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Midtlyng P.J.,VESO | Hendriksen C.,Netherlands Vaccine Institute | Balks E.,Paul Ehrlich Institute | Bruckner L.,Institute fr Viruskrankheiten und Immunprophylaxe | And 10 more authors.
Biologicals | Year: 2011

The workshop on Three Rs Approaches in the Production and Quality Control of Fish Vaccines aimed a) to identify animal tests currently stipulated for the production and quality control of fish vaccines and to highlight animal welfare concerns associated with these tests; b) to identify viable options to replace, reduce, and refine animal use for fish vaccine testing; and c) to discuss the way forward and set out how the Three Rs may be implemented without jeopardizing the quality of the vaccines. The workshop participants - experts from academia, regulatory authorities, a scientific animal welfare organization, and the fish vaccine industry - agreed that efforts should be undertaken to replace the vaccination-challenge batch potency testing with tests based on antigen quantification or antibody response tests. Regulatory requirements of questionable scientific value and relevance for the quality of fish vaccines, such as the re-testing of batches produced outside Europe, or the double-dose batch safety test, should be re-considered. As an immediate measure the design of the current animal tests should be evaluated and modified in the light of refinement and reduction, for example, the number of unprotected control fish in vaccination-challenge tests should be reduced to the minimum. © 2011. Source


Avey M.T.,University of Ottawa | Avey M.T.,Ottawa Hospital Research Institute | Griffin G.,Canadian Council on Animal Care
PLoS ONE | Year: 2016

There are two components to the review of animal based protocols in Canada: review for the merit of the study itself, and review of the ethical acceptability of the work. Despite the perceived importance for the quality assurance these reviews provide; there are few studies of the peer-based merit review system for animal-based protocols for research and education. Institutional animal care committees (ACC)s generally rely on the external peer review of scientific merit for animal-based research. In contrast, peer review for animal based teaching/training is dependent on the review of pedagogical merit carried out by the ACC itself or another committee within the institution. The objective of this study was to evaluate the views of ACC members about current practices and policies as well as alternate policies for the review of animal based teaching/training. We conducted a national web-based survey of ACC members with both quantitative and qualitative response options. Responses from 167 ACC members indicated broad concerns about administrative burden despite strong support for both the current and alternate policies. Participants' comments focused mostly on the merit review process (54%) relative to the efficiency (21%), impact (13%), and other (12%) aspects of evaluation. Approximately half (49%) of the comments were classified into emergent themes that focused on some type of burden: burden from additional pedagogical merit review (16%), a limited need for the review (12%), and a lack of resources (expertise 11%; people/money 10%). Participants indicated that the current system for pedagogical merit review is effective (60%); but most also indicated that there was at least some challenge (86%) with the current peer review process. There was broad support for additional guidance on the justification, criteria, types of animal use, and objectives of pedagogical merit review. Participants also supported the ethical review and application of the Three Rs in the review process. A clear priority from participants in the survey was updating guidance to better facilitate the merit review process of animal-based protocols for education. Balancing the need for improved guidance with the reality of limited resources at local institutions will be essential to do this successfully; a familiar dilemma to both scientists and policy makers alike. © 2016 Avey, Griffin. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source


Griffin G.,Canadian Council on Animal Care | MacArthur Clark J.,Animals in Science Regulation Unit | Zurlo J.,Johns Hopkins Center for Alternatives to Animal Testing | Ritskes-Hoitinga M.,Review Centre
OIE Revue Scientifique et Technique | Year: 2014

The principles of humane experimental technique, first described by Russell and Burch in 1959, focus on minimising suffering to animals used for scientific purposes. Internationally, as these principles became embedded in the various systems of oversight for the use of animals in science, attention focused on how to minimise pain, distress and lasting harm to animals while maximising the benefits to be obtained from the work. Suffering can arise from the experimental procedures, but it can also arise from the manner in which the animals are housed and cared for. Increased attention is therefore being paid to the entire lifetime experience of an animal, in order to afford it as good a quality of life as possible. Russell and Burch were also concerned that animals should not be used if alternatives to such use were available, and that animals were not wasted through poor-quality science. This concept is being revisited through new efforts to ensure that experiments are well designed and properly reported in the literature, that all results - positive, negative or neutral - are made available to ensure a complete research record, and that animal models are properly evaluated through periodic systematic reviews. These efforts should ensure that animal use is truly reduced as far as possible and that the benefits derived through the use of animals truly outweigh the harms. Source


News Article
Site: http://www.nature.com/nature/current_issue/

No statistical methods were used to predetermine sample size. YapΔ/Δ and TazΔ/Δ mice were generated by crossing Yap or Taz floxed mice30 with the villin-cre line (Jackson Laboratory), the villin-creERT2 line (S. Robine, Institut Curie-CNRS) or the Lgr5-creERT line (Jackson Laboratory). The Rosa26-lox-STOP-lox-rtta-IRES-EGFP and Rosa26 lacZ mouse lines were obtained from Jackson Laboratory. The YapTg transgenic line described in this study was generated by introducing a HA-tagged wild-type Yap cDNA downstream of 7 Tet-repressor elements in the pTRE2 vector (J. Whitsett, Cincinnati Children’s Hospital Medical Center). The transgenic Yap construct was linearized and microinjected in ICR embryos. As shown in Extended Data Fig. 3a, activation of Cre deletes a neo cassette and allows for expression of the rtTA gene. In the presence of doxycycline the rtTA activates transcription of HA-Yap. Apc floxed mice were obtained from O. Sansom (Beatson Institute). Lats1 and Lats2 floxed alleles were obtained from R. Johnson (MD Anderson Cancer Center) and crossed with villin-creERT2 mice to obtain Lats1Δ/Δ;Lats2Δ/Δ mice. To measure polyp formation, YapΔ/Δ mice were backcrossed to a Bl/6 background for 4 generations before crossing to ApcMin/+ mice. Polyps from Yap+/+ (Yap+/+;villin-cre;ApcMin/+), Yap+/Δ (Yapfl/+;villin-cre;ApcMin/+) and YapΔ/Δ (Yapfl/fl;villin-cre;ApcMin/+) mice were counted 16 weeks after birth or when animals appeared moribund. Survival of ApcMin mice was measured by the number of days before mice were euthanized due to poor health. In vivo assays comparing control and Yap mutant animals were performed between age- and sex-matched pairs. No method of randomization was followed and no animals were excluded in this study. The investigators were not blinded to allocation during experiments and outcome assessment. Inducible Cre-mediated deletion of genes was performed by intraperitoneal injections of >5-week-old mice with 200 μl tamoxifen in corn oil at 10 mg ml−1. To create mosaic expression of Yap, Yapfl/fl;villin-creERT2 mice were induced with a single injection of 200 μl of tamoxifen at a suboptimal dose typically between 0.5 and 2.0 mg ml−1. For in vivo regeneration assays, mice were given a single dose of 10 or 12 Gy using a GammaCell 40 irradiator. Animals were maintained and handled under procedures approved by the Canadian Council on Animal Care. The immunohistochemistry stainings and standard colorimetric in situ hybridization were carried out according to methods described elsewhere31. Staining experiments were repeated on independent tissue sections prepared from separate mice as indicated by n values in figure legends. The following primary antibodies were used for immunostaining: rat anti-Ki67 (Dako, Cat. no. M7249, 1:1,000), rabbit anti-Yap/Taz (Cell Signaling, Cat. no. 8418, 1:100), rabbit anti-Yap (Cell Signaling, Cat. no. 14074, 1:300), mouse anti-Yap (Santa Cruz, Cat. no. sc-101199, 1:100), rabbit anti-Lef (Cell Signaling, Cat. no. 2230, 1:300), phosphor-Egfr (Tyr1092) (Abcam, Cat. no. ab40815, 1:300), anti-cleaved caspase-3 (Cell Signaling, Cat. no. 9664, 1:300) and anti-lysozyme (Dako, Cat. no. A0099, 1:1,000). Detection of primary antibodies was achieved using the Dako Envision plus system. Multi-colour fluorescence in situ hybridization with tyramide signal amplification (TSA) was done essentially as described elsewhere32, 33, 34. In brief, RNA probes from hybridized sections were detected using appropriate hapten-specific HRP-conjugated antibodies (anti-digoxigenin-HRP (Roche, Cat. no. 11207733910, 1:500), anti-dinitrophenyl-HRP (PerkinElmer, Cat. no. NEL747A001KT, 1:300), and anti-fluorescein-HRP (Life Technologies, A21253, 1:500)). After overnight incubation with antibodies at 4°C (or 2 h at room temperature for anti-fluorescein-HRP detection of cryptin1) sections were washed in PBS, and rinsed twice in 100 mM borate pH 8.5 plus 0.1% BSA. TSA reaction was performed by applying 300 μl per slide of the following mixture: 100 mM borate pH 8.5, 2% dextran sulfate, 0.1% Tween-20 and 0.003% H O , 450 μg ml−1 4-iodophenol: 1:250 Tyramide product (that is, DyLight633-tyramide, Dylight488-tyramide, Dylight 555-tyramide). The TSA reaction was allowed to proceed for 20 min and then terminated by washing slides in 100 mM glycine pH 2.0 for 15 min. Sections were washed further in PBS for the next round of detection. To synthesize tyramide products, the following succinimidyl esters were used for conjugation with tyramine: DyLight 633 NHS-Ester (Thermo Scientific Cat#46414), DyLight 550 NHS-Ester (Thermo Scientific Cat#62262), DyLight 488 NHS-Ester (Thermo Scientific Cat. no. 46402). The synthesis reaction was carried out as described previously32. The following in situ hybridization probes were obtained from the collection of MGC clones at the Lunenfeld Tanenbaum Research Institute: TweakR (BC025860), Ly6c1 (BC092082), Edn1 (BC029547), Areg (BC009138), Ereg (BC027838), Il1rn (BC042532), Il33 (BC003847), Msln (BC023753) and Cyr61 (BC066019). The Olfm4 and cryptdin1 probes were a gift from H. Clevers (Hubrecht Institute). Before fixing organoids, 10 μM Edu was added to the culture media for 1 h. Then organoids were fixed in 10% buffered formalin for 30 min, permeabilized in 0.5% Triton for 20 min and blocked in 2% BSA. Incorporated Edu was detected using the ClickIt EDU Imaging kit (Invitrogen) according to the manufacturer’s instructions. The primary antibodies used for immunostaining were mouse anti-Yap (Santa Cruz, Cat # sc-101199, 1:100), mouse anti-HA (Sigma-Aldrich, Cat. no. H9658, 1:1,000), and chicken anti-β-gal (Abcam, Cat. no. ab9361, 1:300). The secondary antibodies used in immunostaining were: CF555-donkey anti-mouse (Biotium, Cat. no. 20037, 1:400) and CF647 donkey anti-rabbit (Biotium, Cat. no. 20047, 1:400). Organoids were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich) before mounting onto slides for visualization. Images were acquired using a 20×/NA oil immersion objective lens (HCX PL APO, Leica), an EM-CCD camera (ImagEM, Hamamatsu) on an inverted microscope (DMIRE2, Leica) with a spinning disk confocal scanner (CSU10, Yokogawa) and Volocity. De novo crypts were scored as any protrusions, typically containing Paneth cells, budding from the initial sphere formed after seeding isolated crypts. Crypts were counted from bright-field images using Image J. At least four independent cultures derived from four different mice per genotype were used for quantification. Survival of crypts in Fig. 1 was determined by Ki67 staining of cross-sections of proximal portions of the small intestine at 3 days post-irradiation (10 Gy or 12 Gy). Values in Fig. 1b represent average number of fully labelled Ki67+ crypts per intestinal circumference based on counts from at least two sections per mouse and assays were repeated in 6 independent mice per genotype for both 10 Gy and 12 Gy treatments. The percentage of surviving Yap-positive versus negative Lgr5+ ISCs in Fig. 1d was performed by counting 587 β-gal+ crypts from a total of 7 untreated YapΔLgr5-cre mice and 394 β-gal+ crypts from a total of 9 irradiated YapΔLgr5-cre mice. In Yap;ApcΔLgr5-cre mice tumour initiating cells were visualized by staining for the Wnt target gene, Lef. As shown in Extended Data Fig. 10b, Lef is undetected in wild-type crypts and highly upregulated in Apc-null cells and thus serves as a robust marker of Apc deletion31. The percentage of Paneth cells in Lef+ foci (Fig. 4a) was assessed by preparing consecutive sections stained for Lyz, Lef and Yap, respectively. Lysozyme-positive Paneth cells from a total of 207 Yap wild-type and 201 Yap mutant Lef+ foci were counted from 5 Yap;ApcΔLgr5-cre mice (10–16 days after tamoxifen injection) using Image J and the percentage of total cells within the boundaries of a given Lef+ lesion was calculated. Relative activation of Egfr was quantified in consecutive sections from 5 Yap;ApcΔLgr5-cre mice stained for Lef, Yap and phospho-Egfr. For assessing Phospho-Egfr, staining intensity in Lef+ foci was assessed in a blinded fashion. For this, consecutive sections stained for Yap were masked from the observer scoring phospho-Egfr staining intensity. Lef+ foci were scored as ‘+’ if phospho-Egfr expression was elevated compared to wild-type adjacent crypts at comparable levels within the crypt–villus axis (see Extended Data Fig. 10f, panels xi and xii). Lef+ foci were scored as ‘++’ if staining intensity was very strong even relative to the stem cell compartment in normal crypts and/or displayed prominent apical staining (see Yap-positive foci in Fig. 4c, panel iv, and Extended Data Fig. 10f, panels v and vi). Lef+ foci were scored as ‘–’ if staining intensity was undetected or unchanged relative to adjacent wild-type crypts (see Yap mutant foci in Fig. 4c and Extended Data Fig. 10f). In Extended Data Fig. 1, caspase 3 and BrdU positive crypt cells were counted from at least six sections per mouse in 4 independent mice per genotype and expressed as a percentage of total crypt cells. All data are presented as average values with s.e.m. Mann–Whitney (two-tailed) U-test was used to determine statistical significance. Calculations were performed using GraphPad Prism 5 software. RNA was isolated from organoids cultured for 24 h after seeding in Matrigel. RNA samples were pooled from at least three organoid cultures derived from at least three independent mice per genotype (Yapfl/+;villin-cre, Yapfl/fl;villin-cre and YapTg). Quality of RNA was verified by running samples on a Bioanalyzer. High-throughput sequencing was performed using the Illumina HiSeq 2000 at the Lunenfeld Tanenbaum Research Institute (LTRI) sequencing facility. Raw sequencing reads in Fastq formats were mapped onto mouse genome (mm9) using Tophat 1.4.1 and the RPKMs (reads per kilobase of exon model per million mapped reads) were calculated using a customized script. RNA-seq data are presented in Supplementary Table 1. Combined fold change presented in Extended Data Fig. 3d and Supplementary Table 1 was calculated using the following formula: combination fold change = log [(YapΔ/Δ/Yap+/Δ)/(Dox+/Dox−)]. R, Cluster 3.0 and Java TreeView were used for data visualization. Gut organoids were cultured according to a previously described protocol established by Sato and Clevers7. Briefly, crypts were harvested by incubating opened small intestines in PBS containing 2 mM EDTA. The epithelium was released by vigorous shaking and crypts separated using a 70 μm cell strainer. Crypts were seeded in growth factor reduced Matrigel (BD Biosciences) and grown in Advanced DMEM/F12 (Invitrogen) supplemented with 2 mM GlutaMax (Invitrogen), 100 U ml−1 Penicillin/100 μg ml−1 Streptomycin (Invitrogen), N2 Supplement (Invitrogen), B-27 Supplement (Invitrogen Cat), mouse recombinant Egf (R&D Systems), 100 ng ml−1 mouse recombinant Noggin (Peprotech), 150 ng ml−1 human Rsp1 (R&D Systems). Apc-deficient organoids were harvested from Yapfl/+;Apcfl/fl;villin-creERT, Yapfl/fl;Apcfl/fl;villin-creERT or YapTg;Apcfl/fl;villin-creERT mice injected with tamoxifen and seeded 48 h later in basal growth medium without Egf, Rsp1 or Noggin. To induce Yap expression in YapTg organoids, 1.5 μg ml−1 doxycycline was added to the culture medium on day 0. Egf (R&D Systems, Cat. no. AF2028), Areg (R&D Systems, Cat. no. AF989) and Ereg (R&D Systems, Cat. no. 1068-EP-050) were added to the culture medium at a final concentration of 0.5 μg ml−1. The following inhibitors were used: PD153053 (0.5 μM, Tocris Bioscience), U0126 (10 μM, Merck Millipore). To examine pErk1/2 levels, organoids were harvested at day 2 in cold PBS containing 5 mM EDTA, 1 mM NaVO , 1.5 mM NaF and protease inhibitors. Organoids were incubated at 4°C for 30 min to dissolve Matrigel and then lysed in TNTE buffer (50 mM Tris/HCl pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA) containing standard protease and phosphatase inhibitors. Protein concentrations were measured and samples were subjected to SDS–PAGE. Total RNA was extracted by removing culture medium and directly lysing organoids in wells using RTL buffer of the Rneasy Mini Kit (Qiagen). RNA was purified using columns and genomic DNA was removed by treatment with RNase-Free DNase (Qiagen).


Ormandy E.H.,University of British Columbia | Dale J.,Canadian Council on Animal Care | Griffin G.,Canadian Council on Animal Care
ATLA Alternatives to Laboratory Animals | Year: 2013

The genetic engineering of animals for their use in science challenges the implementation of refinement and reduction in several areas, including the invasiveness of the procedures involved, unanticipated welfare concerns, and the numbers of animals required. Additionally, the creation of geneticallyengineered animals raises problems with the Canadian system of reporting animal numbers per Category of Invasiveness, as well as raising issues of whether ethical limits can, or should, be placed on genetic engineering. A workshop was held with the aim of bringing together Canadian animal care committee members to discuss these issues, to reflect on progress that has been made in addressing them, and to propose ways of overcoming any challenges. Although previous literature has made recommendations with regard to refinement and reduction when creating new genetically-engineered animals, the perception of the workshop participants was that some key opportunities are being missed. The participants identified the main roadblocks to the implementation of refinement and reduction alternatives as confidentiality, cost and competition. If the scientific community is to make progress concerning the implementation of refinement and reduction, particularly in the creation and use of genetically-engineered animals, addressing these roadblocks needs to be a priority. Source

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