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: Acta neuropathologica

Article Title: Characterization of prion strains and peripheral prion infectivity patterns in E200K genetic CJD patients.

doi: 10.1007/s00401-025-02903-5

Figure Lengend Snippet: Fig. 1 PrPres western blot profiles in original E200K gCJD isolates and E200K gCJD-inoc- ulated human PrP-expressing mice. Western blot analysis of proteinase K-resistant prion protein (PrPres) profiles was performed on brain homogen- ates from both the original E200K gCJD patient isolates and transgenic mice expressing human PrP with Met129 (tgMet) or Val129 (tgVal) at codon 129. Mice were intracerebrally inoculated with 20 µL of 10% brain homogenate from E200K gCJD patients (n = 6 per group). Two serial passages were carried out in each mouse line (details in Table 1). The PrPres isoform (type 1 or type 2) was identified via SDS-PAGE and Western blot using the anti-PrP monoclonal antibody Sha31 (epitope YEDRYYRE). To control for PrPres isoform, MM1 sCJD (type 1) and VV2 sCJD (type 2) isolates were included in each gel. The PrPres isoform results for each passage and mouse line are summarized in Table 1

Article Snippet: Immunodetection was carried on PVDF membranes out using the monoclonal primary antibody Sha31 (1 μg/mL), which recognizes amino acids 145–152 (YEDRYYRE) of PrP, and an anti-mouse HRP-conjugated secondary antibody (Biorad) [16].

Techniques: Western Blot, Expressing, Transgenic Assay, SDS Page, Control

Journal: Acta neuropathologica

Article Title: Characterization of prion strains and peripheral prion infectivity patterns in E200K genetic CJD patients.

doi: 10.1007/s00401-025-02903-5

Figure Lengend Snippet: Fig. 3 PrPres western blot in the brains of tgMet mice inoculated with brain and peripheral tissues from E200K gCJD Patients. Western blot analysis of proteinase K-resistant prion protein (PrPres) was conducted on brain homogenates from transgenic mice expressing human PrP with Met129 (tgMet) after two serial intracerebral passages (n = 6 per group). The mice were inoculated with 20µL of a 10% homogenate prepared from brain or peripheral tissues of E200K gCJD patients (refer to Tables 1 and 2). PrPres was detected using the anti-PrP mon- oclonal antibody Sha31, targeting the YEDRYYRE epitope. For com- parison, MM1 sCJD (type 1) and VV2 sCJD (type 2) prion isolates were included as controls. PrPres isoform identification results for each passage are summarized in Table 2

Article Snippet: Immunodetection was carried on PVDF membranes out using the monoclonal primary antibody Sha31 (1 μg/mL), which recognizes amino acids 145–152 (YEDRYYRE) of PrP, and an anti-mouse HRP-conjugated secondary antibody (Biorad) [16].

Techniques: Western Blot, Transgenic Assay, Expressing

Journal: Veterinary Research

Article Title: L-BSE experimentally transmitted to sheep presents as a unique disease phenotype

doi: 10.1186/s13567-016-0394-1

Figure Lengend Snippet: WB images from primary passage samples. A and B Nine representative recipients, homozygous for alanine (codon 136) detected by Sha31 ( A ), or P4 ( B ). (Lane 1, 63/11; lane 2, 3/11; lane 3, 457/11; lane 4 26/12; lane 5 112/12; lane 6, 182/12; lane 7, 3/13; lane 8, 98/11; lane 9, 455/11; AB + , donor bovine L-BSE; B + , bovine C-BSE; S + , ovine Scrapie; BS + , experimental ovine CBSE; S-, negative sheep; M, molecular mass markers. A 1 min exposure, B 10 min exposure). C and D Eight representative recipients homozygous or heterozygous for valine (codon 136), detected by Sha31 ( C ) or P4 ( D ), (Lane 1, case 1591/10; lane 2, case 58/11; lane 3, 140/11; lane 4, 267/11; lane 5, 456/11; lane 6, 113/12; lane 7, 4/12; lane 8, 167/12; markers and controls as for A and B . C 1 min exposure, D 10 min exposure). There is low molecular mass migration of the unglycosylated band (arrow), similar to that of the donor bovine L-BSE (AB+), for all but one of the ovine recipients (VRQ/VRQ Lane 8 C , D ) regardless of genotype. Similar di-;mono-glycosylated band ratios are also seen in all cases when detected by mAb Sha31 (see Additional file ). All recipient samples, irrespective of genotype, are also detected with mAb P4 ( B , D ). In contrast the donor L-BSE case (AB+) is not. Following extraction with a Proteinase K digestion step, may contain a mixture of varying molecular mass fragments, partly due to multiple cleavage sites and variability in resistance of PrP Sc to the concentration of the enzyme. The two additional lower bands observed in these profiles are regularly observed in diagnostic samples processed in this way. For diagnostic analysis they are disregarded. Only the standard three bands pertaining to the di, mono and un-glycosylated forms of PrP Sc are considered relevant.

Article Snippet: The primary antibodies were Sha31 (prepared according to the kit protocol) and P4 (0.2 µg/mL, RIDA ® , r-Biopharm).

Techniques: Migration, Concentration Assay, Diagnostic Assay

Journal: Veterinary Research

Article Title: L-BSE experimentally transmitted to sheep presents as a unique disease phenotype

doi: 10.1186/s13567-016-0394-1

Figure Lengend Snippet: WB for ARQ/VRQ subpassage recipients. A Sha31 and P4 blots of the ARQ/VRQ donor and its ARQ/VRQ recipients. (Lane 1, case 1591/10 (ARQ/VRQ Donor), Lane 2, case 6/14; Lane 3, case 73/14; Lane 4, case 76/14 Lane 5, case 78/14; Lane 6, case 79/14 M, Molecular marker; B+, Bovine BSE; S+, Classical Ovine Scrapie. This panel comprises a mixture of 1 and 10 min exposures). B Sha31 and P4 blots of the AFRQ/AFRQ donor and its ARQ/VRQ recipients (Lanes 1 and 2, case 98/11 (AFRQ/AFRQ Donor); Lane 3, case 80/14; Lane 4, case 81/14; Lane 5, case 82/14; Lane 6, case 75/14; M, molecular marker; S+, classical ovine scrapie; B+, bovine BSE. This panel comprises a mixture of 1 and 10 min exposures). In contrast the donor L-BSE case (AB+)molecular characteristics described for primary passage (Figure ; Additional files A–E) are retained on subpassage for all recipient animals regardless of the donor.

Article Snippet: The primary antibodies were Sha31 (prepared according to the kit protocol) and P4 (0.2 µg/mL, RIDA ® , r-Biopharm).

Techniques: Marker