Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet

Unexpected impairment of INa underpins reentrant arrhythmias in a knock-in swine model of Timothy syndrome

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Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices

DP Test in Geotechnical Characterization of Shallow Landslides Source Area:Results and Perspectives

Aiming at improving the knowledge on the source areas of shallow landslides, this research deals with the geotechnical characterization of the debris slope cover of arenaceous formations, by means of low cost dynamic penetration tests (Dynamic Probing - DP). The knowledge of strength parameters of this material is fundamental to understand the triggering mechanisms of the shallow landslides. Therefore, in order to determine the typical values of relative density and internal friction angle, many tests were performed and the results of 132 Dynamic Probing tests, 74 Standard Penetration Tests (SPT) and 14 laboratory shear tests, were analysed. The comparison between the φ' values obtained by DP tests with those resulting from direct shear tests evidenced a good linear correlation. This confirmed the satisfactory and repeatable results of DP tests. Therefore, Dynamic Probing can be an effective, simple, practical and inexpensive tool for the geotechnical characterization of hazardous slopes

Dynamic Probing as an easy and practical tool in geotechnical characterization of shallow landslide source areas: results, problems and perspectives in Northern Tuscany (Italy)

In Northern Tuscany intense and long time rainfalls produce many shallow landslides, mostly soil slip-debris flows, which often cause victims and severe damage. Aiming at contributing to the characterization of the source areas of shallow landslides, this research deals with the geotechnical characterization, by means of low cost dynamic penetration tests (Dynamic Probing, DP), of the debris slope cover of the Macigno Fm. (Tuscan Nappe unit of the Tuscan Domain). This formation, composed of quartz and feldspar sandstones altering with layers of siltstone, constitutes the main Apennines ridge and underlies hundreds of square kilometres of Northern Tuscany. Most of shallow landslides triggered by critical rainfall events really involve the slope cover of the Macigno Fm., which mainly consist of sand and gravel with a minor portion of pelite. The knowledge of physic-mechanical properties of this material is fundamental to understand the triggered mechanisms of the shallow landslides. Therefore, in order to determine the typical values of relative density (Dr) and shear strength angle (φ’), the results of 220 tests were analysed. These tests were subdivided into 91 Medium Dynamic Probing (DPM), 41 Super Heavy Dynamic Probing (DPSH), 74 Standard Penetration Tests (SPT) and 14 samples with direct shear tests. The penetration resistance obtained by DPSH and DPM tests was related to the strength parameters of the debris cover by means of empirical relations. These relations consider the most typical soil categories (silty sand, gravelly sand and sandy gravel) and take into account for the Energy Ratio of the instruments used and the stress conditions of sites. Distribution and variability of shear strength angle and relative density were analysed and related to soil type, test type and probing depth. In general, the Dr and φ’ values show an increase in scattering around the mean values with the increase in grain size and sorting; DPM tests show this trend more clearly then DPSH tests. The presence of pebbles in a silty sand material, even in low percentage, probably can cause a significant increase of N (number of blows, coming from DP), and consequently in the Dr and φ’ values. Furthermore, at the low depth commonly investigated by Dynamic Probing (usually less then 5-6 m in order to investigate the cover materials involved in shallow landslides), Dr and φ’ of each soil type do not show significant increase with depth. In order to validate the results, the values of Dr and φ’ obtained by DP tests were also compared to the results obtained in similar deposits by SPT. This comparison showed small differences: in detail, the SPT provided slightly higher friction angles than DP tests and therefore they seem to be less conservative. Moreover, the φ’ values obtained by DP tests were compared with those resulting from direct shear tests (when available), performed on undisturbed samples taken in the same soil type, close to the DP test sites, at the same depth and stress conditions. The comparison evidenced a good linear correlation between the values resulting from these different tests, so confirming the satisfactory and repeatable results of DP tests. Therefore the DP could be an effective, simple, practical and inexpensive tool for the geotechnical characterization of potentially unstable slope covers. In this way actual and reliable parameters may be achieved, on which shallow landslide susceptibility and hazard maps can be based

EFFICIENT EXPRESSION OF HUMAN ENDOTHELIAL PROTEIN C RECEPTOR AND HUMAN THROMBOMODULIN IN TRANSFECTED PIG PRIMARY hCD55(+)-GAL(-/-) FIBROBLASTS USING F2A EXPRESSION VECTOR

The genetic engineering of the pig genome for xenotransplantation studies requires the insertion of different transgenes to create multi-transgenic pigs. In order to simultaneously add more transgene in a single genetic insertion, we constructed a polycistronic vector using the F2A self-cleaving peptide. Moreover, this solution has the added advantages of preventing possible segregation during breeding of the animals and of guaranteeing an equimolar production of chosen transgenes. The scope of this work was the construction and validation of an ubiquitous F2A-bicistronic expression vector for human thrombomodulin (hTM) and human endothelial protein C receptor (hEPCR) genes in pig primary hCD55-GAL\u2013/\u2013 cells to establish transgenic fibroblasts colonies, to be used for somatic cell nuclear transfer (SCNT) to generate pigs for xenotransplantation research. The expression vector consisted of pCAGGS promoter (CMV-IE+chicken \u3b2 actin) followed by hEPCR-furinF2A-hTM coding sequence. The resulting expression cassette was inserted between 2 insulators obtained from the 5\u2032 MAR region of chicken lysozyme. Outside of this insulated structure, there is a loxable puromycin selection cassette. The resulting purified and linearized expression vector (pEFTM/Lgu I = 5 \u3bcg) was transfected into hCD55-GAL\u2013/\u2013 primary fibroblasts (1 7 106), using Nucleofector (Amaxa, Lonza, Cologne, Germany), in parallel for comparative purposes we cotransfected the 2 pCAGGS-monocistronic vectors for the same transgenes (hEPCR and hTM = 1:3, 5 \u3bcg). Transfected cells were selected with puromycin (1 \u3bcg mL\u20131) for 15 days. After 8 days of selection, resistant colonies were picked up and expanded into 24-well plates for cryopreservation and analyses. Bicistronic transfection produced 20 clones and cotransfection only 8 clones that were analysed by Western blot (WB) and by immunocytochemistry (ICC) using polyclonal antibody anti-EPCR (1:250, R&D) and monoclonal antibody ab6980-Abcam (1:5000, Abcam, Cambridge, UK) in WB; polyclonal antibody RCR252 (1:100, Sigma-Aldrich, St. Louis, MO, USA) and monoclonal antibody ab6980-Abcam (1:100, Abcam) for ICC. Seventeen bicistronic clones (85%) and 2 cotransfected monocistronic clones (25%) were positive for both transgenes using WB. After ICC analyses, only 11 bicistronic colonies (55%) and 1 cotransfected colony (12.5%) uniformly expressed the desired transgenes and were selected for SCNT. The pCAGGS promoter maintained its strong expression also using the hEPCR-FurinF2A-hTM coding sequence and this bicistronic solution permitted us to improve our results obtained with co-transfection. Availability of hEPCR+ hTM+ hCD55+-GAL\u2013/\u2013 colonies will allow us to obtain a new transgenic background for future xenotransplantation projects

215 LIVE PIGLETS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER FOLLOWING TARGETING OF A PORCINE ENHANCED GREEN FLUORESCENT PROTEIN LINE MEDIATED BY ZINC-FINGER NUCLEASES TO ESTABLISH CLONED HYGROMYCIN-RESISTANT PRIMARY CELL LINES SUITABLE FOR Cre-MEDIATED RECOMBINASE-MEDIATED CASSETTE EXCHANGE

Recently, site-specific nucleases (zinc-finger nucleases, ZFN; TAL effector nucleases; and CRISPR) emerged as powerful tools for gene modification of different cells types and enhanced green fluorescent protein (EGFP)-specific ZFN were successfully used in the rat (Geurtz et al. 2010) and in the pig (Watanabe et al. 2010; Whyte et al. 2010). Previously (Brunetti et al. 2008 Clon. Stem Cells), we generated an EGFP transgenic porcine line (Verro2GFP) characterised by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, Mendelian transgene transmission, and expression in F1. The aim of this work was to modify a transcriptionally active GFP-locus into one suitable for Cre-mediated recombinase-mediated cassette exchange (RMCE), using EGFP-specific ZFN. Homology arms for promoter-less targeting vector were derived from pCAGGS-EGFP vector (promoter fragment = left-homology-arm = LHA; polyA sequence = right-homology-arm = RHA). Cloning floxed (lox2272/lox5171) hygromycin resistance coding sequence between LHA and RHA sequences, we generated the targeting/RMCE vector (pB5′3′Hygro-PL) and its positive control (C+) for PCR set-up (100–1000 plasmid copies). Verro2GFP fibroblasts cultured in DMEM+M199(1 : 1) + 10% FCS, bFGF in 5% CO2, 5% O2, were transfected using Nucleofector (V-024 program). In ZFN-mediated gene targeting, 2 μg of each ZFN coding vector (Sigma-CompoZr®) and 2 μg of pB5′3′Hygro-PL/KpnI vector were used to “nucleofect” 1.4 × 106 Verro2GFP fibroblasts in 2 experiments. Transfected cells were plated in 20 Petri dishes (Ø = 150 mm) and cultured under hygromycin selection (200 μg mL–1) for 15 days. After 12 days of drug selection, 82 resistant colonies were picked up and expanded in 24 multiwell plates for SCNT. All colonies were PCR screened and 45 (54.9%) colonies were positive. Four colonies were used in zona-free SCNT experiments with 140 Day 6 compacted morulae/blastocysts transferred into 2 synchronized sows that both became pregnant. One pregnancy went to term and delivered 5 live animals and 5 stillborn with correct hygromycin cassette integration, detected by PCR. The PCR products were sequenced in 7 animals to verify integration of promoterless targeting vector and in all 7 sequenced samples we obtained a correct insertion without any substitution/deletion. Using hygromycin selection in these experiments, we demonstrated that ZFN-mediated gene targeting can be easily done with high efficiency and is compatible with living animals. Moreover, we have validated a feasible SCNT-tested platform for further Cre-mediated site-specific gene modifications