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Background & aims: Clinical pig islet transplantation for the treatment of type I diabetes is still hindered by obvious immunological considerations and less investigated physiological incompatibilities. Cellular encapsulation, donor genetic engineering and host immunomodulation can help improve islet survival in a xenotransplantation context but adequate insulin output from transplanted islets remains the ultimate goal to be achieved for islet transplantation to be clinically efficient. We have previously shown that adenovirus-driven expression of a cassette carrying a dipeptidyl peptidase-resistant form of glucagon-like-peptide-1 (GLP1) and a constitutively activated form of type 3 muscarinic receptor (M3R) in isolated neonatal and adult pig islets significantly increases their secretory response to in vitro glucose stimulation that is otherwise 4-10 times lower than human islets. Our aim in the current study was to replicate our previous results in a transgenic pig model with beta-cell specific expression of our GLP1M3R cassette, to characterize in vivo and in vitro islet function of these pigs and to verify whether their offspring exhibit the same improved insulin secretion as founder animals. Material & methods: Cloned transgenic pigs were produced using Talens technology and validated by TLA sequencing analyses. Pancreata were collected from 14-day old piglets. Insulin secretion in response to different stimuli was evaluated during dynamic islet perifusion experiments. After reaching adult age, selected animals were subjected to IVGTT to study in vivo islet function. Transgenic animals were then bred and islet function of their offspring was studied as for founder animals. Results: Transgenic cloned pig founders (male and female) expressing the GLP1M3R cassette at the beta cell level were successfully obtained. Islets isolated from these piglets showed a 5.5 to 7.5-fold increase of their insulin output upon stimulation with 15 mM glucose. They maintained regul

Recently site specific nucleases (ZFN, Tal Effector and CRISPR) emerged as powerful tools for gene modification of different cells types and EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in 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) characterized 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 RMCE, using EGFP-specific ZFNs. 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) DsRedT4 reporter gene between LHA and RHA sequences, we generated the targeting/RMCE vector (pB50 30 DsRed4-PL) and its positive control (C?) for PCR set-up (100–1,000 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 ZFNs-mediated gene targeting, 2 lg of each ZFNs coding vectors (Sigma-CompoZr) and 2 lg of pB50 30 RedT4-PL/NotI vector were used to 00nucleofect00 1x106 Verro2GFP fibroblasts. Part of transfected cells were plated (3,000 cells/plate) in 10 Petri dishes ([ = 100 mm) and cultured without selection till well growing colonies appeared. Four RFP?/GFPcolonies were identified using epifluorescence, re-plated in two 150 mm dishes/colony and PCR verified. Finally 3 colonies were used in zona-free SCNT to produce reconstructed embryos, and 159 D6 blastocysts/compacted morulae were transferred into two synchronized sows that both became pregnant. One pregnancy is ongoing, another was interrupted at D58 and 7 fetuses (61.14 g ± 17.98 g) were used to generate RFP primary cell lines, finally verified b

Site-specific nucleases (ZFN, Tal effector nucleases and CRISPRs) boosted the genome editing of different species, and EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in pig (Watanabe et al. 2010, Whyte et al. 2010). Previously (Brunetti et al., 2008) we generated an EGFP transgenic porcine line (V2G) characterized 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 generate live cloned piglets using modified cells after two site-specific modifications (ZFNs and RMCE) on the transcriptionally active GFP-locus (V2G), avoiding any cell rejuvenation by SCNT. Homology arms for promoterless targeting vector were derived from pCAGGSEGFP vector (promoter fragment = Left-Homology-Arm = LHA; polyA sequence = Right-Homology-Arm = RHA). Cloning floxed (lox2272/lox5171) puromycin and hygromycin resistance coding sequences between LHA and RHA sequences, and we generated the targeting/RMCE vectors (pB50 30 Puro-PL and pB50 30 Hygro-PL) and theirs positive controls (C+) for PCR set-up (100–1,000 plasmid copies). V2G 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 lg of each ZFNs coding vectors (Sigma-CompoZr ) and 2 lg of pB50 30 Puro-PL/KpnI vector, were used to ‘nucleofect’ 0.9 9 106 Verro2GFP fibroblasts. Transfected cells were cultured under puromycin selection (2 lg/ml) for 8 days, and 0.45 9 106 cells were used for the Cre-mediated cassette exchange (1 lg pB50 30 Hygro-PL/KpnI + 2 lg pCAG:CreEGFP). Transfected cells were plated in 10 Petri dishes (Ø = 150 mm) and cultured under hygromycin selection (200 lg/ml) for 12 days, picked up and expanded in 24 multi-well plates for SCNT. Thirty-one colonies were PCR screened for hygromycin and 16 (51.6 %) colonies were positive for Cre-mediated targeted insertion. Four colonies were use

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of trea

We previously generated EGFP transgenic porcine line (Verro2GFP) characterized by a single integration of pCAGGSEGFP cassette, high ubiquitous EGFP expression, mendelian transgene transmission and expression in F1 (Brunetti et al. 2008). Recently Zinc Finger Nucleases (ZFN) and TALENs emerged as powerful tools for gene modification of different cells types and Recombinase Mediated Cassette Exchange (RMCE) was widely tested in different species. Moreover EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in pig (Watanabe et al. 2010, Whyte et al. 2010). The purposes of our work were: a) to insert a vector suitable for RMCE into a transcriptionally active locus and b) to assess the possibility to obtain good quality cell colonies to be used in Somatic Cell Nuclear Transfer (SCNT) even after two sequential transgenic modifications (ZFN and RMCE), considering the finite life span of Verro2GFP primary fibroblasts. EGFP-specific ZFNs were purchased from Sigma (CompoZr®) and the promoter-less targeting vectors were created using a fragment of the promoter (Left-Homology-Arm = LHA) and the polyA sequence (Right-Homology-Arm = RHA) of pCAGGSEGFP expression vector. Cloning floxed (lox2272/lox5171) reporter genes (PuromycinR, HygromycinR ) between these homology sequences (LHA and RHA), we generated 2 targeting/exchanging vectors (pB53Puro-PL, pB53Hygro-PL) and their positive controls (C+). PCR on C+ was set up to obtain minimal sensitivity of 100–1000 plasmid copies. Verro2GFP fibroblasts (primary and colonies) cultured in DMEM + M199 (1:1) + FBS(10 %), 5 % CO2,5%O2, were transfected using Nucleofector (V-24 program) and selected starting at 48 h after transfection. Colonies were picked up at 8th selection day and expanded in 24-well plates for PCR screening, RMCE and/or SCNT. In ZFNs-mediated gene targeting, 2 μg of each ZFNs coding vectors (pZFN1 and pZFN2) and 2 μg of pB53Puro-PL/ KpnI vector were used to “nucleofect” primary Verro2GFP fibroblasts (3

Introduction. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two forms: sporadic and familial, the latter linked to mutations in the SOD1 gene. As employment of transgenic SOD1 rodent models in ALS research didn’t result in an improvement of patient prognosis, another model, more closely related to human species, is strongly demanded by the scientific community. On this basis, our group produced, by Genetic Engineering and SCNT, transgenic blastocysts and swine carrying the hSOD1G93A mutation, which is the most frequently studied in rodents, since it reproduces patients phenotype progression. Materials and Methods. SCNT blastocysts on fifth day of development were transplanted by midventral laparatomy to the synchronized sows uterus. A cesarean delivery was performed at the 114th day of gestation. In order to achieve a preliminary characterization of our swine model, tissue banking was performed on stillborn piglets and on animals that died soon after birth. Immunocytochemistry on ear biopsy fibroblasts and western blot on homogenized snap-shot frozen tissues (rabbit policlonal antibody 07–403 Millipore, concentration 1:200 and 1:1000 respectively) were performed. To assess SOD1G93A deposition pattern Immunohistochemistry (rabbit policlonal antibody GTX 100659; 1:250) and Immunofluorescence (GTX 100659; 1:250 and NeuN MAB377; 1:1000) were employed on FFPE tissues. Genomic SOD1G93A swine DNA digested by SalI+BglII (10 U/μg DNA) was hybridized with SOD-DIG probes (20 ng/ml) to assess transgene integrations number by Southern Blot. Results and Conclusions. The transfer of 638 embryos to eight recipient sows resulted in four pregnancies and in the birth of 16 vital and 12 stillborn piglets (mean blastocyst development to term efficacy, 8.78%). Five animals developed normally while the remaining piglets died due to events commonly reported in commercial herds. The transgenic protein expression was confirmed by both immunocytochemi

Introduction. ALS is a fatal neurodegenerative disease that occurs in two forms: sporadic and familial, the latter linked to mutations in the SOD1 gene. Pathogenic hypotheses mainly rely on transgenic rodent models however doubts have been raised about their suitability to faithfully reproduce the human disease. Therefore, a more suitable model is strongly demanded to provide a better tool to study the disease and to facilitate the preclinical findings extrapolation. Swine model plays an emerging role in biomedical research as its anatomical, physiological and biochemical features are more closely related to human species. On this basis, our group produced, by Genetic Engineering and SCNT, transgenic swine blastocysts carrying the hSOD1G93A mutation, which is the most frequently studied in rodents, since it reproduces the ALS patients phenotype progression. Material and Methods. Using the Multisite Gateway System (Invitrogen) we obtained the “EntryClone” pENTRL1L2- hSODG93A, containing the cDNA coding the hSOD1G93A gene whose open reading frame was confirmed by sequencing, and the “DestinationVector” pMGOrfA5¢3’MARpuro5171. The exchange reaction between the “DestinationVector” and the “EntryClone” was mediated by LR Clonase-Invitrogen, and was used to transform chemically competent E.coli cells (OneShotMatch1-Invitrogen). The resulting “ExpressionVector” pMG5¢3’MARPuro-hSODG93A was purified and analyzed. The same construct was successfully used to develop an ubiquitous EGFP expression vector that maintains high expression level through the next generation of pigs. This vector carries the pCAGGS hybrid promoter (CMV-IE enhancer + chicken β-actin promoter) inserted between two insulators (5′MAR of chicken lysozyme gene) to prevent positional or copy number silencing effects. 5 µg of linearized vector were used to transfect 1 × 106 Pig Adult Fibroblast (PAF) cultured in DMEM/ M199[1:1] + 10%FCS+1% β-FGF by Nucleofector (Amaxa Biosystem). Transgene expression was detect

Due to the increasing demand of organ for transplantation and the shortage of human donors several areas of research, including regenerative medicine and xenotransplantation, are currently explored to provide different clinical solutions to a varieties of pathological conditions. While regenerative medicine will relay on stem cell auto-regeneration and/or cell transplantation in partially compromised tissues and organs, xenotransplantation would provide ready to use tissues or organs for more severe pathologies or organ failures. Genetic engineering of porcine genome lies at the heart of xenotransplantation research since the pig is currently considered, on the risk/benefit ratio, the most appropriate species. The stepsinvolved in the creation of candidate animals for xenotransplantation research include the selection of a somatic cell line (usually fibroblasts) that is engineered using current technologies, either for the inactivation or for the insertion of candidate genes, followed by somatic cell nuclear transfer (SCNT) procedure to generate live animals. The founder animals obtained can be suitable directly for xenotransplantation and/or their cells can be further engineered. In our laboratory we have been working primarily with a male GAL-KO miniature pig cell line (provided by D. Sachs, MGH, Boston USA) with the aim of overexpressing under an ubiquitous promoter (pCGACGS) several transgenes controlling complement mediated lysis and inflammation (hCD55, hCD39), and coagulation (hEPCR, hTM). To speed up the process we cotransfected by nucleofection two transgenes at the same time, one of which carried the selectable marker (hCD55HygromycinR ? hCD39; hEPCRPuromycinR ? hTM). Forty-eight hours later the cells were subjected to drug selection for 15 days to isolate resistant cell clones that were expanded in duplicates for SCNT and phenotypic characterisation by Western blot and immunocitochemistry to assess the presence of the protein and the uniform expression of

It is important to understand the relative contribution of primary and secondary particles to regional and global aerosol so that models can attribute aerosol radiative forcing to different sources. In large-scale models, there is considerable uncertainty associated with treatments of particle formation (nucleation) in the boundary layer (BL) and in the size distribution of emitted primary particles, leading to uncertainties in predicted cloud condensation nuclei (CCN) concentrations. Here we quantify how primary particle emissions and secondary particle formation influence size-resolved particle number concentrations in the BL using a global aerosol microphysics model and aircraft and ground site observations made during the May 2008 campaign of the European Integrated Project on Aerosol Cloud Climate Air Quality Interactions (EUCAARI). We tested four different parameterisations for BL nucleation and two assumptions for the emission size distribution of anthropogenic and wildfire carbonaceous particles. When we emit carbonaceous particles at small sizes (as recommended by the Aerosol Inter-comparison project, AEROCOM), the spatial distributions of campaign-mean number concentrations of particles with diameter >50 nm (N(50)) and >100 nm (N(100)) were well captured by the model (R(2)>= 0.8) and the normalised mean bias (NMB) was also small (-18% for N(50) and -1% for N(100)). Emission of carbonaceous particles at larger sizes, which we consider to be more realistic for low spatial resolution global models, results in equally good correlation but larger bias (R(2)>= 0.8, NMB = -52% and -29%), which could be partly but not entirely compensated by BL nucleation. Within the uncertainty of the observations and accounting for the uncertainty in the size of emitted primary particles, BL nucleation makes a statistically significant contribution to CCN-sized particles at less than a quarter of the ground sites. Our results show that a major source of uncertainty i, authorCount :41

Over-expression of human CD39 in transgenic pigs is a potential strategy to bypass acute vascular rejection in xenotransplantation. The aim of this work is the production of transgenic cloned pigs using a Gal -/- CD55/CD39 cell line. A neonatal pig Gal -/- fibroblast line cultured in DMEM/M199 1:1 + 10% FCS + 5 ng/ml bFGF was co-transfected by nucleofection with two ubiquitous expression vectors, the first carrying hCD55 under Elongation Factor promoter and a HygroR cassette; the second carrying hCD39 under pCAGGS promoter and a 3¢ MAR region. After nucleofection, cells were plated in Petri dishes and selected with Hygromycin B for 8 days. Drug resistant colonies were isolated and expanded for transgene expression analysis. We used immunohistochemistry (IHC) to detect the expression of the proteins. For hCD55 we used IA10 and for hCD39 BU61. Cells co-expressing CD55–CD39 were serum starved for 24 h before being fused to enucleated oocytes. Following electric activation, embryos were grown in vitro up to compact morula/blastocyst and all (n=144) were transplanted in two synchronized sows. PAEC and fibroblasts derived from delivered piglets were analysed with FACS, using the following antibodies: BRIC110, IH4, 2G2, and MEM-118 for hCD55 and TU66 for hCD39 detection respectively. Using a double transgenic CD55/CD39 Gal -/- colony in a Somatic Cell Nuclear Transfer (SCNT) experiment we have obtained 35.4% compacted morula/blastocyst development. One of two sows resulted in a pregnancy. At day 117 of gestation, this sow was induced to farrowing and delivered two stillborn piglets that were probably too immature and died from respiratory failure. Nevertheless, IHC analysis performed on PAEC and fibroblasts derived from these piglets showed strong expression of CD55– CD39, as in the original colony. FACS analysis confirmed robust human CD39 expression but showed a very low level of hCD55 expression. Two Gal -/- CD55/CD39 piglets were obtained. Over-expression of hCD39 seem