329 FLOW CYTOMETRIC ANALYSIS OF SPERMATOZOA FROM REPORTER TRANSGENIC BOARS DERIVED BY PRECISION GENETIC ENGINEERING

2013 ◽  
Vol 25 (1) ◽  
pp. 312
Author(s):  
W. Garrels ◽  
S. Holler ◽  
N. Cleve ◽  
S. Klein ◽  
Z. Ivics ◽  
...  
Keyword(s):  
Germ Line ◽  
Flow Cytometric Analysis ◽  
Embryonic Stem ◽  
Sleeping Beauty ◽  
Large Animal Model ◽  
Large Animal ◽  
Reporter Protein ◽  
Cassette Exchange ◽  
Flow Cytometric

Recently, we produced 2 founder boars with a non-autonomous Sleeping Beauty (SB) system carrying 3 monomeric integrations of a Venus transposon cassette and showing transgene segregation during meiosis (Garrels et al. 2011 PLoS One 6, e27563). It was possible to show transmission of the reporter protein to fertilized oocytes by confocal microscopy. The aim of this study was to assess the suitability of different fluorophore reporters for in vivo labelling of pig spermatozoa. Therefore, we used Venus transposon fibroblasts from a F1 boar, which carry a single integration of the transposon cassette and used these fibroblasts for a Cre-mediated cassette exchange against an mCherry reporter. These cells were used for somatic cell nuclear transfer (SCNT) to derive a syngene clone cohort of boars, which differ only in the fluorophore reporter cDNAs (either Venus or mCherry). Importantly, this methodology did not request any antibiotic selection cassette and allows precise genetic modifications in a livestock species where no authentic embryonic stem cells are available (Garrels et al. 2012 Trends in Biotechnology 30, 386–393). A total of 8 male piglets carrying the Venus transposon, and 4 male piglets carrying the mCherry reporter were born. Three Venus boars and 2 mCherry boars were raised to sexual maturity, and ejaculated sperm was obtained with the help of a phantom. A detailed flow cytometric analysis revealed that the spermatozoa samples were specifically Venus or mCherry positive [Gallios, Beckmann Coulter, Krefeld, Germany; solid-state laser (488 nm; 22 mW), filter for green fluorescence (525 BP); filter for red fluorescence: (620/30)], respectively. In direct comparative measurements, the spermatozoa samples from transgenic boars (Venus and Cherry) and wildtype controls could be discriminated. Interestingly, spermatozoa were uniformly Venus- or mCherry-positive and gave a distinct fluorescence peak in flow-cytometric measurements. The monomeric transgenes were transmitted through the germ line according to Mendelian rules with the expected ratio of 50% transgenic and 50% nontransgenic piglets. Fluorescence microscopic analysis and Western blotting confirmed the uniform presence of Venus and mCherry in boar spermatozoa, respectively. This is the first characterisation of spermatozoa from a pig cohort carrying a targeted cassette exchange. This large animal model may help to elucidate the function of paternally transmitted components to fertilized oocytes.

429 GENERATION OF Oct4-EGFP TRANSGENIC PIGS FOR MONITORING REPROGRAMMING AND PLURIPOTENCY

2010 ◽  
Vol 22 (1) ◽  
pp. 371 ◽  
Author(s):  
M. Nowak-Imialek ◽  
W. A. Kues ◽  
B. Petersen ◽  
A. Lucas-Hahn ◽  
D. Herrmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Embryonic Stem ◽  
Large Animal Model ◽  
Large Animal ◽  
Transgenic Pigs ◽  
Egfp Fluorescence

The Oct4 gene is an essential transcription factor for maintenance of pluripotency in mammals. Here, we report the production of cloned transgenic pigs carrying a genomic construct encompassing murine Oct4 regulatory regions and driving an enhanced green fluorescent protein (Oct4-EGFP) construct. We employed fetal porcine fibroblasts, stably co-transfected with neomycin and the mouse Oct4-EGFP construct, for somatic cell nuclear transfer to reconstruct transgenic embryos. The cloned embryos (811 embryos) were surgically transferred into the oviducts of 8 recipient animals. Two pregnancies were terminated at Day 25 for recovery of fetuses and the others delivered a total of 23 piglets, of which 11 survived the postpartum period. A detailed analysis showed that the Oct4-EGFP construct was active in cloned pig blastocysts from Days 5 to 6. EGFP fluorescence was found exclusively in the primordial germ cells of Day 25 fetuses, whereas somatic tissues did not express the transgene. We could also detect expression of Oct4-EGFP in individual cells of the postnatal testis. Testis-specific expression was confirmed by Northern blotting. We fused transgenic porcine fibroblasts with murine embryonic stem cells to analyze reactivation of the Oct4-EGFP transgene under experimental reprogramming conditions. The fused hybrids displayed stem cell morphology and a high proliferation rate and started to express EGFP fluorescence 72 h after fusion. In conclusion, we report the production of viable Oct4-EGFP transgenic piglets that express EGFP exclusively in germ line and pluripotent cells. This transgenic pig line is a valuable tool for derivation and maintenance of porcine embryonic stem cells and will be of utmost interest for reprogramming studies and for preclinical testing of stem cell therapies in a large animal model. Funded by BMBF.

scholarly journals A patient-like swine model of gastrointestinal fibrotic strictures for advancing therapeutics

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ling Li ◽  
Mohamad I. Itani ◽  
Kevan J. Salimian ◽  
Yue Li ◽  
Olaya Brewer Gutierrez ◽  
...  
Keyword(s):  
Argon Plasma Coagulation ◽  
Argon Plasma ◽  
Large Animal Model ◽  
Swine Model ◽  
Large Animal ◽  
High Grade ◽  
Gi Tract ◽  
Endoscopic Techniques ◽  
Approved Drugs

AbstractGastrointestinal (GI) strictures are difficult to treat in a variety of disease processes. Currently, there are no Food and Drug Administration (FDA) approved drugs for fibrosis in the GI tract. One of the limitations to developing anti-fibrotic drugs has been the lack of a reproducible, relatively inexpensive, large animal model of fibrosis-driven luminal stricture. This study aimed to evaluate the feasibility of creating a model of luminal GI tract strictures. Argon plasma coagulation (APC) was applied circumferentially in porcine esophagi in vivo. Follow-up endoscopy (EGD) was performed at day 14 after the APC procedure. We noted high grade, benign esophageal strictures (n = 8). All 8 strictures resembled luminal GI fibrotic strictures in humans. These strictures were characterized, and then successfully dilated. A repeat EGD was performed at day 28 after the APC procedure and found evidence of recurrent, high grade, fibrotic, strictures at all 8 locations in all pigs. Pigs were sacrificed and gross and histologic analyses performed. Histologic examination showed extensive fibrosis, with significant collagen deposition in the lamina propria and submucosa, as well as extensive inflammatory infiltrates within the strictures. In conclusion, we report a porcine model of luminal GI fibrotic stricture that has the potential to assist with developing novel anti-fibrotic therapies as well as endoscopic techniques to address recurring fibrotic strictures in humans.

scholarly journals Niobium Carbide Loaded by Macrophage for Targeted Phototherapy Ablation of Breast Cancer

2020 ◽  
Author(s):  
Chiara Da Pieve ◽  
Gabriela Kramer Marek ◽  
Jolanta Saczko ◽  
Anant Shah ◽  
Florian Raes
Keyword(s):  
Animal Study ◽  
Human Body ◽  
Targeted Delivery ◽  
Near Infrared ◽  
Niobium Carbide ◽  
Reactive Oxygen Species Generation ◽  
Large Animal Model ◽  
Large Animal ◽  
Photodynamic Effect

ABSTRACTAltough nanomaterial-mediated phototherapy has been extensively studied, the major antitumor success is limited to treating subcutaneous tumor on nude, lacking of clinically-relevant big animal study. Therefore, it is urgent to make further investigation on the typical big model, which is more closely related to the human body. In this work, niobium carbide (NbC) was selected as photoactive substance in virtue of its outstanding near infrared (NIR) absorption properties and resultantly NIR-triggered hyperthemia and reactive oxygen species generation for the synergetic photothermal and photodynamic effect. Moreover, macrophage was used as bio-carrier for the targeted delivery of NbC and the phagocytosis of macrophages was proved to be able to retain the photothermal/photodynamic effect of NbC. Resultantly, macrophage loaded NbC could realize complete removal of solid tumor on both of nude mice and big animal of rabbits. Meanwhile, two-dimensional ultrasound, shave wave elastography (SWE) and contrast-enhanced ultrasound (CEUS) have been applied for monitoring the physiological evolutions of in vivo tumor post treatment, which clearly disclosed the photoablation process of tumor and provided a new way for the surveillance of tumor on the big animal study. Hence, large animal model study in this work presented higher clinical significance than the previous studies.SignificanceFindings show that niobium carbide carried by macrophages can be used for targeted phototherapy. At the same time, we applied the rabbit tumor model which is closer to the human body microenvironment.

scholarly journals Intracoronary Delivery of Porcine Cardiac Progenitor Cells Overexpressing IGF-1 and HGF in a Pig Model of Sub-Acute Myocardial Infarction

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2571
Author(s):  
Cristina Prat-Vidal ◽  
Verónica Crisóstomo ◽  
Isabel Moscoso ◽  
Claudia Báez-Díaz ◽  
Virginia Blanco-Blázquez ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Large Animal Model ◽  
Cardiac Progenitor Cells ◽  
Large Animal ◽  
Insulin Growth Factor ◽  
Decellularized Extracellular Matrix ◽  
Preclinical Animal Models

Human cardiac progenitor cells (hCPC) are considered a good candidate in cell therapy for ischemic heart disease, demonstrating capacity to improve functional recovery after myocardial infarction (MI), both in small and large preclinical animal models. However, improvements are required in terms of cell engraftment and efficacy. Based on previously published reports, insulin-growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) have demonstrated substantial cardioprotective, repair and regeneration activities, so they are good candidates to be evaluated in large animal model of MI. We have validated porcine cardiac progenitor cells (pCPC) and lentiviral vectors to overexpress IGF-1 (co-expressing eGFP) and HGF (co-expressing mCherry). pCPC were transduced and IGF1-eGFPpos and HGF-mCherrypos populations were purified by cell sorting and further expanded. Overexpression of IGF-1 has a limited impact on pCPC expression profile, whereas results indicated that pCPC-HGF-mCherry cultures could be counter selecting high expresser cells. In addition, pCPC-IGF1-eGFP showed a higher cardiogenic response, evaluated in co-cultures with decellularized extracellular matrix, compared with native pCPC or pCPC-HGF-mCherry. In vivo intracoronary co-administration of pCPC-IGF1-eGFP and pCPC-HFG-mCherry (1:1; 40 × 106/animal), one week after the induction of an MI model in swine, revealed no significant improvement in cardiac function.

A novel in vivo large animal model of lumbar spinal joint degeneration

2018 ◽  
Vol 18 (10) ◽  
pp. 1896-1909 ◽  
Author(s):  
Tian Wang ◽  
Matthew H. Pelletier ◽  
Chris Christou ◽  
Rema Oliver ◽  
Ralph J. Mobbs ◽  
...  
Keyword(s):  
Animal Model ◽  
Large Animal Model ◽  
Large Animal ◽  
Joint Degeneration ◽  
Lumbar Spinal

scholarly journals Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 356-364 ◽  
Author(s):  
RF Carter ◽  
AC Abrams-Ogg ◽  
JE Dick ◽  
SA Kruth ◽  
VE Valli ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Autologous Transplantation ◽  
Large Animal Model ◽  
Multiple Infections ◽  
Large Animal ◽  
Retroviral Infection ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.

Exploration of cytotoxic and genotoxic endpoints following sub-chronic oral exposure to titanium dioxide nanoparticles

2019 ◽  
Vol 35 (9) ◽  
pp. 577-592 ◽  
Author(s):  
Srijita Chakrabarti ◽  
Danswrang Goyary ◽  
Sanjeev Karmakar ◽  
Pronobesh Chattopadhyay
Keyword(s):  
Titanium Dioxide ◽  
Titanium Dioxide Nanoparticles ◽  
Flow Cytometric Analysis ◽  
Raw 264.7 Cells ◽  
Oral Exposure ◽  
Chromosomal Breakage ◽  
Flow Cytometric ◽  
Higher Doses

Health hazards of titanium dioxide nanoparticles (TiO2-NPs) have raised severe concerns because of the paucity of information regarding the toxic effects among the population. In the present research, the in vitro and in vivo cytotoxic potential of TiO2-NPs were evaluated using flow cytometric techniques. Further, in vitro and in vivo genotoxic endpoints were estimated by means of comet, micronucleus (MN), and chromosomal aberration (CA) assays. In vitro analysis was performed at the concentration range of 10–100 µg/mL using murine RAW 264.7 cells. In vivo experiments were conducted on Albino mice (M/F) by exposing them to 200 and 500 mg/kg TiO2-NPs for 90 days. Decreased percentage of cell viability with higher doses of TiO2-NPs was evident in both in vitro and in vivo flow cytometric analysis. Further, an impaired cell cycle (G0/G1, S, and G2/M) was reflected in the present investigation following the exposure to TiO2-NPs. Increased comet scores such as tail length, % DNA in tail, tail moment, and olive moment were also observed with the higher doses of TiO2-NPs in vitro and in vivo comet assays. Finally, the in vivo MN and CA assays revealed the formation of MN and chromosomal breakage following the exposure to TiO2-NPs.

scholarly journals Light-degradable hydrogels as dynamic triggers for gastrointestinal applications

2020 ◽  
Vol 6 (3) ◽  
pp. eaay0065 ◽  
Author(s):  
Ritu Raman ◽  
Tiffany Hua ◽  
Declan Gwynne ◽  
Joy Collins ◽  
Siddartha Tamang ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Implantable Devices ◽  
Large Animal Model ◽  
Large Animal ◽  
Gi Tract ◽  
Biomedical Device ◽  
Precise Tool ◽  
And Performance

Triggerable materials capable of being degraded by selective stimuli stand to transform our capacity to precisely control biomedical device activity and performance while reducing the need for invasive interventions. Here, we describe the development of a modular and tunable light-triggerable hydrogel system capable of interfacing with implantable devices. We apply these materials to two applications in the gastrointestinal (GI) tract: a bariatric balloon and an esophageal stent. We demonstrate biocompatibility and on-demand triggering of the material in vitro, ex vivo, and in vivo. Moreover, we characterize performance of the system in a porcine large animal model with an accompanying ingestible LED. Light-triggerable hydrogels have the potential to be applied broadly throughout the GI tract and other anatomic areas. By demonstrating the first use of light-degradable hydrogels in vivo, we provide biomedical engineers and clinicians with a previously unavailable, safe, dynamically deliverable, and precise tool to design dynamically actuated implantable devices.

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