merkel cell polyomavirus mcv dna  (Thermo Fisher)

Journal: Viruses

Article Title: Replication Kinetics for a Reporter Merkel Cell Polyomavirus

doi: 10.3390/v14030473

Figure Lengend Snippet: Development of a Merkel cell polyomavirus (MCV) molecular clone using minicircle (mc) technology. ( A ) Electrophoresis of MCV molecular clone re-circularized by T-4 DNA-ligase. DNA size markers are shown at the left of the gel image. ( B ) Schematic of recombinase-mediated re-circularization (minicircle technology) of MCV molecular clone (MCVmc). ( C ) Electrophoresis of MCV DNA extracted before and after recombination. DNA size markers are indicated at the left of the gel image. ( D ) Western blot of MCV-encoded proteins (LT, sT and VP1) from MCV-ligated and MCVmc-transfected 293 cells 2 and 4 days post transfection. Untransfected 293 cells were used as a negative control. The LT and VP1 expression constructs transfected in 293 cells were used as a positive control (indicated as +), while α-tubulin was used as an endogenous protein control. Protein molecular weight markers are shown on the left. The result is representative of three independent experiments. ( E ) Real-time PCR (qPCR) of the DpnI-resistant MCV genome from MCV-ligated or MCVmc-transfected 293 cells 2 and 4 days post transfection. Untransfected 293 cells were used as a negative control (indicated as −). The ΔΔCT method was used to determine MCV genome copy number fold change; GAPDH was used as an endogenous control, while the MCV-ligated 2 days post transfection group was used as the experimental control. Error bars indicate the ± SD of three independent experiments. ( F – G ) Immunofluorescence of LT-AF488 (pseudo color green), VP1-AF488 (pseudo color red) and DAPI (blue) in MCV-ligated and MCVmc-transfected 293 or U2OS cells 5 days post transfection. The numbers of VP1-positive cells per coverslip were counted with a Cytation 5 cell imaging multi-mode reader and are shown on the right. Images were originally acquired at 40× magnification.

Article Snippet: A denatured, biotin-11dUTP-labeled MCV DNA probe generated with the Bio-prime Array CGH kit (cat. no. 45-0048, Invitrogen, Waltham, MA, USA) using a full-length MCV genome as template was then added to the prehybridization buffer and incubated overnight at 65 °C.

Techniques: Electrophoresis, Western Blot, Transfection, Negative Control, Expressing, Construct, Positive Control, Molecular Weight, Real-time Polymerase Chain Reaction, Immunofluorescence, Imaging

Journal: Viruses

Article Title: Replication Kinetics for a Reporter Merkel Cell Polyomavirus

doi: 10.3390/v14030473

Figure Lengend Snippet: MCVmc is infectious in primary cells. ( A ) Western blot of MCV-ligated and MCVmc virions fractions from Opti-Prep (iodixanol) gradient purification. Fractions 1–13 (top to bottom) are indicated. * Indicates VP1 dimer. Fractions 10 through 13 were mixed and MCV genome copy numbers per µL are shown at the bottom of virion-positive fractions. Protein molecular weight markers are shown on the right. Results are representative of three independent experiments. ( B ) Quantification of the number of VP1-positive cells per 4× magnification field in BJ-hTert, HFF-1, MSC-bm and MSC-a cells infected with MCV-ligated or MCVmc virus from ( A ), 6 days post infection, by IFA of VP1. Four fields from one cover slip for each cell line was counted for MCVmc and MCV-ligated. ( C ) Representative immunofluorescence images showing LT-AF488 (pseudo color green), VP1-AF568 (pseudo color red), together with DAPI (blue) in MCV-ligated and MCVmc-infected HFF-1 cells 6 days post infection. Images were originally acquired at 40× magnification. Images ( C ) were acquired and the number of VP1 positive cells ( B ) was counted using a Cytation 5 cell imaging multi-mode reader. Results represent three independent experiments. ( D ) Southern blot of MCV genome from BJ-hTert cells infected with MCV-ligated or MCVmc. Mock-infected cells were used as a negative control. The relative amount of total DNA loaded is shown by EtBr staining and DNA size markers are shown in the left. Results represent three independent experiments.

Article Snippet: A denatured, biotin-11dUTP-labeled MCV DNA probe generated with the Bio-prime Array CGH kit (cat. no. 45-0048, Invitrogen, Waltham, MA, USA) using a full-length MCV genome as template was then added to the prehybridization buffer and incubated overnight at 65 °C.

Techniques: Western Blot, Purification, Molecular Weight, Infection, Immunofluorescence, Imaging, Southern Blot, Negative Control, Staining

Journal: Viruses

Article Title: Replication Kinetics for a Reporter Merkel Cell Polyomavirus

doi: 10.3390/v14030473

Figure Lengend Snippet: Generation of an mScarlet reporter MCVmc. ( A ) Schematic of VP1 fluorescent fusion constructs representing VP1 (black) containing mScarlet (red) tag in the presence (cyan) or absence of a P2A linker. Arrow indicates the P2A ribosome-skipping site and the whole P2A peptide sequence (cyan) is shown with four flanking amino acids from VP1 (black) and mScarlet (red). ( B ) Immunoblot of MCV-encoded proteins (LT and VP1) and mScarlet in 293 cells transfected with MCVmc, MCVmc.VP1-mS and MCVmc. VP1-P2A-mS after 2 and 4 days post transfection. Un-transfected 293 cells were used as a negative control (−), while 293 cells transfected with LT or VP1 expression construct were used as a positive control (+). α-tubulin was used as an endogenous protein-loading control. Protein molecular weight markers are shown on the left. ( C ) Quantification of DpnI-resistant replicated MCV DNA in 293 cells transfected with MCVmc, MCVmc.VP1-mS or MCVmc. VP1-P2A-mS 2 and 4 days post transfection qPCR results. The ΔΔCT method was used to calculate relative MCV DNA levels; GAPDH was used as the endogenous loading control, while MCVmc was used as the experimental control. Error bars indicate ± SD of three independent replicates. ( D ) Confocal images of mScarlet expression (pseudo color red) and LT-AF488 (pseudo color green) or VP1-AF488 (pseudo color Green) immunofluorescence in MCVmc, MCVmc.VP1-mS and MCVmc. VP1-P2A-mS-transfected 293 cells 5 days post transfection. LT, VP1 or mScarlet expression construct-transfected 293 cells were used as positive controls. Images were originally acquired at ×40 magnification.

Article Snippet: A denatured, biotin-11dUTP-labeled MCV DNA probe generated with the Bio-prime Array CGH kit (cat. no. 45-0048, Invitrogen, Waltham, MA, USA) using a full-length MCV genome as template was then added to the prehybridization buffer and incubated overnight at 65 °C.

Techniques: Construct, Sequencing, Western Blot, Transfection, Negative Control, Expressing, Positive Control, Molecular Weight, Immunofluorescence

Journal: Viruses

Article Title: Replication Kinetics for a Reporter Merkel Cell Polyomavirus

doi: 10.3390/v14030473

Figure Lengend Snippet: Virus production and infection using MCV mScarlet reporter virus. ( A ) Western blot and qPCR analysis of fractions from MCVmc, MCVmc.VP1-mS, and MCVmc.VP1-P2A-mS virions purified over an Opti-Prep gradient (Iodixanol) (concentrations noted on top). In the qPCR quantification, the blue curve shows total MCV DNA copy number, while the red curve shows Benzonase-protected MCV DNA copy numbers. Results represent two independent experiments. ( B ) Image of MCVmc virions (from ( A )) by negative staining electron microscopy. ( C ) Western blot and qPCR analysis in Opti-Prep fractions from heterologous VP1/VP2 packaged MCVmc, MCVmc.VP1-mS, and MCVmc.VP1-P2A-mS. Results represent one-time experiments. Iodixanol concentration for each fraction is noted on top. qPCR quantification: blue curves show total MCV DNA copy number, while red curves show the number of Benzonase-resistant MCV DNA copies.

Article Snippet: A denatured, biotin-11dUTP-labeled MCV DNA probe generated with the Bio-prime Array CGH kit (cat. no. 45-0048, Invitrogen, Waltham, MA, USA) using a full-length MCV genome as template was then added to the prehybridization buffer and incubated overnight at 65 °C.

Techniques: Infection, Western Blot, Purification, Negative Staining, Electron Microscopy, Concentration Assay

Journal: Viruses

Article Title: Replication Kinetics for a Reporter Merkel Cell Polyomavirus

doi: 10.3390/v14030473

Figure Lengend Snippet: Kinetics of MCVmc reporter viruses. ( A ) A model of MCV replication kinetics, early/late gene expression as well as viral DNA replication is depicted. LT-Ag and VP1 expression kinetics is based on the Western blots in . The MCV genome replication kinetics is hypothetical. ( B ) A map of MCVmcs with sites of fluorescent protein cassettes and minicircle vector insertion is indicated. ( C ) A detailed schematic of fluorescent-tagged MCV viral proteins that can be expressed from ( A ). Arrows in ( B ) indicate ribosome-skipping sites for F2A and P2A peptide sequences. ( D ) A table of tested MCVmc fluorescent reporter genomes. +: detected in transfected cells; −: not detected in transfected cells.

Article Snippet: A denatured, biotin-11dUTP-labeled MCV DNA probe generated with the Bio-prime Array CGH kit (cat. no. 45-0048, Invitrogen, Waltham, MA, USA) using a full-length MCV genome as template was then added to the prehybridization buffer and incubated overnight at 65 °C.

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection