Engineering large animal models of human disease:Domesticated Animal Models of Human Disease

The recent development of gene editing tools and methodology for use in livestock enables the production of new animal disease models. These tools facilitate site‐specific mutation of the genome, allowing animals carrying known human disease mutations to be produced. In this review, we describe the various gene editing tools and how they can be used for a range of large animal models of diseases. This genomic technology is in its infancy but the expectation is that through the use of gene editing tools we will see a dramatic increase in animal model resources available for both the study of human disease and the translation of this knowledge into the clinic. Comparative pathology will be central to the productive use of these animal models and the successful translation of new therapeutic strategies. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

MODULATING KEY GENES INVOLVED IN PANCREAS FORMATION AND INSULIN SIGNALING USING CRISPR/CAS9 IN THE PIG

Among the metabolic diseases, diabetes remains a “pressing problem” as recognized by World Health Organization, not only due to the impact on individuals’ lives, but also because of the rapid increase in newly diagnosed patients. To better understand the mechanisms of diabetes, this dissertation investigates the role of NGN3 in pancreas development using CRISPR/Cas9 gene targeting in the pig model. NGN3 was selected for study because of its critical role in endocrine pancreas formation. Our research demonstrates that the targeted ablation of NGN3 blocks development of the endocrine pancreas, a finding supported through gene expression analysis. Furthermore, follow-up studies show that clonal piglets derived from NGN3-ablated animals lack the major endocrine islet cell types and subsequent expression of key endocrine hormones. This porcine model provides valuable insights into the study of type 1 diabetes in early post-natal life and future applications of human-to-pig chimeric organ development for transplant surgery. Expanding upon this porcine model for diabetes, we sought to apply this approach to the study of type 2 diabetes using a novel pig model, thus bridging the gap between mouse and human. For this endeavor, we identified GRB10 as a potential critical mediator in insulin signaling, development, and growth potential following an extensive literature review. The potential for dual applications in both agriculture and medicine was also identified as an objective. Analysis of qPCR data from in vitro overexpression studies supports that GRB10 modulates insulin signaling through the canonical insulin pathway. Additional data from two in vivo gene editing trials targeting the GRB10 locus in both Ossabaw and domestic pig breeds show a supportive qualitative trend towards growth regulation in the Ossabaw pig breed. Further evidence is required to determine whether GRB10 plays the same role in the domestic pig, as a limited cohort size of mutants precluded an extensive analysis of phenotypes. Together, our assessment of NGN3 and GRB10 offer significant potential for modeling of both type 1 and type 2 diabetes as well as modeling of growth traits in the pig through application of advanced genome engineering technology

Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs

The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools such as clustered regularly interspaced short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9), it is now possible to perform site-specific alterations with relative ease, and will likely help realize the potential of this valuable model. In this article, we investigated for the first time a combination of somatic cell nuclear transfer (SCNT) and direct injection of CRISPR/Cas ribonucleoprotein complex targeting GRB10 into the reconstituted oocytes to generate GRB10 ablated Ossabaw fetuses. This strategy resulted in highly efficient (100%) generation of biallelic modifications in cloned fetuses. By combining SCNT with CRISPR/Cas9 microinjection, genome edited animals can now be produced without the need to manage a founder herd, while simultaneously eliminating the need for laborious in vitro culture and screening. Our approach utilizes standard cloning techniques while simultaneously performing genome editing in the cloned zygotes of a large animal model for agriculture and biomedical applications

Relative mRNA expression of pancreas development transcription factors by CD133+ cells isolated after 4 days of exocrine tissue culture.

<p>Mean ± SEM relative expression level of genes in CD133+ cells compared to the CD133-depleted (CD133D) population shown on Y-axis as fold difference. Significance determined by Student’s t-test from 2^-Δct values. ***, p<0.001, **, p<0.01, *, p<0.05 (n = 4 exocrine cultures). Genes ranked in order of overexpression in the CD133+ population. Inset shows gene expression using an enlarged scale. Genes are: Neurogenin 3 (NGN3), One cut homeobox 2 (ONECUT2), NK6 homeobox 1 (NKX6.1), GLIS family zinc finger 3 (GLIS3), motor neuron and pancreas homeobox 1 (MNX1), HNF1 homeobox B (HNF1B), pancreatic and duodenal homeobox 1 (PDX1), SRY (sex determining region Y)-box 9 (SOX9), Hairy enhancer of split 1 (HES1), One cut homeobox 1 (ONECUT1), Forkhead box A2 (FOXA2), Forkhead box O1 (FOXO1), v-maf avian musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB), GATA binding protein 4 (GATA4), Pancreas specific transcription factor 1A (PTF1A), Neuronal differentiation 1 (NEUROD1), ISL LIM homeobox 1 (ISL1), paired box 6 (PAX6). No expression of NK2 homeobox 2 or paired box 4 was detected.</p

Expression of Notch pathways genes.

<p><b>A</b>, Western blot analyses of Notch intercellular domain (NICD), hairy enhancer of split 1 (HES1) and endogenous control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells isolated from human exocrine tissue. Whole cell lysates from CD133+ (+) and CD133-depleted (D) cells. Nuclear (N) and cytoplasmic (C) extracts from CD133+ cells. <b>B</b>, Volcano plot of Notch pathway gene mean ± SEM mRNA level (n = 3 exocrine cultures) differences in expression level from CD133+ cells compared to CD133D shown on X-axis as Log2 of fold difference. Significance determined by Student’s t-test shown on Y-axis as p value. Magenta vertical lines mark a 2-fold difference in expression. Blue horizontal line marks the significance cutoff (p<0.05). Selected gene names shown. Genes are: receptor tyrosine-protein kinase erbB-2 (ERBB2), frizzled class receptor 7 (FZD7), E1A binding protein p300 (EP300), MFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase (MFNG), H19, imprinted maternally expressed transcript (H19), LIM domain only 2 (rhombotin-like 1) (LMO2), inhibitor of DNA binding 1 (ID1), hairy enhancer of split 4 (HES4), cyclin D1 (CCND1), matrix metallopeptidase 7 (MMP7), mastermind-like 2 (MAML2), jagged 1 (JAG1), Notch 2 (NOTCH2), hes-related family bHLH transcription factor with YRPW motif-like (HEYL), snail family zinc finger 2 (SNAI2), recombination signal binding protein for immunoglobulin kappa J region-like (RBPJL). <b>C</b>, Normalized mRNA expression level of neurogenin 3 (NGN3), HES1 and pancreas transcription factor 1 subunit alpha (PTF1A) in exocrine tissue after 4 days of culture in the presence of 20 μM Notch inhibitor DAPT. Results reported as mean ± SEM percent of levels in DMSO carrier control. mRNA levels normalized to the level of cyclophillin A. Significance determined by Student’s t-test, ***, p<0.001 (n = 3 exocrine cultures). <b>D</b>, Expression of NGN3 protein following treatment with 20 ∞M DAPT and 47 ∞M Notch agonist JAG-1 peptide (JAG-1). Mean ± SEM percent of DMSO carrier only control or 47 ∞M scrambled JAG-1 peptide, respectively indicated on Y-Axis. Significance determined by Student’s t-test, ***, p<0.001 (n = 3 exocrine cultures). <b>E-H</b>, Orthogonal analysis of colocalized HES1 and NGN3 in nuclei of exocrine tissue after 4 days of culture. Nuclei counterstained with Hoechst 33342 (H). <b>E</b>, Overlay of 3 channels. 0.5 ∞m confocal section. Scale bar is 50 ∞m. <b>F-H</b>, Higher magnification of crosshair region in all three channels shown at right. Scale bars are 20 ∞m. <b>I</b>, Coprecipitation of ID proteins with HES1. Whole cell lysate from exocrine tissue after 4 days of culture immunoprecipitated with antibody to HES1. ID1, 2 and 4 detected following SDS PAGE and western blotting. Predicted molecular weights of ID proteins (ID) and immunoglobulin heavy chain used for precipitation (HC) shown at right. Molecular weight marker positions shown at left in kDa.</p

Expression of hormones, chromogranin A and PDX1 by CD133+ cells following <i>in vitro</i> differentiation.

<p><b>A</b>, Phase microscopic image of pancospheres on day 6 of formation. Scale bar is 100 μm. <b>B</b>, Pancreatic and duodenal homeobox 1 (PDX1) expression in a day 6 pancosphere. Scale bar is 50 μm. <b>C,D</b>, Orthogonal analyses of PDX1 / glucagon (GCG) and PDX1 / insulin C-peptide (CPEP) coexpression in day 21 pancospheres. 1 μm optical sections, scale bar is 50 μm. Inset box in D is magnified and rotated confocal reconstruction of cells indicated by lines. <b>E</b>, Coexpression of GCG and chromogranin A (CHGA) by cells within a day 21 pancosphere. Scale bar is 20 μm. <b>F</b>, Coexpression of CPEP, CHGA and PDX1 by cells within a day 21 pancosphere. Scale bar is 10 μm. B-F, Nuclei stained with Hoechst 33342 (H).</p