Livestock 2.0 – genome editing for fitter, healthier, and more productive farmed animals

Abstract The human population is growing, and as a result we need to produce more food whilst reducing the impact of farming on the environment. Selective breeding and genomic selection have had a transformational impact on livestock productivity, and now transgenic and genome-editing technologies offer exciting opportunities for the production of fitter, healthier and more-productive livestock. Here, we review recent progress in the application of genome editing to farmed animal species and discuss the potential impact on our ability to produce food

Inactivation of DAP12 in PMN inhibits TREM1-mediated activation in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by dysregulated and chronic systemic inflammatory responses that affect the synovium, bone, and cartilage causing damage to extra-articular tissue. Innate immunity is the first line of defense against invading pathogens and assists in the initiation of adaptive immune responses. Polymorphonuclear cells (PMNs), which include neutrophils, are the largest population of white blood cells in peripheral blood and functionally produce their inflammatory effect through phagocytosis, cytokine production and natural killer-like cytotoxic activity. TREM1 (triggering receptor expressed by myeloid cells) is an inflammatory receptor in PMNs that signals through the use of the intracellular activating adaptor DAP12 to induce downstream signaling. After TREM crosslinking, DAP12's tyrosines in its ITAM motif get phosphorylated inducing the recruitment of Syk tyrosine kinases and eventual activation of PI3 kinases and ERK signaling pathways. While both TREM1 and DAP12 have been shown to be important activators of RA pathogenesis, their activity in PMNs or the importance of DAP12 as a possible therapeutic target have not been shown. Here we corroborate, using primary RA specimens, that isolated PMNs have an increased proportion of both TREM1 and DAP12 compared to normal healthy control isolated PMNs both at the protein and gene expression levels. This increased expression is highly functional with increased activation of ERK and MAPKs, secretion of IL-8 and RANTES and cytotoxicity of target cells. Importantly, based on our hypothesis of an imbalance of activating and inhibitory signaling in the pathogenesis of RA we demonstrate that inhibition of the DAP12 signaling pathway inactivates these important inflammatory cells

MicroRNA-155 governs SHIP-1 expression and localization in NK cells and regulates subsequent infiltration into murine AT3 mammary carcinoma.

NK cell migration and activation are crucial elements of tumor immune surveillance. In mammary carcinomas, the number and function of NK cells is diminished, despite being positively associated with clinical outcome. MicroRNA-155 (miR-155) has been shown to be an important regulator of NK cell activation through its interaction with SHIP-1 downstream of inhibitory NK receptor signaling, but has not been explored in regard to NK cell migration. Here, we explored the migratory potential and function of NK cells in subcutaneous AT3 in mice lacking miR-155. Without tumor, these bic/miR-155-/- mice possess similar numbers of NK cells that exhibit comparable surface levels of cytotoxic receptors as NK cells from wild-type (WT) mice. Isolated miR-155-/- NK cells also exhibit equivalent cytotoxicity towards tumor targets in vitro compared to isolated WT control NK cells, despite overexpression of known miR-155 gene targets. NK cells isolated from miR-155-/- mice exhibit impaired F-actin polymerization and migratory capacity in Boyden-chamber assays in response chemokine (C-C motif) ligand 2 (CCL2). This migratory capacity could be normalized in the presence of SHIP-1 inhibitors. Of note, miR-155-/- mice challenged with mammary carcinomas exhibited heightened tumor burden which correlated with a lower number of tumor-infiltrating NK1.1+ cells. Our results support a novel, physiological role for SHIP-1 in the control of NK cell tumor trafficking, and implicate miR-155 in the regulation of NK cell chemotaxis, in the context of mammary carcinoma. This may implicate dysfunctional NK cells in the lack of tumor clearance in mice

Blocking DAP12 reduces the increased signaling and cytokine production brought on by increased expression of DAP12 in PMN from RA patients.

<p>(A) Quantitative RT-PCR was used to determine the relative levels of DAP12 in isolated PMN from RA primary specimens (n = 19) compared with healthy controls (n = 6). Error bars denote the SEM of duplicate determinations per sample. P value shown. (B) Healthy PMN (n = 4) were isolated and cultured in the presence of either TNFα (1ng/ml), IL-6 (1ng/ml), or IL-17 (20ng/ml) for 24 and 48 hours before DAP12 analysis by flow cytometry. (C) Healthy PMN (n = 4) were isolated and cultured in the presence of TNFα after the addition of TNFα blocking antibody (0.01ug/ml to 0.1ug/ml) for 24 and 48 hours before DAP12 analysis by flow cytometry. For both B and C error bars denote the SEM of duplicate readings of 4 treated primary healthy specimens. Asterisk denote p<0.05 against control untreated cells. (D) HEK293 cells co-transfected with TREM-1 and either of the FLAG-tagged DAP12 constructs: wild type (WT, lanes 5, 6), or dnDAP12 (lane 7) were cross-linked with anti-TREM-1 antibody followed by western blot analysis for the activation of p42/p44 MAPK and compared against total ERK. Single transfections with TREM-1 and WTDAP12 are included as negative controls (lanes 3 and 4). Supernatants from this experiment were also used to analyze (E) IL-8 and (F) RANTES by ELISA were error bars denote the SEM of triplicate measurements of each sample and asterisk p<0.05 measured against * normal, ** Mock or ***CD56.</p

Increased expression of TREM-1 in PMN from RA patients.

<p>(A) PMN cells isolated from the synovial fluid of RA patients and healthy donors were analyzed by flow cytometry for the cell surface expression of TREM-1. A representative dot plot of CD66/TREM-1 expression is shown. (B) Representative figure of the expression of TREM-1 on the isotype stained cells, healthy PMN and the PMN from a primary RA specimen analyzed by flow cytometry. Legend shows the percent of total TREM 1 cells from the gate shown. (C) The same analysis shown in B was used to determine the relative levels of TREM-1 in isolated PMN from RA primary specimens compared with healthy controls (n = 19 RA and n = 6 controls). The MFI of all the samples was also obtained and averaged. However there were not significant changes (numbers shown). (D) Quantitative RT-PCR was used to determine the relative levels of TREM-1 in isolated PMN from RA primary specimens compared with healthy controls (n = 19 RA and n = 6 controls). (E) Surface expression of TREM-1 was compared between PMNs (blue), NK cells (red), T cells (green) and monocytes through flow cytometric analysis of isolated (n = 6). Error bars denote the standard error of duplicate determinations of 5 samples per group and p values are shown between figures except in E where it is shown as an asterisk to denote p = 0.0013 of normal versus RA while the other ones were not significant.</p