Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-0

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>ked by the human cytomegalovirus immediate early promoter/enhancer region (CMV) and the bovine growth hormone poly(A) signal (pA). Angled black arrows mark the transcriptional start point. The pIGvector contains intron A and flanking untranslated exonic regions E1 and E2 of the cytomegalovirus immediate early gene. In pIΔIGthe exon boundaries were precisely fused by deleting the intron. B) Northern blot analysis. Cells were cotransfected with the indicated VSV-G expression plasmids, a codon optimised HIV-1 expression plasmid (Hgp) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Poly(A) RNA was isolated from transfected cells and analysed by Northern blot with a probe spanning the transcribed region of the BGH poly(A) signal present on all VSV-G transcripts and the positive 5 kb HIV-1 transcript. C) Western blot analyses. Cells were cotransfected with the indicated VSV-G expression plasmids, an SIV expression plasmid (SgpΔ2) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Monoclonal antibodies to HIV-1 p24 capsid protein, which is cross reactive to SIV p27, or to VSV-G, respectively, were used for detection of the viral proteins in lysates of transfected cells

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-2

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p> of primers used for the PCR analyses. The scale indicates the distance to the transcriptional start site. AATAAA: consensus signal for polyadenylation. B) Characterisation of splicing. 293T cells were transfected with pIF. Cytoplasmic RNA was isolated from transfected cells and reverse transcribed by oligo-dT priming (pIFcDNA). A PCR spanning the splice sites was performed with primers: 5'UTR-s and RSV-F-ia. The size of the PCR-products was compared to the size obtained in parallel PCR using pIΔIFand pIFplasmid-DNA (pDNA) as templates. HO: negative control. C) Characterisation of poly(A) signal usage. 293T cells were transfected with the indicated plasmids. Cytoplasmic RNA was isolated from transfected cells and reverse transcribed by priming with Oligo(dT)Add-a. As a control for DNA contamination, the reverse transcription reaction was also performed without the enzyme (-). The cDNA was amplified by PCR with primers 5' UTR-s and Oligo(dT)Add-a and the size of the PCR products was determined by agarose gel electrophoresis. D) PCR products from pIFtransfected cells were cloned and sequenced. The 3' end of the sequence obtained in 9 of 10 clones (RSV-F mRNA exp.) is shown aligned to the RSV-F sequence of the parental plasmid (RSV-F mRNA theor.)

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-4

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>taining an inframe 3'-terminal myc-tag. Similar transfection efficiencies were controlled by measuring the luciferase activity in cell supernatants (not shown). Six hours following transfection cells were infected with the GFP expressing recombinant RSV at an MOI of 2 (+). As negative control, cells were also left uninfected (-). GFP expression analysis revealed similar infection efficiencies (data not shown). Equal amounts of protein were analysed in non-reducing Western blot analysis using a monoclonal antibody to the myc-tag 48 h after transfection

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-6

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>ked by the human cytomegalovirus immediate early promoter/enhancer region (CMV) and the bovine growth hormone poly(A) signal (pA). Angled black arrows mark the transcriptional start point. The pIGvector contains intron A and flanking untranslated exonic regions E1 and E2 of the cytomegalovirus immediate early gene. In pIΔIGthe exon boundaries were precisely fused by deleting the intron. B) Northern blot analysis. Cells were cotransfected with the indicated VSV-G expression plasmids, a codon optimised HIV-1 expression plasmid (Hgp) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Poly(A) RNA was isolated from transfected cells and analysed by Northern blot with a probe spanning the transcribed region of the BGH poly(A) signal present on all VSV-G transcripts and the positive 5 kb HIV-1 transcript. C) Western blot analyses. Cells were cotransfected with the indicated VSV-G expression plasmids, an SIV expression plasmid (SgpΔ2) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Monoclonal antibodies to HIV-1 p24 capsid protein, which is cross reactive to SIV p27, or to VSV-G, respectively, were used for detection of the viral proteins in lysates of transfected cells

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-3

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>d in figure legend 3C. Plasmids pIFΔ2 and pIFΔ24 contain mutations in the second or the second and forth consensus poly(A) signal, respectively. B) Northern blot analysis of cytoplasmic RNA of 293T cells transfected with the indicated plasmids or HEp2 cells infected with RSV. Size separated RNA was stained with ethidiumbromide (EtBr) revealing non-degraded 18S and 28S ribosomal RNA bands (18S shown as representative). A DIG-labelled probe from the RSV-F ORF was used for hybridisation. C, D) Western blot analysis. 293T cells were cotransfected with an EGFP expression plasmid and the indicated RSV-F expression plasmids and analysed by Western blot using an anti-RSV-F monoclonal antibody. The lysate of pIFtransfected cells was diluted from 1:10 to 1:10, while the lysates from the other transfected cell were loaded at a 1:1 dilution. Similar transfection efficiencies were controlled for by measuring the fluorescence activity of cell lysates (data not shown)

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-5

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>cate mutated consensus poly(A) signals. B) Western blot analysis of RSV-F protein levels after transfection of the indicated expression plasmids. An expression plasmid for EGFP was cotransfected. Undiluted lysates had equal protein content and a similar amount of fluorescent activity revealing constant transfection efficiency. Numbers above the lanes indicate the dilution at which the cell lysates were loaded on the gel

Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8 T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity-9

). His: C-terminal tag; L-GM: leader peptide of GM-CSF; m: murine; rh: rhesus monkey; "": [Gly4Ser]-linker. (B) Ovalbumin-specific Western Blot analysis of supernatants of 293T cells transiently transfected with plasmids containing the indicated expression cassettes. GFP: supernatant of cells transfected with a GFP expression plasmid. (C) GM-CSF dependent FDCP-1 proliferation. 293 cells were infected with adenoviral vectors containing the indicated expression cassettes and the GM-CSF levels in the supernatants were determined 24 h post infection. Serial dilutions of the supernatants were co-cultured with FDCP-1 cells for 2 days. Proliferation was measured by a MTT assay.<p><b>Copyright information:</b></p><p>Taken from "Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity"</p><p>http://www.biomedcentral.com/1471-2172/9/13</p><p>BMC Immunology 2008;9():13-13.</p><p>Published online 11 Apr 2008</p><p>PMCID:PMC2324072.</p><p></p

Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8 T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity-6

Es with their SEM (n = 6) from the vaccinated animals. Immune sera were diluted 1 to 10 and 1 to 1000 for determination of IgG2a and IgG1 antibody levels, respectively. Only statistically significant differences between immunization regimens expressing either ovalbumin or GM-OVA in the one-way ANOVA test are indicated.<p><b>Copyright information:</b></p><p>Taken from "Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity"</p><p>http://www.biomedcentral.com/1471-2172/9/13</p><p>BMC Immunology 2008;9():13-13.</p><p>Published online 11 Apr 2008</p><p>PMCID:PMC2324072.</p><p></p

Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8 T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity-5

E cells were gated (A) and analysed for CD62L expression (B, D). D) Mean percentage and SEM of CD62L-low cells from the CD8 and Tetramer double positive cells from three mice per group are shown. Panel C gives a representative example of CD62L expression levels of the CD8+, but tetramer negative population.<p><b>Copyright information:</b></p><p>Taken from "Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity"</p><p>http://www.biomedcentral.com/1471-2172/9/13</p><p>BMC Immunology 2008;9():13-13.</p><p>Published online 11 Apr 2008</p><p>PMCID:PMC2324072.</p><p></p

Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8 T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity-2

GM-CSF-ovalbumin fusion protein (GM-OVA). One week after a single injection or the second injection the percentage of OT-I tetramer positive CD8cells (A) and IFN-γ and CD107a double-positive CD8+ cells after stimulation with the OT-I peptide (B) were determined. All percentages are percent of CD8+ lymphocytes. Mean percentages with SEM of three independent experiments each with three animals per group are shown. Only statistically significant differences between immunization regimens expressing ovalbumin or GM-OVA in the one-way ANOVA test are indicated.<p><b>Copyright information:</b></p><p>Taken from "Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity"</p><p>http://www.biomedcentral.com/1471-2172/9/13</p><p>BMC Immunology 2008;9():13-13.</p><p>Published online 11 Apr 2008</p><p>PMCID:PMC2324072.</p><p></p