this sequence are shown as vertical black bars, whereas deletions are shown in red. Nucleotide insertions in a given sequence are shown as blue bars, and register as gaps in the backbone of the remaining genomes. Genomes that were isolated from MCC or MCC-derived cell lines are marked by an asterisk; the mutations which lead to the truncation of LT-Ag sequences in these genomes are likewise marked.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Sequencing, Isolation, Derivative Assay

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: Cell lines used to study MCVSyn replication, early and late transcription as well as particle formation.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques:

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: (A) 100 ng of intramolecular religated SV40 viral DNA was transfected in CV-1 cells and cells were lysed 12 h, 24 h, 36 h, 2d and 7d post transfection. Protein lysates were subsequently analyzed for SV40 LT-Ag (Pab419 antibody) and VP1 expression (α-VP1 polyclonal rabbit serum) by SDS-page and Western Blotting. Staining of actin was used to ensure that equal protein amounts were loaded per lane. (B) Low molecular weight DNA was isolated from SV40 DNA transfected CV-1 cells at the indicated time points by HIRT extraction, 1 µg DNA was DpnI and EcoRI digested; DNA was separated on an agarose gel and stained with EtBr (left panel), followed by southern blotting and detection of viral DNA using a 32 PdCTP-labeled SV40 LT-Ag PCR fragment as a probe. The blot was exposed for 30 min. Numbers below the lanes correspond to the quantification of newly replicated DNA using a Fuji phosphoimager FLA7000 and MultiGauge software.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Transfection, Expressing, SDS Page, Western Blot, Staining, Molecular Weight, Isolation, Extraction, Agarose Gel Electrophoresis, Southern Blot, Labeling, Software

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: 5×10 4 H1299, PSFK-1 or 293 cells were transfected with 100 ng re-circularized MCVSyn DNA or equivalent amounts of pUC18 DNA (Mock control). At the indicated time points, cells were lysed and analyzed by immunoblotting for MCPyV LT-Ag expression using the monoclonal LT-Ag antibody Cm2B4. Equal protein loading was confirmed by re-incubating the membrane with an anti-actin antibody. An LT-Ag expression control (pos. control; transient transfection with a CMV-promoter driven LT-Ag expression construct for 48 h) was loaded as an internal control.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Transfection, Control, Western Blot, Expressing, Membrane, Construct

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: RNA was isolated at the indicated time points after transfection, DNAse I digested and used for cDNA synthesis followed by real time PCR using a LT-Ag or VP1 specific primer set. Results were normalized against GAPDH transcript levels.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Isolation, Transfection, cDNA Synthesis, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: (A) Double staining of CV-1 cells transfected with SV40 viral DNA. 4d p.t. the cells were fixed, and VP1 was detected with a polyclonal anti-VP1 antibody. LT-Ag was visualized with the monoclonal anti-LT antibody Pab419. Z-stack pictures were taken using confocal microscopy. Each picture represents an individual Z-stack. VP1 staining was observed primarily in speckles close to or at the nuclear membrane. LT-Ag staining was observed throughout the nucleoplasm with the nucleoli excluded. In some cells granular LT-Ag staining was observed. The panel on the lower right represents a 3× zoomed picture of a CV1 transfected cell with the two channels merged. Double staining of Merkel cell polyomavirus VP1 and LT-Ag in H1299 cells (B) and PFSK-1 cells (C) 4d p.t. reveals inner peripheral nuclear localization of MCVSyn VP1 protein. VP1 was visualized with a polyclonal anti-VP1 serum and anti-rabbit FITC, while LT-Ag was visualized with the monoclonal antibody Cm2B4 specifically recognizing MCPyV LT-Ag. 40 Z-stack pictures were taken scanning through the cells using a 63× magnification and 2fold zoom on a confocal microscope. The picture shown represents an individual image from the center of a Z-stack.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Double Staining, Transfection, Confocal Microscopy, Staining, Membrane, Microscopy

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: Optiprep™ gradient centrifugation was performed with cell lysates from CV1 (A) and H1299 (B) cells 4d after transfection with viral DNA. 15×250 µl fractions were collected (fraction 1 represents the fraction with the highest density and fraction 15 represents the lowest density fraction). (A) Left panel: Real time PCR of micrococcal nuclease treated fractions was performed using SV40 VP1 primer sequences. 20 µl of each gradient fraction was loaded on a 10% SDS-page followed immunoblotting using anti-VP1 serum. Right panel: Negative EM staining of SV40 particles identified in fraction 9. (B) Left panel: Real time PCR results of H1299 MCVSyn gradient fractions after micrococcal nuclease treatment using MCPyV VP1-specific primers. Right panel: Negative EM staining of particles identified in fractions 10 and 6.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Gradient Centrifugation, Transfection, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Staining

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: Images were prepared from PFSK-1 cultures at 8 days post transfection with MCVSyn DNA. (A and B) ∼40 µm electron dense particles were observed in approximately 1 out of 50 cells with the particles localizing in the nucleus close to membrane structures (additional particles in B that are located outside of the enlarged inset are marked by arrows). (C) Membrane-attached MCPyV particles, reminiscent of the structures observed in SV40 infected cells as shown in .

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Transfection, Membrane, Infection

Journal: Cell Reports Medicine

Article Title: A genetically modified minipig model for Alzheimer’s disease with SORL1 haploinsufficiency

doi: 10.1016/j.xcrm.2022.100740

Figure Lengend Snippet: Increased Aβ and tau levels in CSF from SORL1 -deficient Göttingen minipigs (A–F) Quantification of the APP processing products Aβ38 (A), Aβ40 (B), Aβ42 (C), Aβ42/Aβ40 ratio (D), and soluble APPα (E) and APPβ (F) in CSF from SORL1 -deficient (n = 6) and age-matched wt (n = 9/10) Göttingen minipigs. The average ages of the two groups of pigs were similar. The group of SORL1 het minipigs is depicted, including data obtained from the ko pig (6304) shown in red and statistical analysis shown for data excluding (black) or including (red) the data point for the SORL1 ko pig. Quantifications were performed using MSD assays for human APP fragments because of 100% conservation of the 42 amino acids comprising the Aβ sequence. (G) WB analysis of tau in CSF (first-time isolation) from wt and het SORL1 minipigs at 5, 18, 24, or 30 months of age. Identification numbers of individual minipigs are provided below every lane. Detection was performed with the 5E2 anti-tau antibody that binds to a region of tau that is 100% conserved between the human and pig protein ( Figure S5 A). (H) Quantification of CSF tau WB analysis for the 18-month-old (1 wt / het pair), 24-month-old (2 wt / het pairs), and 30-month-old (2 wt / het pairs) SORL1 minipigs. The signal for SORL1 wt pigs was set to 100% for each age, and the signal for the paired SORL1 het was expressed relative to this. (A–F and H) Two-tailed unpaired Student’s t test was used for all statistical analyses, with p values below 0.05 considered significantly changed. Data are expressed as mean ± SEM. (I) RT-PCR analysis of genes involved in generation of amyloid and tau. Gene expression of APP , α-secretases ( ADAM10 and ADAM17 ), β-secretase ( BACE1) , subunits of the γ-secretase ( PSEN1 and PSEN2 ), and MAPT (encoding tau) were analyzed in Cx tissue from a wt (6475), a het (6469), and the ko (6304) SORL1 minipig, validating that CRISPR-Cas9 had no detrimental effect on these genes. GAPDH served as a ctrl for successful cDNA synthesis, whereas −RT samples and water were used as a negative ctrl.

Article Snippet: The human codon-optimized Cas9 (kindly made available by George Church, Addgene plasmid # 41815) and the sgRNA (encoded by a pFUS-U6 vector) were expressed from two individual plasmids.

Techniques: Sequencing, Isolation, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Gene Expression, CRISPR, cDNA Synthesis

Journal: Cell Reports Medicine

Article Title: A genetically modified minipig model for Alzheimer’s disease with SORL1 haploinsufficiency

doi: 10.1016/j.xcrm.2022.100740

Figure Lengend Snippet:

Article Snippet: The human codon-optimized Cas9 (kindly made available by George Church, Addgene plasmid # 41815) and the sgRNA (encoded by a pFUS-U6 vector) were expressed from two individual plasmids.

Techniques: Recombinant, Plasmid Preparation, Software, CRISPR

Journal: eLife

Article Title: Group II truncated haemoglobin YjbI prevents reactive oxygen species-induced protein aggregation in Bacillus subtilis

doi: 10.7554/eLife.70467

Figure Lengend Snippet:

Article Snippet: * 1 NBRP: National BioResource Project, Japan. * 2 DNA fragments were synthesised with codon optimisation by GenScript. Bold letters indicate restriction enzyme sites. Initiation and termination codons are underlined. , , .

Techniques: Plasmid Preparation, Gene Expression, Over Expression, Sequencing

Journal: bioRxiv

Article Title: Divergent acute versus prolonged in vivo GLP-1R responses in β-arrestin 2-deleted primary beta cells

doi: 10.1101/2022.04.21.489075

Figure Lengend Snippet: ( a ) Relative β-arrestin 2 gene expression in β-arrestin 2 (Barr2) KD vs control INS-1 832/3 cells (n = 3). ( b ) Ex-4 dose-response AUC curves for Nb37-SmBit and CAAX-LgBiT (plasma membrane) or Endofin-LgBiT (endosomal) signalling complementation assays (n = 5). ( c ) LogEC50 and Emax values calculated from (b). ( d ) Representative images of CFP and YFP channels from INS-1 832/3 cells transfected either with global (cytoplasmic) or TGN-targeted T Epac VV cAMP biosensor constructs. ( e ) Changes in FRET (YFP/CFP) over time in INS-1 832/3 Barr2 KD vs control cells expressing global or TGN-targeted T Epac VV cAMP biosensor constructs in response to 100 nM Ex-4 followed by 100 μM IBMX + 10 μM forskolin (n = 5). ( f ) AUCs calculated for the Ex-4 treatment period from (e). TGN-localised normalised to global cAMP AUCs are depicted for each cell type. ( g ) Representative blots for pERK1/2, total ERK1/2, pCREB, and total CREB in Barr2 KD and control INS-1 832/3 cells at baseline (0 min) or after treatment with 100 nM Ex-4 for the indicated time-points. ( h ) Quantification of pERK1/2 over total ERK1/2 using densitometry analysis. Values normalised to baseline (fold vs vehicle) are also shown (n = 4). ( i ) Quantification of pCREB over total CREB using densitometry analysis. Values normalised to baseline (fold vs vehicle) are also shown (n = 3). Comparisons were made using t-tests, or two-way ANOVA with Sidak’s post hoc tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs control group. Data are presented as mean ± SEM.

Article Snippet: Nb37 cDNA (synthesised by GenScript with codon optimisation) was C-terminally fused to SmBiT with a 15-amino-acid flexible linker (GGSGGGGSGGSSSGGG), and the resulting construct referred to as Nb37-SmBiT.

Techniques: Gene Expression, Control, Clinical Proteomics, Membrane, Transfection, Construct, Expressing