Journal:

Article Title: Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

doi: 10.1182/blood-2005-02-0638

Figure Lengend Snippet: Engineering an in vivo system for conditional overexpression of genes in the megakaryocyte/platelet lineage. The scheme shown illustrates the Tet-on/off inducible gene expression system, involving the interaction between 2 transgenes of interest. The tissue-specific PF4 promoter drives the expression of the transactivator tTA (fused to the enhancing factor VP16). This line is crossbred to a tetracycline responsive element (TRE) bidirectional CMV line. The use of the TRE bidirectional CMV promoter allows the simultaneous regulation of both Aurora-B and β-gal by one central TRE. The TRE is a stretch of 7 copies of the 42-bp Tet operator sequences that bind to tTA-VP16 fusion protein in the absence of tetracycline/doxycycline. To achieve inducible transcription of 2 transgenes, the TRE is sandwiched between 2 minimal CMV promoters (directing the transcription of Aurora-B and β-gal in opposite directions, presumably in the absence of tetracycline).

Article Snippet: Gels were blotted and reacted with mouse monoclonal anti-VP16 antibody (1:1000-fold dilution; BD Biosciences, San Jose, CA) and anti–β-galactosidase ( E coli, rabbit, 1:200-dilution, catalog no. 200-4136; Rockland Immunochemical, Gilbertsville, PA).

Techniques: In Vivo, Over Expression, Expressing

Journal:

Article Title: Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

doi: 10.1182/blood-2005-02-0638

Figure Lengend Snippet: Generation of PF4-tTA-VP16 transgenic lines and confirmation of transgene expression. (A) Transgene integration. PCR of genomic DNA was used to screen for specific transgene integration (yielding a 460-bp product shown here) following protocols detailed in “Materials and methods.” Three founder lines, PF4-tTA-VP16 (referred to as PF4-tTA) nos. 38, 42, and 54, were identified, as confirmed by Southern blot analysis. (B-C) Transgene expression. After obtaining homozygous PF4-tTA-VP16 lines, we used Western blot analysis (B) to identify a line with the highest expression of the tetracycline responsive enhancer fusion protein (tTA-VP16). Bone marrow cells were subjected to Western blotting with anti-VP16 antibody to detect expression of tTA-VP16. Actin levels were determined to confirm equal loading of samples. Tissue-specific expression of tTA-VP16 was determined by immunohistochemical analysis of bone marrow cells (C) using anti-VP16 as a primary antibody and FITC-conjugated anti–mouse IgG as a secondary antibody. Since transgenic line no. 42 displays high expression of tTA-VP16 fusion protein in megakaryocytes (indicated by arrows), we decided to use this line for subsequent crossbreeding and experiments. Shown is a representative experiment in which the cells were viewed at a magnification of × 20. Images were captured using an Olympus ×70 microscope with Hamamatsu CCD camera C4742-95. Images were acquired with Openlab 3.1.2 software.

Article Snippet: Gels were blotted and reacted with mouse monoclonal anti-VP16 antibody (1:1000-fold dilution; BD Biosciences, San Jose, CA) and anti–β-galactosidase ( E coli, rabbit, 1:200-dilution, catalog no. 200-4136; Rockland Immunochemical, Gilbertsville, PA).

Techniques: Transgenic Assay, Expressing, Southern Blot, Western Blot, Immunohistochemical staining, Microscopy, Software

Journal:

Article Title: Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

doi: 10.1182/blood-2005-02-0638

Figure Lengend Snippet: Generation of CMV–Aurora-B/β-gal transgenic lines and double-transgenic lines (PF4-tTA-VP16/CMV–Aurora-B/β-gal). (A) Generation of CMV–Aurora-B/β-gal transgenic lines. We generated 3 transresponder lines, nos. 32, 34, and 41, carrying the CMV–Aurora-B/β-gal transgene. Genomic integration was confirmed by Southern blot analysis and PCR of genomic tail DNA. Shown is a representative Southern blot analysis of DNA derived from F1 offspring of each of the lines and probed with a 1.1-kb DNA sequence encoding the rat Aurora-B gene. After obtaining homozygous lines by crossbreeding the confirmed F1 transgenic mice, we crossbred each of the 3 transresponder lines to the transactivator line no. 42 (Figure 2). (B) Confirmation of double-transgenic mice PF4-tTA-VP16/CMV–Aurora-B/β-gal. Shown is PCR screening for double-transgenic mice carrying PF4-tTA-VP16 and CMV–Aurora-B/β-gal transgenes, using primer sets that amplified a 460-bp and 1.1-kb DNA fragment coding for both transgenes, respectively. Lines that show both PCR products are double transgenic. More details are provided in “Materials and methods.”

Article Snippet: Gels were blotted and reacted with mouse monoclonal anti-VP16 antibody (1:1000-fold dilution; BD Biosciences, San Jose, CA) and anti–β-galactosidase ( E coli, rabbit, 1:200-dilution, catalog no. 200-4136; Rockland Immunochemical, Gilbertsville, PA).

Techniques: Transgenic Assay, Generated, Southern Blot, Derivative Assay, Sequencing, Amplification

Journal:

Article Title: Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

doi: 10.1182/blood-2005-02-0638

Figure Lengend Snippet: Conditional and reversible expression of transgenes in megakaryocytes. (A) Induced β-galactosidase expression. Shown are immunohistochemistry analyses of freshly isolated bone marrow cells from double-transgenic littermates PF4-tTA-VP16/CMV–Aurora-B/β-gal no. 41/42 shown in Figure 3B and age- and sex-matched wild-type mice. Tet-off mice were removed from doxycycline treatment (referred as Tet-off to keep with a terminology that refers to tetracycline inducible promoter) 4 weeks prior to analysis, whereas Tet-on mice were continuously supplemented with doxycycline. To detect β-galactosidase (β-gal)–induced expression, mouse monoclonal antibody against E coli β-gal was used (red). The same preparations were also stained for CD41 (specific marker for megakaryocytes) using anti–CD41-FITC (green). Images were captured with an Olympus microscope using a × 60 objective. DAPI indicates 4,6 diamidino-2-phenylindole. (B) Reversible transgene expression. To demonstrate that conditional gene expression was reversible, half of the Tet-off mouse population received doxycycline for 4 weeks (Bvi), while the other half of its littermates remained Tet-off (Bii). Similarly, half of the Tet-on transgenic mouse population (doxycycline supplement) was removed from doxycycline treatment for 4 weeks (Bv), while the other half remained on doxycycline supplement (Bvi). β-galactosidase was followed as in panel A (not shown) as well as by enzyme activity (shown here as a representative). Fresh bone marrow cells derived from the double-transgenic mice (PF4-tTA-VP16/CMV–Aurora-B/β-gal no. 41/42) and equivalent wild-type cells were stained for β-gal as outlined in “Materials and methods” (visualized via phase-contrast, × 40 original magnification). Megakaryocytes were clearly identified based on their size and morphology. Images were captured using an Olympus ×70 microscope with Hamamatsu CCD camera C4742-95. Images were acquired with Openlab 3.1.2 software.

Article Snippet: Gels were blotted and reacted with mouse monoclonal anti-VP16 antibody (1:1000-fold dilution; BD Biosciences, San Jose, CA) and anti–β-galactosidase ( E coli, rabbit, 1:200-dilution, catalog no. 200-4136; Rockland Immunochemical, Gilbertsville, PA).

Techniques: Expressing, Immunohistochemistry, Isolation, Transgenic Assay, Staining, Marker, Microscopy, Activity Assay, Derivative Assay, Software

Journal:

Article Title: Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

doi: 10.1182/blood-2005-02-0638

Figure Lengend Snippet: β-Gal activity is retained in platelets of the inducible Tet-on/off double-transgenic mouse model. (A) β-Galactosidase expression in platelets. To demonstrate the feasibility of retaining the expression of transgenes (β-gal in this case) after megakaryocytes fragmented into platelets, we isolated platelets from plasma of double-transgenic mice with Tet-on/off conditions along with age- and sex-matched wild-type mice. Shown are phase-contrast micrographs at × 60 objective of platelets stained for β-galactosidase activities. (B-C) Usage of the double-transgenic model (PF4-tTA-VP16/CMV–Aurora-B/β-gal no. 41/42) to trace platelets at sites of accumulation. Identifying and quantifying platelet accumulation at sites of injury or stress is a goal common to various experimental pursuits. As a model system, we used endotoxic shock using LPS injection as detailed in “Materials and methods.” Shown are livers, lungs, and spleen stained for β-gal activity. Whole tissues (B) as well as tissue sections (C) (spleen and liver shown as representatives) displayed accumulated platelets. Thin sections were stained only with eosin and visualized by light phase contrast; × 60 original magnification. Arrows indicate areas of platelet accumulation. Images were captured using an Olympus ×70 microscope with Hamamatsu CCD camera C4742-95. Images were acquired with Openlab 3.1.2 software.

Article Snippet: Gels were blotted and reacted with mouse monoclonal anti-VP16 antibody (1:1000-fold dilution; BD Biosciences, San Jose, CA) and anti–β-galactosidase ( E coli, rabbit, 1:200-dilution, catalog no. 200-4136; Rockland Immunochemical, Gilbertsville, PA).

Techniques: Activity Assay, Transgenic Assay, Expressing, Isolation, Staining, Injection, Microscopy, Software

Journal:

Article Title: Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

doi: 10.1182/blood-2005-02-0638

Figure Lengend Snippet: Inducible expressions of Aurora-B kinase in megakaryocytes. Primary bone marrow cell mRNA was isolated from double-transgenic mice (PF4-tTA-VP16/CMV–Aurora-B/β-gal no. 41/42) treated with and without doxycycline (Tet-on or Tet-off) and reverse transcribed to generate cDNA. PCR was subsequently conducted using primer sets specifically designed to amplify FLAG-tagged rat Aurora-B kinase. GAPDH was amplified to show that the RT-PCR was successfully conducted in all samples. GAPDH-specific primers and PCR cycles were performed, as indicated in “Materials and methods.”

Article Snippet: Gels were blotted and reacted with mouse monoclonal anti-VP16 antibody (1:1000-fold dilution; BD Biosciences, San Jose, CA) and anti–β-galactosidase ( E coli, rabbit, 1:200-dilution, catalog no. 200-4136; Rockland Immunochemical, Gilbertsville, PA).

Techniques: Isolation, Transgenic Assay, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

doi: 10.1182/blood-2005-02-0638

Figure Lengend Snippet: Platelet and ploidy levels in the inducible double-transgenic mice (PF4-tTA-VP16/CMV–Aurora-B/β-gal no. 41/42). Double-transgenic mice and age- and sex-matched wild-type mice were killed after 4 weeks of treatment with and without doxycycline. Blood was withdrawn and subjected to platelet counts (A) as indicated in “Materials and methods.” Data are presented as platelet count averages ± SD for 10 mice in each group. To determine megakaryocyte ploidy status (B), primary bone marrow cells were isolated from the same population of mice, fixed, stained with anti-CD41 (FITC) antibody and propidium iodide, and subjected to flow cytometry analysis. A representative histogram is shown.

Article Snippet: Gels were blotted and reacted with mouse monoclonal anti-VP16 antibody (1:1000-fold dilution; BD Biosciences, San Jose, CA) and anti–β-galactosidase ( E coli, rabbit, 1:200-dilution, catalog no. 200-4136; Rockland Immunochemical, Gilbertsville, PA).

Techniques: Transgenic Assay, Isolation, Staining, Flow Cytometry

Journal: Antimicrobial Agents and Chemotherapy

Article Title: NP-1, a Rabbit α-Defensin, Prevents the Entry and Intercellular Spread of Herpes Simplex Virus Type 2

doi: 10.1128/aac.47.2.494-500.2003

Figure Lengend Snippet: FIG. 3. Effects of NP-1 and heparin on binding of HSV-2 or recombinant gB-2 to cells. (a) Cells were exposed to twofold dilutions of purified HSV-2(G) (equivalent to an MOI of 1, 0.5, 0.25, 0.12, 0.06, and 0.03 PFU/cell; lanes 1 to 6, respectively) for 5 h at 4°C in the absence (upper gel) or presence of 25 g of NP-1/ml (middle gel) or 100 g of heparin/ml (lower gel). After washing away unbound virus, cell lysates were prepared and separated by SDS-PAGE, and cell-bound virus was visualized by Western blotting with anti-gD monoclonal Ab. The gels are representative of at least two independent experiments. (b) The relative particle numbers of viral input and bound virus were quantified by densitometry scanning of the Western blots of equal portions of input and bound virus. (c) Alternatively, cells were exposed to recombinant gB-2 (105 g/cell) for 1 h at 37°C in the absence or presence of the indicated concentrations of heparin or 100 g of NP-1/ml, and the bound gB-2 was compared by Western blotting of cell lysates probed with anti-gB monoclonal Ab.

Article Snippet: The cells were blocked overnight with 10% goat serum and 1% bovine serum albumin in PBS, then incubated with a 1:50 dilution of anti-HSV monoclonal Ab (mouse anti-VP16; Santa Cruz Biotechnology), washed extensively, and subsequently incubated with a 1:500 dilution of a goat anti-mouse Ab conjugated to fluorescein isothiocyanate (FITC; Calbiochem).

Techniques: Binding Assay, Recombinant, Virus, SDS Page, Western Blot

Journal: Antimicrobial Agents and Chemotherapy

Article Title: NP-1, a Rabbit α-Defensin, Prevents the Entry and Intercellular Spread of Herpes Simplex Virus Type 2

doi: 10.1128/aac.47.2.494-500.2003

Figure Lengend Snippet: FIG. 4. Effect of NP-1 or acyclovir on VP-16 translocation to the host cell nucleus. Cells were mock infected (a) or inoculated with HSV-2(G) that had been preincubated for 1 h at 37°C with 0.1% acetic acid (b), 25 g of NP-1/ml (c), or 100 g of acyclovir/ml (d). After 4 h at 37°C, cells were fixed, permeabilized, and incubated with mouse monoclonal anti-VP16 Ab, followed by incubation with goat anti-mouse IgG conjugated to FITC. Cells were visualized using a Zeiss Axioskop fluorescence microscope. The number of cells expressing VP16 in the nucleus as a percentage of the total cells visualized per high-power field were counted in four to eight fields per experiment. The graph depicts the mean standard deviation obtained from three to four independent experiments; the asterisk indicates a significant reduction in VP16 translocation, by the Mann-Whitney test (P 0.0001).

Article Snippet: The cells were blocked overnight with 10% goat serum and 1% bovine serum albumin in PBS, then incubated with a 1:50 dilution of anti-HSV monoclonal Ab (mouse anti-VP16; Santa Cruz Biotechnology), washed extensively, and subsequently incubated with a 1:500 dilution of a goat anti-mouse Ab conjugated to fluorescein isothiocyanate (FITC; Calbiochem).

Techniques: Translocation Assay, Infection, Incubation, Microscopy, Expressing, Standard Deviation, MANN-WHITNEY