Bruce Whitelaw

A future for transgenic livestock

The techniques that are used to generate transgenic livestock are inefficient and expensive. This, coupled with the fact that most agriculturally relevant traits are complex and controlled by more than one gene, has restricted the use of transgenic technology. New methods for modifying the genome will underpin a resurgence of research using transgenic livestock. This will not only increase our understanding of basic biology in commercial species, but might also lead to the generation of animals that are more resistant to infectious disease.

Characterisation and differentiation potential of bone marrow derived canine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have potential for use in regenerative therapeutics, since they are capable of multi-lineage differentiation. In this study, primary canine MSCs (cMSCs) were isolated from bone marrow aspirates and characterised using marker expression and morphology. cMSCs expressed CD44 and STRO-1, but not CD34 or CD45. Morphologically, cMSCs were similar to previously described MSCs and were capable of chondrocyte differentiation towards articular type cartilage, characterised by increased collagen type II vs. collagen type I expression and expression of Sox-9. cMSCs demonstrated no significant alterations in marker profiles and failed to differentiate into cardiomyocytes in response to a cardiac differentiation protocol or when co-cultured with canine cardiac stem cells. The study indicated that cMSCs can be derived readily from bone marrow and are capable of differentiation into articular cartilage, but appear to have limited ability to differentiate into cardiomyocytes using current protocols.

Characterisation and cardiac directed differentiation of canine adult cardiac stem cells

This study describes the isolation and characterisation of adult canine cardiac stem cells, and explores their ability to differentiate into cardiac myocytes. Direct comparisons are also made with available human data. Atrial cardiac explants were taken from dogs post-mortem and cultured to isolate adult stem cells. Cells were able to survive successive passages in serum-free media, were able to form cardiospheres, and under controlled culture conditions were capable of clonal expansion, demonstrating their ability for self-renewal. Characterisation of these cells demonstrated the following marker profile: c-kit, GATA 4 and flk-1 positive; cardiac troponin T and NKx2.5 low. Cardiac lineage directed differentiation was performed based on the published literature. Gene expression studies demonstrated that cardiac directed differentiation was partially achieved, with up-regulation of cardiac troponin T and NKx2.5, and down-regulation of c-kit and endothelial lineage markers. However the cells did not express the ryanodine receptor or β(1)-adrenergic receptors and did not contract spontaneously.

A high resolution atlas of gene expression in the domestic sheep (Ovis aries)

Sheep are a key source of meat, milk and fibre for the global livestock sector, and an important biomedical model. Global analysis of gene expression across multiple tissues has aided genome annotation and supported functional annotation of mammalian genes. We present a large-scale RNA-Seq dataset representing all the major organ systems from adult sheep and from several juvenile, neonatal and prenatal developmental time points. The Ovis aries reference genome (Oar v3.1) includes 27,504 genes (20,921 protein coding), of which 25,350 (19,921 protein coding) had detectable expression in at least one tissue in the sheep gene expression atlas dataset. Network-based cluster analysis of this dataset grouped genes according to their expression pattern. The principle of ‘guilt by association’ was used to infer the function of uncharacterised genes from their co-expression with genes of known function. We describe the overall transcriptional signatures present in the sheep gene expression atlas and assign those signatures, where possible, to specific cell populations or pathways. The findings are related to innate immunity by focusing on clusters with an immune signature, and to the advantages of cross-breeding by examining the patterns of genes exhibiting the greatest expression differences between purebred and crossbred animals. This high-resolution gene expression atlas for sheep is, to our knowledge, the largest transcriptomic dataset from any livestock species to date. It provides a resource to improve the annotation of the current reference genome for sheep, presenting a model transcriptome for ruminants and insight into gene, cell and tissue function at multiple developmental stages.

Recommendations for minimum information for publication of experimental pathology data: MINPEPA guidelines

Animal models are essential research tools in modern biomedical research, but there are concerns about their lack of reproducibility and the failure of animal data to translate into advances in human medical therapy. A major factor in improving experimental reproducibility is thorough communication of research methodologies. The recently published ARRIVE guidelines outline basic information that should be provided when reporting animal studies. This paper builds on ARRIVE by providing the minimum information needed in reports to allow proper assessment of pathology data gathered from animal tissues. This guidance covers aspects of experimental design, technical procedures, data gathering, analysis, and presentation that are potential sources of variation when creating morphological, immunohistochemical (IHC) or in situ hybridization (ISH) datasets. This reporting framework will maximize the likelihood that pathology data derived from animal experiments can be reproduced by ensuring that sufficient information is available to allow for replication of the methods and facilitate inter‐study comparison by identifying potential interpretative confounders. Copyright

Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR.

Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for differences in RTL measurements from samples extracted by different DNA extraction methods within a study.

Lentiviral transgenesis in livestock

The application of lentivirus vectors (LV) to deliver transgenes to the germline of animals has been established in rodents, livestock and birds. This technology is presented as offering a very efficient route to germline transgenesis (Pfeifer 2004; Sang 2004; Whitelaw 2004). As such, and especially in non-rodent species where the standard methodology is either technically difficult or expensive, LV transgenesis is a very attractive addition to the tools available for transgene delivery. While the majority of publications to date have been with HIV-1-based vectors, alternative systems based on lentiviruses such as EIAV (Vasey et al. 2009) or SIV (Hiripi et al. 2010) are also used. However, both published concerns over transgene silencing (Hofmann et al. 2006) and ‘grapevine’ comments on variable efficiency limit widespread uptake of the technology. We now wish to make the comment that in our hands this technology can be applied efficiently. In the sheep season of 2008/2009 our aim was to utilise HIV-1-based lentiviral vectors to deliver 7 transgenes, under the control of either tissue-specific or short viral promoters, by perivitelline injection (Ritchie et al. 2009). We transferred 230 embryos, produced 61 lambs and identified 32 transgenic founder animals. This translates into a transgenesis rate of 52% of live births. We produced transgenic animals for 6/7 transgenes; we have reasons to believe the lack of success with the remaining transgene may reflect embryo lethality as a consequence of transgene expression. To put these efficiencies in context, since 1985 when the first transgenic sheep were produced at Roslin up and until this season, we have successfully introduced 8 different transgenes resulting in 25 lines of transgenic sheep. In our first study way back in 1984–1985, one transgene was introduced, 92 zygotes were transferred and 23 lambs born of which only one was identified as transgenic. So in this recent season we produced more transgenic founder animals than in the previous 25 years combined. Moreover, unlike the early days when in vivo produced zygotes were required, the method now allows the use of slaughterhouse derived material and IVF/IVM (Ritchie et al. 2005) refining the procedure resulting in reducing significantly the number of animals used. We maintain that the ability to deliver many transgenes efficiently is attractive to both public and commercial funders. The authors recognise the support provided by the BBSRC, University of Edinburgh IKTF award and Scottish Funding Council SRDG.

A Csf1r-EGFP Transgene Provides a Novel Marker for Monocyte Subsets in Sheep

Expression of Csf1r in adults is restricted to cells of the macrophage lineage. Transgenic reporters based upon the Csf1r locus require inclusion of the highly conserved Fms-intronic regulatory element for expression. We have created Csf1r-EGFP transgenic sheep via lentiviral transgenesis of a construct containing elements of the mouse Fms-intronic regulatory element and Csf1r promoter. Committed bone marrow macrophage precursors and blood monocytes express EGFP in these animals. Sheep monocytes were divided into three populations, similar to classical, intermediate, and nonclassical monocytes in humans, based upon CD14 and CD16 expression. All expressed EGFP, with increased levels in the nonclassical subset. Because Csf1r expression coincides with the earliest commitment to the macrophage lineage, Csf1r-EGFP bone marrow provides a tool for studying the earliest events in myelopoiesis using the sheep as a model.

CD13/aminopeptidase N is a negative regulator of mast cell activation

Antigen‐induced mast cell (MC) activation via cross‐linking of IgE‐bound high‐affinity receptors for IgE (FcεRI) underlies type I allergy and anaphylactic shock. Comprehensive knowledge of FcεRI regulation is thus required. We have identified a functional interaction between FcεRI and CD13 in murine MCs. Antigen‐triggered activation of IgE‐loaded FcεRI results in cocapping and cointernalization of CD13 and equivalent internalization rates of up to 40%. Cointernalization is not unspecific, because ligand‐driven KIT internalization is not accompanied by CD13 internalization. Moreover, antibody‐mediated cross‐linking of CD13 causes IL‐6 production in an FcεRI‐dependent manner. These data are indicative of a functional interaction between FcεRI and CD13 on MCs. To determine the role of this interaction, CD13‐deficient bone marrow‐derived MCs (BMMCs) were analyzed. Intriguingly, antigen stimulation of CD13‐deficient BMMCs results in significantly increased degranulation and proinflammatory cytokine production compared to wild‐type cells. Furthermore, in a low‐dose model of passive systemic anaphylaxis, antigen‐dependent decrease in body temperature, reflecting the anaphylactic reaction, is substantially enhanced by the CD13 inhibitor bestatin (–5.9 ± 0.6°C) and by CD13 deficiency (–8.8 ± 0.6°C) in contrast to controls (–1.2 ± 1.97°C). Importantly, bestatin does not aggravate anaphylaxis in CD13‐deficient mice. Thus, we have identified CD13 as a novel negative regulator of MC activation in vitro and in vivo.—Zotz, J. S., Wölbing, F., Lassnig, C., Kauffmann, M., Schulte, U., Kolb, A., Whitelaw, B., Müller, M., Biedermann, T., Huber, M. CD13/aminopeptidase N is a negative regulator of mast cell activation. FASEB J. 30, 2225–2235 (2016). www.fasebj.org

Developing welfare protocols for transgenic large animals

Abstracts from the UC Davis Transgenic Animal Research Conference XIs from the UC Davis Transgenic Animal Research Conference XI