Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Design of MLV-Based Gag.MS2.CRISPR/Cas9 All-in-One Chimera (A) Schematic illustration of Gag.MS2.CRISPR/Cas9 all-in-one particles. Chimeric Gag.MS2 proteins bind to non-viral RNAs containing two copies of a high-affinity MS2 target site (TS) hairpin structure. Hypothetically, redirection of the MLV-based packaging machinery should now allow specific packaging of different RNA species. In the case of CRISPR/Cas9, TS-containing Cas9 mRNA and Pol III-driven sgRNA transcripts should be co-packaged into one Gag.MS2 particle. (B) Design of the Gag.MS2 expression construct. The MLV-based wild-type Gag-Pol expression construct is depicted on top. In this plasmid, expression of Gag and Gag-Pol precursor proteins is initiated by enhancer and promoter sequences of the cytomegalovirus (CMV) and terminated by the bovine growth hormone poly(A) signal (pA). During particle maturation, Gag and Gag-Pol proteins are further processed into matrix (MA), p12, capsid (CA), NC, protease (PR), reverse transcriptase (RT), and integrase (IN) subunits. To generate the Gag.MS2 expression plasmids, coding sequences for Gag and Pol were separated on two CMV-driven plasmids. Subsequently, NC was replaced by a genetically fused MS2 heterodimer. All viral protease sites separating Gag and Pol subunits were maintained. (C) SpCas9 expression plasmids with or without 2 MS2 TS copies. SpCas9 was co-expressed with EGFP, and separation of both proteins was achieved via the P2A cleavage site from porcine teschovirus. PRE, woodchuck hepatitis virus post-transcriptional regulatory element. (D) Non-viral sgRNA expression plasmids targeting Tet2 , Tp53 , PTEN , TP53 , CXCR4 , or Renilla luciferase (Luc). sgRNAs with or without TS hairpins were generated. Two TS hairpins were either incorporated (TS.inc) into or cloned adjacent (TS.adj) to the sgRNA scaffold. To control for transfection efficiencies, a CMV-driven red-fluorescent DsRedexp expression cassette was included. hU6, human RNA polymerase III U6 promoter. (E) Detailed overview of TS.inc and TS.adj expression cassettes. The scaffold sequence of TS.inc is depicted on top, and the scaffold sequence of TS.adj is at the bottom. Both sgRNAs contain a 105 bp long stuffer sequence flanked by BsmBI restriction sites (red). The design of the TS.inc scaffold was adapted from Konermann et al., and positioning of the two TS hairpins in the TS.adj constructs was based on the work of Mali et al. with differences in TS linker and spacer sequences. The positions of the two TS hairpins (TS 1 and TS 2) are marked in blue. The arrows point to the cutting sites of BsmBI. Expression of the sgRNAs is terminated by the TTTTTT motif.

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: CRISPR, Expressing, Construct, Plasmid Preparation, Reverse Transcription, Virus, Luciferase, Generated, Clone Assay, Control, Transfection, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Individual Transfer of CRISPR/Cas9 RNAs by Gag.MS2 Particles (A) Highly efficient delivery of SpCas9.TS mRNA into target cells. Gag.MS2.SpCas9.TS particles were used to transduce NIH3T3-based RFP657.Tet2+sgRNA.Tet2 reporter cells (see the scheme above the graph) with the depicted supernatant volumes. The percentage of RFP657-negative cells was determined 6 days post-transduction. The results of 8 independent supernatants are shown. (B) Inefficient transfer of sgRNAs Tet2.TS.inc and Tet2.TS.adj by Gag.MS2 particles. RFP657.Tet2+SpCas9 NIH3T3 reporter cells were transduced with Tet2.TS.inc or Tet2.TS.adj Gag.MS2 particles. RFP657 knockout was determined by flow cytometry 6 days post-transduction. The results of 10 (Tet2.TS.inc) to 13 (Tet2.TS.adj) independently packaged supernatants are shown. (C) Co-transduction of separately packaged SpCas9.TS mRNA and sgRNA.TS resulted in poor RFP657 knockout rates. NIH3T3-based RFP657.Tet2 reporter cells were transduced with 6–8 individually packaged SpCas9.TS and Tet2.TS.inc or Tet2.TS.adj Gag.MS2 supernatants from (A) and (B) at the depicted volume ratios. Flow cytometry was performed 6 days post-transduction.

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: CRISPR, Transduction, Knock-Out, Flow Cytometry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Efficient RFP657 Knockout by Gag.MS2.CRISPR/Cas9 All-in-One Particles SpCas9, Tet2 sgRNA, Gag.MS2, Pol, and VSVg expression plasmids were co-transfected into the HEK293T packaging cell line. The resulting supernatants SpCas9+Tet2, SpCas9.TS+Tet2.TS.inc, or SpCas9.TS+Tet2.TS.adj were harvested and concentrated 50-fold. Transfection efficiencies were determined for all produced supernatants (see also A and S4B). (A and B) Efficient delivery of CRISPR/Cas9 RNA components depends on the presence of the TS hairpin dimer. Supernatants from HEK293T cells that exhibited transfection efficiencies <50% were used to transduce NIH3T3-based (A) or HT1080-based (B) RFP657.Tet2 reporter cells. The graphs show RFP657 knockout rates mediated by 100 μL supernatant for the individual experiments 5–6 days post-transduction. Results from 4–7 independently generated supernatants are displayed (SpCas9+Tet2 = 6 supernatants, SpCas9.TS+Tet2.TS.inc = 7 supernatants, and SpCas9.TS+Tet2.TS.adj = 4 supernatants) (see also Figure S4 A). (C and D) Non-specific packaging of CRISPR/Cas9 RNAs and/or RNPs into Gag.MS2 particles. Experiments were similar to those shown in (A) and (B). However, NIH3T3 (C) and HT1080 (D) RFP657.Tet2 reporter cells were transduced with supernatants that were obtained from transfections with efficiencies >50%. The depicted results were generated with 100 μL of 10–12 individually packaged supernatants (SpCas9+Tet2 = 10 supernatants, SpCas9.TS+Tet2.TS.inc = 10 supernatants, and SpCas9.TS+Tet2.TS.adj = 12 supernatants) (see also Figure S4 B). The graph depicted in (D) also displays RFP657 knockout rates induced by different volumes of 50-fold-concentrated integrating RIT.CRISPR/Cas9.Tet2 all-in-one supernatants. The respective MOIs are given in the gray box. Each data point represents an individually generated supernatant (n = 4) with an average functional titer of 7.2 × 10 7 t.u./mL.

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: Knock-Out, CRISPR, Expressing, Transfection, Produced, Transduction, Generated, Functional Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Characterization of Gag.MS2.CRISPR/Cas9 Particles (A) SpCas9 and SpCas9.TS mRNA content of individually packaged Gag.MS2 particles. Absolute mRNA copies per 50 μL supernatant are shown. (B) Tet2 sgRNA quantification. The differences of incorporated Tet2 (no TS), Tet2.TS.inc, or Tet2.TS.adj sgRNAs are shown. The graph depicts the sgRNA copies per 50 μL. Absolute amounts were calculated with the help of individual standards. (C) Gag.MS2.CRISPR/Cas9 supernatants contain SpCas9 protein. SpCas9, SpCas9.TS, SpCas9.TS+Tet2.TS.inc (TS.inc), and SpCas9.TS+Tet2.TS.adj (TS.adj) Gag.MS2 supernatants were subjected to immunoblot analysis, and membranes were successively stained with Ponceau S for total, SpCas9 (163 kDa), and MS2 dimer (27 kDa) proteins. The 82 kDa band on the MS2 blot represents the immature Gag.MS2 precursor polyprotein. (D) Quantification of SpCas9 protein by ELISA within different particle types. Each data point represents one independent supernatant. (E) Chimeric Gag.MS2.CRISPR/Cas9 all-in-one particles do not integrate. The genomic DNA of transduced cells was harvested 10 days post-transduction and analyzed for the occurrence of stable integration events. The graph depicts the determined mean vector copy numbers (VCNs) of two independently generated supernatants. An integrating RIT.CRISPR/Cas9.Tet2 all-in-one vector served as a positive control (average transduction rate: 14% EGFP-positive cells). Each data point represents the mean value of 3 independently determined C t values (technical replicates).

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: CRISPR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation, Generated, Positive Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Detailed Analysis of Gag.MS2.CRISPR/Cas9 All-in-One Particles

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: CRISPR, Knock-Out, Functional Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Stable and High-Dose SpCas9 Expression Is Cytotoxic (A) Reduced cell numbers in RIT.SpCas9-treated cultures. NIH3T3 cells were transduced with integrating RIT.EGFP or RIT.SpCas9 particles or non-integrating Gag.MS2.SpCas9.TS particles. Based on titration via real-time qRT-PCR, RIT.EGFP and RIT.SpCas9 supernatants were adjusted before transduction (see also Figure S6 D). Gag.MS2.SpCas9.TS supernatants were not adjusted. Cell counts were determined 4 days post-transduction. Non-treated NIH3T3 cells served as Mock control. (B) Prolonged and high-dose SpCas9 expression decreases the metabolic activity of NIH3T3 cells. To assess the metabolic activity, transduced cells of (A) were subjected to the colorimetric MTS cell proliferation and cytotoxicity assay. The graph shows the absorbance (490 nm) of purple formazan, which originated from the reduction of MTS by cellular NAD(P)H. Results are shown as mean ± SD. (C) High-dose SpCas9 expression induces apoptosis. Transduced cells of (A) were co-stained with Annexin V and propidium iodide (PI) and analyzed for the occurrence of apoptotic (Annexin V + ) and dead (PI + or Annexin V + /PI + ) cells. The left graph depicts the total percentage of Annexin V + cells in the respective samples. The right graph shows Annexin V + , PI + and Annexin V + /PI + cells of non-transduced Mock cells and cultures that were transduced with RIT.SpCas9. The error bars in the right graph indicate the SD of the mean. (D) High-dose SpCas9 expression resulted in a substantial G0/G1 cell-cycle arrest. NIH3T3 cells were freshly transduced with the depicted supernatants and volumes. Cultures were subjected to cell-cycle analysis 6 days post-transduction. The percentages of cells in G0/G1 and G2/M of transduced cultures were shown relative to the respective phase of non-transduced Mock cultures. Results depicted in (A)–(C) were generated with 2–3 independently packaged supernatants (RIT.EGFP = 2–3 supernatants, RIT.SpCas9 = 3 supernatants, and Gag.MS2.SpCas9.TS = 3 supernatants). Each data point presented in (D) was obtained from independently packaged supernatants (n = 3 biological replicates).

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: Expressing, Transduction, Titration, Quantitative RT-PCR, Control, Activity Assay, Cytotoxicity Assay, Staining, Cell Cycle Assay, Generated

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Functional Knockout of Endogenous CXCR4 in Human Jurkat Cells (A) Scheme of the functional CXCR4 knockout experiment. Human Jurkat cells sorted for high CD4 and CXCR4 expression were transduced with LIT or Gag.MS2 CRISPR/Cas9 all-in-one particles for targeted CXCR4 knockout. In a second round of transduction, CXCR4 knockout cultures were transduced with a CXCR4-tropic LIT.EGFP vector. Only cells that still carry the CXCR4 receptor will be susceptible to transduction with CXCR4-tropic LIT.EGFP. The flow cytometric plots show CD4 and CXCR4 expression in Jurkat cells before and after sorting. (B) Schemes of LIT.CRISPR/Cas9.CXCR4 all-in-one and LIT.EGFP vectors. Depicted is the vector configuration after integration. All vectors are self-inactivating (SIN) due to deletion of viral enhancer and promoter sequences within the long terminal repeats (LTRs), and transgene expression ( SpCas9.P2A.dTomato or EGFP ) is driven by an internal spleen focus-forming virus (SFFV) promoter. In the LIT.CRISPR/Cas9.CXCR4 all-in-one vector, the hU6 promoter drives expression of sgRNA.CXCR4. Ψ, packaging signal; RRE, rev responsive element; cPPT, central polypurine tract; PRE, post-transcriptional regulatory element from woodchuck hepatitis virus. (C) Efficient CXCR4 knockout by Gag.MS2.CRISPR/Cas9 all-in-one particles. Sorted CD4 + /CXCR4 + Jurkat cells were transduced with LIT.CRISPR/Cas9.CXCR4 all-in-one particles (MOI 5) or either 25 μL of 100-fold concentrated (CXCR4.TS.inc and CXCR4.TS.adj) or 50 μL of 50-fold concentrated (Tp53.TS.inc and Tp53.TS.adj) Gag.MS2.CRISPR/Cas9 all-in-one particles. The knockout rate was determined by flow cytometry 5 days after transduction. Each data point reflects one independently generated supernatant. (D) Genome targeting efficiencies of LIT.CRISPR/Cas9.CXCR4 and Gag.MS2.CRISPR/Cas9.CXCR4.TS.adj all-in-one particles. The gDNA of CXCR4 knockout cultures was subjected to a T7 endonuclease I assay. On-target activity is indicated by the cleavage of the 940 bp CXCR4 PCR amplicon into 580 and 360 bp fragments. The InDel rate of each sample is depicted as a percentage. (E) Reduced CXCR4-tropic LIT.EGFP transduction rates in cultures that were treated with Gag.MS2.CRISPR/Cas9.CXCR4.TS.adj all-in-one particles. LIT.CRISPR/Cas9.CXCR4 all-in-one and Gag.MS2.CRISPR/Cas9.CXCR4.TS.adj cultures from (C) were individually transduced with CXCR4-tropic LIT.EGFP vector particles (9 days after CRISPR/Cas9 treatment). Three days later, cultures were analyzed by flow cytometry for EGFP and CXCR4 expression. The right graph depicts the ratios EGFP + /EGFP − cells in CXCR4 + or CXCR4 − fractions expressed as mean ± SD. Representative flow cytometry plots are shown on the left (MFIs in red).

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: Functional Assay, Knock-Out, Expressing, Transduction, CRISPR, Plasmid Preparation, Virus, Flow Cytometry, Generated, T7EI Assay, Activity Assay, Amplification

Journal: Molecular Therapy. Nucleic Acids

Article Title: Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

doi: 10.1016/j.omtn.2018.09.006

Figure Lengend Snippet: Growth Advantage of NUFF TP53 Knockout Cells (A) Generation of DsRedexp + NUFF cells. Freshly thawed NUFF cells (passage 9) were stably transduced with LIT.DsRedexp.IRES.Zeo encoding for DsRedexp and the Zeocin resistance gene (Zeo) at MOI 0.5. Co-expression of DsRedexp and Zeo was achieved by the internal ribosomal entry site (IRES) of endomycarditis virus and was initiated by enhancer and promoter sequences of SFFV. Transduced NUFF cells were selected with 100 μg/mL Zeocin for 10 days. The flow cytometric plots depict the percentage of DsRedexp + NUFF cells before and after Zeocin selection. (B) Knockout of TP53 , but not PTEN , resulted in a growth advantage. DsRedexp + NUFF cells were transduced in a single round with LIT.CRISPR/Cas9 all-in-one vector particles for targeted knockout of PTEN , TP53 , or Luc at MOI 10. Cells were mixed with non-treated wild-type NUFF cells 3 days post-transduction, and co-cultures were analyzed for DsRedexp expression for up to 67 days. The control co-culture of non-treated DsRedexp + and wild-type NUFF cells is marked in red. Exp 1, experiment 1; Exp 2, experiment 2. (C) Outgrowth of DsRedexp + NUFF cells that were treated with Gag.MS2.CRISPR/Cas9.TP53 all-in-one particles. DsRedexp + NUFF cells were transduced once with 50 μL of the depicted non-integrating Gag.MS2.CRISPR/Cas9 all-in-one particles. In TS.inc and TS.adj sgRNA combined samples, the respective supernatants were mixed at a ratio of 1:1 before transduction. (D) Transduction of DsRedexp + NUFF cells twice with Gag.MS2.CRISPR/Cas9.TP53 all-in-one particles slightly accelerates outgrowth of TP53 knockout cells. The experiment was similar to that in (C), but DsRedexp + NUFF cells were transduced twice with Gag.MS2.CRISPR/Cas9 all-in-one particles on 2 consecutive days before co-culture. (E) Double knockout of PTEN and TP53 by Gag.MS2.CRISPR/Cas9 all-in-one particles did not accelerate outgrowth of DsRedexp + NUFF cells. Before co-culture, DsRedexp + NUFF cells were co-transduced with either LIT or Gag.MS2 CRISPR/Cas9 all-in-one particles targeting TP53 or PTEN . LIT.CRISPR/Cas9 all-in-one supernatants were each applied at MOI 10, and 50 μL of each Gag.MS2.CRISPR/Cas9 all-in-one supernatant was used. The curves were obtained from individual transductions with independently generated supernatants. Numbers 1–9 (in blue) displayed in graphs (B)–(E) indicate selected cultures that were expanded for gDNA harvest and subjected to InDel analyses (see also ).

Article Snippet: After staining all blotted proteins with Ponceau S (Sigma-Aldrich), the membrane was successively probed with a rabbit polyclonal anti-enterobacteriophage MS2 coat protein antibody (1:5,000) (Merck Millipore, Darmstadt, Germany) and a monoclonal mouse purified anti-CRISPR (Cas9) antibody (1:1,500) (BioLegend, Koblenz, Germany) in Tris-buffered saline with 0.05% Tween and 3% milk powder at 4°C overnight (both Roth, Karlsruhe, Germany).

Techniques: Knock-Out, Stable Transfection, Transduction, Expressing, Virus, Selection, CRISPR, Plasmid Preparation, Control, Co-Culture Assay, Double Knockout, Generated

Journal: Frontiers in Microbiology

Article Title: Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

doi: 10.3389/fmicb.2016.01911

Figure Lengend Snippet: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE – left part of the picture) and Western blot ( right part of the picture) results. 1,6, E. coli BL21 (DE3) negative control; 2,5, IPTG-non-induced culture; 3,4, IPTG-induced culture. 13 kDa MS2 coat protein was massively produced in the IPTG-induced culture. M, marker Spectra multicolor broad range protein ladder (Fermentas), the marker values are in kDa.

Article Snippet: To verify the production of MS2 coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis was carried out using a phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) as primary antibody and Goat Anti-Rabbit IgG, Fc specific fragment (Jackson ImmunoResearch, UK) as secondary antibody.

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Negative Control, Produced, Marker

Journal: Frontiers in Microbiology

Article Title: Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

doi: 10.3389/fmicb.2016.01911

Figure Lengend Snippet: Transmission electron microscopy (TEM) photograph of the intact MS2 phage-like particles (MS2 PLP) present in the supernatant after ultrasonic disruption of E. coli production cells. MS2 PLP are about 27 nm in diameter. The scale is 100 nm.

Article Snippet: To verify the production of MS2 coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis was carried out using a phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) as primary antibody and Goat Anti-Rabbit IgG, Fc specific fragment (Jackson ImmunoResearch, UK) as secondary antibody.

Techniques: Transmission Assay, Electron Microscopy, Disruption

Journal: Frontiers in Microbiology

Article Title: Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

doi: 10.3389/fmicb.2016.01911

Figure Lengend Snippet: Result of agarose gel electrophoresis (1%) detection of MS2 phage-like particles (MS2 PLP) in three layers removed after ultracentrifugation from the tube. 1, top layer; 2, middle layer; 3, lower layer. M, marker 2-log DNA ladder (New England Biolabs, UK), the marker values are in kb. The top layer contained the highest amount of MS2 PLP (the band on the gel around 1.5 kb).

Article Snippet: To verify the production of MS2 coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis was carried out using a phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) as primary antibody and Goat Anti-Rabbit IgG, Fc specific fragment (Jackson ImmunoResearch, UK) as secondary antibody.

Techniques: Agarose Gel Electrophoresis, Marker

Journal: Frontiers in Microbiology

Article Title: Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

doi: 10.3389/fmicb.2016.01911

Figure Lengend Snippet: Transmission electron microscopy (TEM) photograph of the intact MS2 phage-like particles (MS2 PLP) after purification steps. The scale is 100 nm.

Article Snippet: To verify the production of MS2 coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis was carried out using a phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) as primary antibody and Goat Anti-Rabbit IgG, Fc specific fragment (Jackson ImmunoResearch, UK) as secondary antibody.

Techniques: Transmission Assay, Electron Microscopy, Purification

Journal: Frontiers in Microbiology

Article Title: Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

doi: 10.3389/fmicb.2016.01911

Figure Lengend Snippet: Transmission electron microscopy photograph from TEM MS2 phage-like particles (MS2 PLP) quantification against latex standard. The large black “dot” is the latex standard and the white dots are MS2 PLP. The scale is 500 nm.

Article Snippet: To verify the production of MS2 coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis was carried out using a phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) as primary antibody and Goat Anti-Rabbit IgG, Fc specific fragment (Jackson ImmunoResearch, UK) as secondary antibody.

Techniques: Transmission Assay, Electron Microscopy

Journal: Frontiers in Microbiology

Article Title: Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

doi: 10.3389/fmicb.2016.01911

Figure Lengend Snippet: The results of MS2 phage-like particles (MS2 PLP) quantification experiments.

Article Snippet: To verify the production of MS2 coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis was carried out using a phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) as primary antibody and Goat Anti-Rabbit IgG, Fc specific fragment (Jackson ImmunoResearch, UK) as secondary antibody.

Techniques: Spectrophotometry, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

doi: 10.3389/fmicb.2016.01911

Figure Lengend Snippet: Transmission electron microscopy photograph of homogenous and non-aggregated MS2 phage-like particles (MS2 PLP). The scale is 200 nm.

Article Snippet: To verify the production of MS2 coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis was carried out using a phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) as primary antibody and Goat Anti-Rabbit IgG, Fc specific fragment (Jackson ImmunoResearch, UK) as secondary antibody.

Techniques: Transmission Assay, Electron Microscopy

Journal: Frontiers in Microbiology

Article Title: Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

doi: 10.3389/fmicb.2016.01911

Figure Lengend Snippet: Extraction efficiency of MS2 phage-like particles (MS2 PLP) isolated from different types of matrices artificially contaminated with 5 × 10 6 MS2 PLP per sample.

Article Snippet: To verify the production of MS2 coat protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis was carried out using a phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) as primary antibody and Goat Anti-Rabbit IgG, Fc specific fragment (Jackson ImmunoResearch, UK) as secondary antibody.

Techniques: Extraction, Isolation

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: Bioconjugation of the B19 viral protein 1 unique region (VP1u) with the bacteriophage MS2 capsid, and internalization of the bioconjugate on erythroleukemic UT7/Epo cells: ( A ) Schematic depiction of the bioconjugation and labeling strategy of the bacteriophage MS2 capsid (PDB ID: 1MST ). The recombinant ∆C126 VP1u protein with a C-terminal metal affinity tag (MAT), FLAG tag, and a unique cysteine was coupled to the MS2 capsid using the heterobifunctional maleimide-PEG 24 -N-hydroxysuccinimide ester (NHS-PEG 24 -maleimide) crosslinker. The NHS-ester was conjugated to the surface lysines of the MS2 capsid and the maleimide group reacted with the unique cysteine in the VP1u. In addition, the capsid was labeled with the NHS-Atto 488 (green) and NHS-Atto 633 (blue) dyes; ( B ) Migration of the wild-type (WT) and bioconjugated MS2 capsids by agarose gel electrophoresis and detection of encapsidated RNA with GelRed. Capsid proteins are labeled with NHS-Atto dyes (green-blue); ( C ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the MS2 capsids, recombinant VP1u proteins, and the bioconjugates. Recombinant VP1u and MS2-VP1u conjugates were detected with an anti-FLAG antibody. In the Western blot, the transfer of low molecular weights proteins was less efficient; therefore, the detected signal did not quantitatively reflect the original and relative input of the proteins; ( D ) Uptake of the fluorescent MS2-∆C126 VP1u into UT7/Epo cells and detection of the internalized capsids in living cells by fluorescence microscopy and fluorescence-activated cell sorting (FACS); ( E ) Fluorescence microscopy images of the MS2-∆C126 VP1u and the Atto-labeled but internalization-deficient controls MS2-∆N30/∆C126 VP1u and MS2 (63× magnification). Scale bar: 15 µm.

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Labeling, Recombinant, FLAG-tag, Migration, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Fluorescence, Microscopy, FACS

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: Competition of native B19V internalization with the MS2-VP1u bioconjugate. ( A ) Schematic comparison of the native B19V (three VP1u) with the MS2-∆C126 VP1u bioconjugate (20–30 PEG 24 -linked ∆C126 VP1u per capsid). Nucleic acids are encapsidated in the icosahedral assembled coat proteins (blue). The VP1u contains a phospholipase A 2 (PLA 2 ) motif (green) and a receptor-binding domain (red); ( B ) Internalization of B19V into UT7/Epo cells was measured in presence of increasing concentrations of MS2-VP1u as competitor. After 30 min incubation at 37 °C, cells were washed, trypsinized and internalized B19V DNA was quantified by quantitative polymersaase chain reaction (qPCR; ( C ) Competition of B19V uptake in presence of equimolar concentrations of MS2-∆C126 or internalization-deficient MS2-∆N30/∆C126 ( n = 3). Samples kept at 4 °C instead of incubation at 37 °C show the background signal without internalization.

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Comparison, Binding Assay, Incubation

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: Native B19V and MS2-VP1u specifically target erythroid cell lines. ( A ) MS2-∆C126 VP1u and native B19V were incubated with the same mixed cell culture of non-adherent UT7/Epo cells, and adherent HeLa and MRC-5 cells. Cells were fixed and immunostained with an anti-B19V capsid antibody (860-55D ). Cell-specific internalization of MS2-∆C126 VP1u (green) and native B19V (magenta) was visualized by fluorescence microscopy. Scale bar: 100 µm (20× magnification), 15 µm (63× magnification); ( B ) Cellular uptake of native B19V into the different cell lines measured by qPCR ( n = 3); ( C ) Internalization of MS2-VP1u on normal and phorbol myristate acetate (PMA)-differentiated erythroleukemic K562 cells, visualized by fluorescence microscopy. Scale bar: 30 µm (63× magnification).

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Incubation, Cell Culture, Fluorescence, Microscopy

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: VP1u receptor expression correlates with B19V internalization and infection along the erythroid differentiation: ( A ) cluster of differentiation (CD) 34+ hematopoietic stem cells were expanded ex vivo to differentiate into the erythroid lineage and tested for MS2-∆C126 VP1u uptake (63× magnification). MS2-∆C126 VP1u internalization was further tested on peripheral blood cells, containing mature erythrocytes and ~1% reticulocytes, representing the final erythroid differentiation stages; ( B ) Immunostaining of glycophorin A (63× magnification) and May–Grünwald–Giemsa staining (100× magnification) indicates the successful transition to the terminal erythroid differentiation; B19V internalization ( C ) and infection ( D ) along the erythroid differentiation was quantified by qPCR ( n = 3); ( E ) Internalization of B19V into erythroid progenitor/precursor cells (day 8) was competed with ∆C126 VP1u or dysfunctional ∆N30/∆C126 VP1u; ( F ) After VP1u-competed uptake of B19V, cells (day 8) were incubated for further 24 h to measure the effect of the competition on the viral infection ( n = 3). Notably, B19V internalization was performed with more cells (2×) and higher multiplicity of infection (MOI) (7.5×) compared with infectivity assays, explaining the relatively high detection signal of uptake versus infection. Scale bar: 15 µm.

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Expressing, Infection, Ex Vivo, Immunostaining, Staining, Incubation

Journal: Viruses

Article Title: The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

doi: 10.3390/v8100265

Figure Lengend Snippet: The VP1u receptor is expressed on erythropoietin (EPO)-dependent erythroid differentiation stages: CD34+ hematopoietic stem cells were expanded in StemCell medium and then stimulated with 3 U/mL EPO. VP1u receptor expression, B19V uptake and infection were tested at different days after EPO-stimulation. ( A ) Fluorescence microscopy images and ( B ) flow cytometry analysis of MS2-∆C126 VP1u uptake into differentiated cells; ( C ) Histograms of the FACS data, showing the mean fluorescence of MS2-VP1u internalization per cell and ( D ) number of VP1u receptor-positive cells; ( E ) Quantification of B19V uptake and ( F ) infection before (day 0) or after stimulation with EPO ( n = 3). Scale bar: 15 µm.

Article Snippet: The MS2 coat protein sequence was obtained from GenScript (Nanjing, China) and transformed as plasmid into E. coli BL21(DE3).

Techniques: Expressing, Infection, Fluorescence, Microscopy, Flow Cytometry

Journal: Scientific Reports

Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

doi: 10.1038/s41598-017-17951-5

Figure Lengend Snippet: Transmission electron microscopy (TEM) photograph of purified His-tagged MS2 phage-like particles (His-tagged MS2 PLP). The scale is 100 nm.

Article Snippet: The membranes were incubated with Anti-Enterobacterio Phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) at a dilution of 1:1500 for 1 hour at room temperature, washed in PBS with 0.05% Tween-20 (PBS/T, Serva, Germany), and then incubated for 1 hour at room temperature with goat anti-rabbit IgG conjugated with HRP diluted 1:1500 (Jackson ImmunoResearch, UK).

Techniques: Transmission Assay, Electron Microscopy, Purification

Journal: Scientific Reports

Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

doi: 10.1038/s41598-017-17951-5

Figure Lengend Snippet: Characterization of the single-chain version of the coat protein dimer containing the His-tag. ( A ) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), molecular weight of wild type MS2 bacteriophage coat protein was about 13 kDa (A1) whereas single chain version of the MS2 coat protein dimer containing the His-tag was about 28 kDa (A2); ( B ) western blot analysis using primary Anti-Enterobacterio Phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) and secondary goat anti-rabbit IgG conjugated with HRP (Jackson ImmunoResearch, UK) antibodies at wild type MS2 bacteriophage coat protein (B1) and single chain version of the MS2 coat protein dimer containing the His-tag (B2); ( C ) western blot analysis using primary anti-HisTag antibody (Pierce, Thermo Scientific, USA) and secondary goat anti-mouse IgG conjugated with HRP (Jackson ImmunoResearch) antibodies at wild type MS2 bacteriophage coat protein (C1) and single chain version of the MS2 coat protein dimer containing the His-tag (C2); ( D , E ) laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) analysis of wild-type bacteriophage MS2 coat protein and single-chain version of the MS2 coat protein dimer containing the His-tag; M, marker, spectra multicolor broad range protein ladder (Fermentas); the marker values are in kDa. The figures were cropped, full length gel and blots are presented in Supplementary Figures , and .

Article Snippet: The membranes were incubated with Anti-Enterobacterio Phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) at a dilution of 1:1500 for 1 hour at room temperature, washed in PBS with 0.05% Tween-20 (PBS/T, Serva, Germany), and then incubated for 1 hour at room temperature with goat anti-rabbit IgG conjugated with HRP diluted 1:1500 (Jackson ImmunoResearch, UK).

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Molecular Weight, Western Blot, Mass Spectrometry, Marker

Journal: Scientific Reports

Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

doi: 10.1038/s41598-017-17951-5

Figure Lengend Snippet: Temperature stability testing of particles. ( A ) Temperature stability testing of MS2 phage-like particles (MS2 PLP) and ( B ) His-tagged MS2 phage-like particles (His-tagged MS2 PLP) using agarose gel electrophoresis; temperature values are in °C; M, marker, 2-log DNA ladder (New England Biolabs, UK); the marker values are in kb. The figures were cropped, full length gels are presented presented in Supplementary Figures and .

Article Snippet: The membranes were incubated with Anti-Enterobacterio Phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) at a dilution of 1:1500 for 1 hour at room temperature, washed in PBS with 0.05% Tween-20 (PBS/T, Serva, Germany), and then incubated for 1 hour at room temperature with goat anti-rabbit IgG conjugated with HRP diluted 1:1500 (Jackson ImmunoResearch, UK).

Techniques: Agarose Gel Electrophoresis, Marker

Journal: Scientific Reports

Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

doi: 10.1038/s41598-017-17951-5

Figure Lengend Snippet: RT-qPCR testing of optimal method of thermal lysis of His-tagged MS2 phage-like particles (His-tagged MS2 PLP).

Article Snippet: The membranes were incubated with Anti-Enterobacterio Phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) at a dilution of 1:1500 for 1 hour at room temperature, washed in PBS with 0.05% Tween-20 (PBS/T, Serva, Germany), and then incubated for 1 hour at room temperature with goat anti-rabbit IgG conjugated with HRP diluted 1:1500 (Jackson ImmunoResearch, UK).

Techniques: Lysis, Concentration Assay

Journal: Scientific Reports

Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

doi: 10.1038/s41598-017-17951-5

Figure Lengend Snippet: The results of RT-qPCR purity testing of His-tagged MS2 phage-like particles (His-tagged MS2 PLP) and MS2 phage-like particles (MS2 PLP).

Article Snippet: The membranes were incubated with Anti-Enterobacterio Phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) at a dilution of 1:1500 for 1 hour at room temperature, washed in PBS with 0.05% Tween-20 (PBS/T, Serva, Germany), and then incubated for 1 hour at room temperature with goat anti-rabbit IgG conjugated with HRP diluted 1:1500 (Jackson ImmunoResearch, UK).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

doi: 10.1038/s41598-017-17951-5

Figure Lengend Snippet: Specific oligonucleotides used in present study.

Article Snippet: The membranes were incubated with Anti-Enterobacterio Phage MS2 Coat Protein polyclonal antibody (Merck Millipore, USA) at a dilution of 1:1500 for 1 hour at room temperature, washed in PBS with 0.05% Tween-20 (PBS/T, Serva, Germany), and then incubated for 1 hour at room temperature with goat anti-rabbit IgG conjugated with HRP diluted 1:1500 (Jackson ImmunoResearch, UK).

Techniques: Sequencing