Journal: PLoS ONE

Article Title: Investigating CRISPR/Cas9 gene drive for production of disease-preventing prion gene alleles

doi: 10.1371/journal.pone.0269342

Figure Lengend Snippet: (A) Sanger sequencing chromatograms for mice derived from fertilized FVB/NJ oocytes that were electroporated with recCas9/ Prnp gRNA–3 RNP complexes. The numbers below the automated base calls indicate the position in the sequencing read rather than the Prnp ORF. Changes to the PrP C amino acid sequence for the lines with disrupted Prnp are indicated in the bottom box. (B) Agarose gel image showing the products of T7E1 mismatch cleavage assay reactions for negative (homoduplex) and positive (heteroduplex) control DNA solutions and DNA obtained from the founder of line 34. The presence of the bands indicated by the red arrows confirms that line 34 has a disrupted Prnp allele. (C) Capillary western images confirming that PrP C expression is undetectable in brain homogenates from homozygous line 33 mice. Sha31 and 12B2 are two different PrP C antibodies. New abbreviation: KO, knockout.

Article Snippet: Primary antibodies were Sha31 anti-PrP (Spi-Bio Inc., A03213; diluted 1:10000), 12B2 anti-PrP (see Ref. [ ]; diluted 1:500) and anti-beta-tubulin (Novus Biologicals, NB600-936; diluted 1:200).

Techniques: Sequencing, Derivative Assay, Agarose Gel Electrophoresis, Cleavage Assay, Control, Western Blot, Expressing, Knock-Out

Journal: PLoS ONE

Article Title: Emergence of Classical BSE Strain Properties during Serial Passages of H-BSE in Wild-Type Mice

doi: 10.1371/journal.pone.0015839

Figure Lengend Snippet: Mice inoculated with with H-BSE (lanes 1–3 and 5–7 from isolates 01-2604 and 03-2095 respectively), at first (lanes 1, 5) or second passage (lanes 2–3 and 6–7), were compared to mice infected with classical BSE (lanes 4, 8) in panels A–C. At second passage, PrP res exhibited either H-type features, from mice that died later (491 and 504 d.p.i. in lanes 2 and 6 respectively), or “C-BSE like” features (464 and 322 d.p.i. in lanes 3 and 7 respectively). The brain tissue equivalents loaded per lane are indicated (in tenth of mg). D - Glycoform proportions (means +/− standard deviations) of PrP res from H-BSE (isolate 03-2095)-infected mice detected using Sha31 antibody. Mice exhibited PrP res with a molecular mass either similar to that of H-BSE (full squares) or to classical BSE (full diamonds). E - PrP d extracted in the absence of protease digestion and deglycosylated by PNGase treatment from H-BSE infected mice at first (lane 1) or second passage (lanes 2-3 from mice that died at 497 d.p.i. and 464 d.p.i. respectively), compared to a classical BSE control in lane 4 (0.3 mg brain tissue equivalent per lane). Full-length PrP d (FL) and C1 and C2 fragments are indicated. Panels A and B were revealed by monoclonal antibodies Sha31 and 12B2 respectively and panels C and E by HRP-labelled SAF84 antibody. Bars to the left indicate the 29.0, 20.1 and 14.3 kDa marker positions.

Article Snippet: PrP res or PrP d were detected using the monoclonal antibodies anti-PrP Sha31 (1/10 from kit TeSeE sheep/goat Biorad), SAF84 (500 ng/ml)(SPI-Bio, France) or 12B2 (340 ng/ml) against the 144-WEDRYYRE-151, 163-RPVDQY-168 and 88-WGQGG-92 murine PrP sequences respectively.

Techniques: Infection, Control, Bioprocessing, Marker

Journal: PLoS ONE

Article Title: Emergence of Classical BSE Strain Properties during Serial Passages of H-BSE in Wild-Type Mice

doi: 10.1371/journal.pone.0015839

Figure Lengend Snippet: A - Detection of PrP res from spleens of mice inoculated with H-BSE isolates 01-2604 (lanes 1–3) or 03-2095 (lanes 4–9) at first passage. B - Detection of PrP res from spleens of mice inoculated with H-BSE at third passage in mice inoculated with a brain homogenate with “C-BSE like” PrP res (lanes 4–8). PrP res from the brain of one of these mice (lanes 3, 9) and from brains of C506M3 scrapie (lanes 1, 11) or classical BSE (lanes 2, 10) controls are shown for comparison. C - PrP res from spleens of mice infected with H-BSE at first passage (lanes 2, 4)(isolates 03-1928 and 03-2095), second passage in a mouse with “C-BSE like” PrP res (lane 6) and third passage from mice inoculated with a brain homogenate with “C-BSE like” PrP res (lanes 8, 10). PrP res from spleens of classical BSE (lanes 3, 5, 7, 9) and C506M3 (lanes 1, 11) controls are shown. Panels A and B were revealed by monoclonal antibody Sha31 and panel C by HRP-labelled SAF84 antibody Bars to the left indicate the 29.0 and 20.1 kDa marker positions in panels A and B or the 20.1 and 14.3 kDa marker positions in panels C and D.

Article Snippet: PrP res or PrP d were detected using the monoclonal antibodies anti-PrP Sha31 (1/10 from kit TeSeE sheep/goat Biorad), SAF84 (500 ng/ml)(SPI-Bio, France) or 12B2 (340 ng/ml) against the 144-WEDRYYRE-151, 163-RPVDQY-168 and 88-WGQGG-92 murine PrP sequences respectively.

Techniques: Comparison, Infection, Marker

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Relative pixel intensity analysis of PrP C expression in facial integumentary clarified 10% (w/v) gland homogenates of mule deer (A, C, E) and white-tailed deer (B, D, F). Background-adjusted average pixel intensities of PrP C bands of forehead (a-b), preorbital (c-d), and vestibular nasal glands were compared between lanes of SDS-PAGE PVDF membranes probed with anti-PrP Sha31. Unclarified white-tailed deer whole brain homogenate was used for reference. Sample size, mean, 95% confidence intervals, and significance by Mann-Whitney tests are shown.

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Expressing, SDS Page, MANN-WHITNEY

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Protein concentration-adjusted PrP C protein expression in deer exocrine glands. Capillary electrophoresis immunoassay chemiluminescence sample size, mean, and 95% confidence intervals of clarified 10% (w/v) deer gland homogenates prepared in RIPA buffer. Deer facial (a-e) and leg (f-h) tissue and gland homogenate samples were adjusted to final protein concentrations of 1.5 μg/μL for the immunoassay. PrP C signal was detected by anti-PrP SHA31 antibody.

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Protein Concentration, Expressing, Electrophoresis

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Species and sex influence on PrP C detection by anti-PrP SHA31 capillary gel electrophoresis assay with total protein concentration-standardized 10% (w/v) clarified gland homogenates

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Nucleic Acid Electrophoresis, Concentration Assay

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Quantified PrP C protein concentration in deer gland homogenates. Total PrP C concentrations of individuals, means, and 95% confidence intervals of clarified 10% (w/v) mule deer (MD) and white-tailed deer (WT) facial (a-e) and leg (f-h) gland homogenates prepared in RIPA buffer as determined by SHA31-N5 sandwich ELISA. Protein concentration was calculated using a full-length recombinant deer prion protein standard curve.

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Protein Concentration, Sandwich ELISA, Recombinant

Journal: Prion

Article Title: Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

doi: 10.1080/19336896.2022.2079888

Figure Lengend Snippet: Mean PrP C concentrations and 95% confidence intervals of clarified 10% (w/v) gland homogenates as determined by SHA31-N5 sandwich ELISA

Article Snippet: PrP C was probed with the mouse IgG 1 anti-prion protein SHA31 monoclonal primary antibody (1:10,000) (Cayman Chemical, USA) which binds to an epitope of 148–155 ( Odocoileus amino acid sequence).

Techniques: Concentration Assay

Journal: Prion

Article Title: Identification of H-type BSE in Portugal

doi: 10.1080/19336896.2014.997615

Figure Lengend Snippet: Phenotypical features of the first Portuguese H-BSE. (a) PrPres immunolabelling in the reticular formation of the brainstem: linear pattern (arrow) and the predominant intraglial pattern as single granules in the white matter (arrowheads) (b). The box in picture (a) outlines the area shown in picture (b) at higher magnification. Specific PrPres signal was visualized with Vectastain–peroxidase, DAB (Vector). All sections were counterstained with Mayer's haematoxylin. The magnifications used are indicated. (c) Western blot analysis of PrPres after proteinase K treatment using Sha31 and P4 immunolabelling: Sha31 shows a higher molecular mass migration (orange arrow) by comparison to C-BSE; with P4 there is immunolabelling in the Portuguese H-BSE (PT H-BSE) similar to that presented by the H-BSE control. Absence of signal in C-BSE and L-BSE. H-BSE, C-BSE and L-BSE controls from AHVLA proficiency testing. Scrapie represents classical scrapie control. M, molecular mass marker (kDa).

Article Snippet: The confirmation of the H-type BSE phenotype was carried out using the discriminatory molecular method with the C-terminal anti-PrP antibody Sha31 and the N- terminal anti-PrP monoclonal antibody P4 (aa 101–107 bovine PrP protein, 15 R-Biopharm, 0.1mg/ml) diluted 1/500 as recommended by the European Union Reference Laboratory (AHVLA Bio-Rad TeSeE- based Hybrid Western Blot method).

Techniques: Plasmid Preparation, Western Blot, Migration, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Studies in bank voles reveal strain differences between chronic wasting disease prions from Norway and North America

doi: 10.1073/pnas.2013237117

Figure Lengend Snippet: Immunohistochemical patterns of PrP Sc deposition in Bv109I inoculated with CWD isolates. Immunohistochemical detection of PrP Sc in the thalamus, visual cortex (third layer), and hippocampus of Bv109I inoculated with the CWD isolates indicated on the Left . Norwegian moose isolates (M-NO1, M-NO2, and M-NO3) were characterized by strong PrP Sc deposition in all brain areas, which was mainly intraneuronal, particularly in the case of M-NO1 and M-NO2. In contrast, PrP Sc deposition was milder in Bv109I inoculated with Canadian isolates M-CA1 and E-CA2 and was mainly detected as diffuse deposits in the thalamus. The only Bv109I that was positive after inoculation of R-NO1 was characterized by a strong and diffuse PrP Sc deposition in the visual cortex. PrP Sc was detected by mAb SAF84. (Scale bar: 50 µm.)

Article Snippet: Purified PrP-specific mAbs were as follows: 12B2 (WBVR), Sha31 and SAF84 (SpiBio), and L42 (R-Biopharm).

Techniques: Immunohistochemical staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Studies in bank voles reveal strain differences between chronic wasting disease prions from Norway and North America

doi: 10.1073/pnas.2013237117

Figure Lengend Snippet: Different patterns of PrP Sc deposition in Bv109I-adapted CWD isolates. PET blot detection of protease-resistant PrP Sc in coronal sections of the forebrain, representing telencephalon (1), diencephalon (2), midbrain (3), and hindbrain (4) from representative Bv109I following two passages with the CWD isolates indicated on the Left . PrP Sc was detected with mAb SAF84.

Article Snippet: Purified PrP-specific mAbs were as follows: 12B2 (WBVR), Sha31 and SAF84 (SpiBio), and L42 (R-Biopharm).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Studies in bank voles reveal strain differences between chronic wasting disease prions from Norway and North America

doi: 10.1073/pnas.2013237117

Figure Lengend Snippet: Different patterns of PrP Sc deposition in cortical layers of Bv109I-adapted CWD isolates. Immunohistochemical detection of PrP Sc in the third, fourth, and fifth cortical layers of the somatosensory cortex of Bv109I after second passage with the CWD isolates indicated on the Left . PrP Sc was not detected in Bv109I infected with the Canadian CWD isolates but was found as diffuse and punctate deposits in all layers of Bv109I infected with R-NO1. PrP Sc was detected by mAb SAF84. (Scale bar: 50 µm.)

Article Snippet: Purified PrP-specific mAbs were as follows: 12B2 (WBVR), Sha31 and SAF84 (SpiBio), and L42 (R-Biopharm).

Techniques: Immunohistochemical staining, Infection

Journal: Scientific Reports

Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

doi: 10.1038/s41598-018-32581-1

Figure Lengend Snippet: PrP-NGLuc and PrP-CGLuc expression in RK13-DC cells results in bioluminescence. ( a ) Western blot analysis with the Sha31 antibody against PrP shows that RK13-DC cells stably express PrP-NGLuc and PrP-CGLuc from a bicistronic expression vector (lanes 3 and 4). Non-transfected RK13 cells do not express PrP and do not show a detectable signal for PrP (lanes 1 and 2), whereas N2a cells show the characteristic signal for wild-type PrP (lanes 5 and 6). Deglycosylation of cell lysates with peptide-N-glycosidase F (PNGase F, lanes 2, 4, and 6) resulted in lower molecular weight bands suggesting that mature PrP-NGLuc and PrP-CGLuc are properly glycosylated in RK13-DC cells as PrP is in N2a cells. Detection of tubulin on the same blot served as a loading control. Additional lanes were excised for presentation purposes. ( b ) Western blot analysis with the Sha31 antibody against PrP shows that treatment of RK13-DC cells with increasing amounts of phosphoinositide phospholipase C (PI-PLC) increases the amount of detectable protein released into cell culture medium suggesting that mature PrP-NGLuc and PrP-CGLuc are anchored to the outer cell membrane by GPI. Additional lanes were excised for presentation purposes. ( c ) Densitometric quantification of the PrP signal from three different experiments as shown in ( b ). ( d ) Life cell immunofluorescence staining with the Sha31 antibody to PrP (left panels) shows that PrP-NGLuc and PrP-CGLuc are expressed in RK13-DC cells (top row), similar to wild-type PrP in N2a cells (bottom row). Bright-field microscopy (centre panels) shows the contours of the imaged cells. An overlay of fluorescence and bright-field images (right panels) shows that PrP-NGLuc, PrP-CGLuc, and PrP are located at the cell membrane. Bar = 20 µm. ( e ) The bioluminescence measured from RK13-DC cells expressing both PrP-NGLuc and PrP-CGLuc was more than 10-fold above background levels measured from RK13 cells stably expressing only PrP-NGLuc or PrP-CGLuc, or both NGLuc and CGLuc together each lacking the PrP moiety. A functional luciferase consisting of its N- and C-terminal halves was only formed in RK13-DC cells where dimers between the PrP moieties of PrP-NGLuc and PrP-CGLuc could form. In comparison to PrP-NGLuc and PrP-CGLuc in RK13-DC cells, bioluminescence of full-length GLuc in RK13 cells was almost 17-fold higher. In ( c , e ) error bars indicate SD.

Article Snippet: Additional lanes were excised for presentation purposes. ( c ) Densitometric quantification of the PrP signal from three different experiments as shown in ( b ). ( d ) Life cell immunofluorescence staining with the Sha31 antibody to PrP (left panels) shows that PrP-NGLuc and PrP-CGLuc are expressed in RK13-DC cells (top row), similar to wild-type PrP in N2a cells (bottom row).

Techniques: Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Transfection, Molecular Weight, Control, Cell Culture, Membrane, Immunofluorescence, Staining, Microscopy, Fluorescence, Functional Assay, Luciferase, Comparison