scholarly journals Sperm-mediated gene transfer

2006 ◽  
Vol 18 (2) ◽  
pp. 19 ◽  
Author(s):  
Marialuisa Lavitrano ◽  
Marco Busnelli ◽  
Maria Grazia Cerrito ◽  
Roberto Giovannoni ◽  
Stefano Manzini ◽  
...  
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Small Animal ◽  
Ease Of Use ◽  
Large Animal ◽  
Transgenic Pigs ◽  
Exogenous Dna ◽  
Large Animal Models

Since 1989, a new method for the production of transgenic animals has been available, namely sperm-mediated gene transfer (SMGT), based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA molecules and to transfer them into the oocyte at fertilisation. We first described the SMGT procedure in a small animal model, with high efficiency reported in the mouse. In addition, we successfully adapted and optimised the technique for use in large animals; it was, in fact, highly efficient in the generation of human decay accelerating factor transgenic pig lines, as well as multigene transgenic pigs in which three different reporter genes, namely enhanced green fluorescent protein, enhanced blue fluorescent protein and red fluorescent protein, were introduced. The major benefits of the SMGT technique were found to be its high efficiency, low cost and ease of use compared with other methods. Furthermore, SMGT does not require embryo handling or expensive equipment. Sperm-mediated gene transfer could also be used to generate multigene transgenic pigs that would be of benefit as large animal models for medical research, for agricultural and pharmaceutical applications and, in particular, for xenotransplantation, which requires extensive genetic manipulation of donor pigs to make them suitable for grafting to humans.

scholarly journals Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 259-272 ◽  
Author(s):  
Francisco A García-Vázquez ◽  
Salvador Ruiz ◽  
Carmen Matás ◽  
M José Izquierdo-Rico ◽  
Luis A Grullón ◽  
...  
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Reactive Oxygen Species Generation ◽  
Reca Protein ◽  
Transgenic Animals ◽  
Membrane Lipid ◽  
Transgenic Pigs ◽  
Exogenous Dna

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA–DNA complexes at 5 μg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT–ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia

Blood ◽  
2003 ◽  
Vol 102 (7) ◽  
pp. 2403-2411 ◽  
Author(s):  
Anja Ehrhardt ◽  
Hui Xu ◽  
Aaron M. Dillow ◽  
Dwight A. Bellinger ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Adenoviral Vector ◽  
High Efficiency ◽  
Liver Toxicity ◽  
Normal Liver ◽  
Adenoviral Vectors ◽  
Factor Ix ◽  
Hemophilia B ◽  
Large Animal ◽  
Large Animal Models

Abstract Many approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Recombinant adenoviral vectors offer high-efficiency transfer of an episomal vector but have been plagued by the cytotoxicity/immunogenicity of early-generation vectors that contain viral genes. In our current study, we have used a nonintegrating helper-dependent (HD) adenoviral vector for liver-directed gene transfer to achieve hemostatic correction in a dog with hemophilia B. We measured plasma canine factor IX (cFIX) concentrations at a therapeutic range for up to 2.5 months and normalization of the whole blood clotting time (WBCT) for about a month. This was followed by a decrease and stabilized partial correction for 4.5 months. Hepatic gene transfer of a slightly lower dose of the HD vector resulted in WBCTs that were close to normal for 2 weeks, suggesting a dose threshold effect in dogs. In sharp contrast to other studies using first- or second-generation adenoviral vectors, we observed no vector-related elevation of liver enzymes, no fall in platelet counts, and normal liver histology. Taken together, this study demonstrates that injection of an adenoviral HD vector results in complete but transient phenotypic correction of FIX deficiency in canine models with no detectable toxicity. (Blood. 2003;102:2403-2411)

Animal models of hypoxic-ischemic encephalopathy: optimal choices for the best outcomes

2017 ◽  
Vol 28 (1) ◽  
pp. 31-43 ◽  
Author(s):  
Lan Huang ◽  
Fengyan Zhao ◽  
Yi Qu ◽  
Li Zhang ◽  
Yan Wang ◽  
...  
Keyword(s):  
Animal Models ◽  
Small Animal ◽  
Underlying Disease ◽  
Therapeutic Interventions ◽  
Hypoxic Ischemic Encephalopathy ◽  
Large Animal ◽  
Large Animal Models ◽  
Neurological Research ◽  
Serious Disease ◽  
Ischemic Encephalopathy

AbstractHypoxic-ischemic encephalopathy (HIE), a serious disease leading to neonatal death, is becoming a key area of pediatric neurological research. Despite remarkable advances in the understanding of HIE, the explicit pathogenesis of HIE is unclear, and well-established treatments are absent. Animal models are usually considered as the first step in the exploration of the underlying disease and in evaluating promising therapeutic interventions. Various animal models of HIE have been developed with distinct characteristics, and it is important to choose an appropriate animal model according to the experimental objectives. Generally, small animal models may be more suitable for exploring the mechanisms of HIE, whereas large animal models are better for translational studies. This review focuses on the features of commonly used HIE animal models with respect to their modeling strategies, merits, and shortcomings, and associated neuropathological changes, providing a comprehensive reference for improving existing animal models and developing new animal models.

61 EFFICIENCY OF TWO ENUCLEATION METHODS FOR THE PRODUCTION OF TRANSGENIC PIG EMBRYOS BY HANDMADE CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 148 ◽  
Author(s):  
J. Li ◽  
Y.-H. Zhang ◽  
Y.-T. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
...  
Keyword(s):  
High Efficiency ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Transgenic Pig ◽  
Transgenic Pigs ◽  
Uv Illumination ◽  
Polar Bodies ◽  
Transgenic Embryos ◽  
Handmade Cloning

Since the successful production of transgenic pigs by somatic nuclear transfer (Lai et al. 2002 Science 295, 1089–1092), more efficient reproduction technologies for transgenic pigs have been in demand. The purpose of our work was to develop an efficient method for production of transgenic embryos by handmade cloning (HMC; Vajta et al. 2001 Cloning 3, 89–95) connected to oriented enucleation to eliminate potential harm of staining and UV illumination at cytoplast selection. After 41–42 h of in vitro maturation, oocytes were further cultured with or without 0.4 µg mL−1 demecolcine for 45 min (i.e. chemically assisted handmade enucleation (CAHE) vs. oriented handmade enucleation (OHE)). Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with visible extrusion cones or polar bodies attached to the surface were subjected to oriented bisection. The putative cytoplasts without extrusion cones or polar bodies, containing the major part of cytoplasm, were selected as the recipients. Two cytoplasts were electro-fused with one transgenic fibroblast expressing either amyloid precursor protein (APP) or green fluorescent protein (GFP), while non-transgenic fibroblasts were used as control nuclear donors. After activation (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205), reconstructed embryos were cultured in porcine zygote medium-3 for 7 days. The rates of cleavage and blastocyst cell numbers were recorded on Day 2 and Day 7, respectively. In 5 replicates, the correct bisection efficiency achieved with CAHE was higher compared to that with the OHE method (93 ± 1% vs. 82 ± 2%, respectively; P < 0.05). Table 1 shows that blastocyst rates with APP and GFP transgenic fibroblasts as nuclear donors after CAHE were lower (P < 0.05) compared to those with the OHE method; in contrast, cleavage rates of embryos from different fibroblast donors were similar and so were blastocyst rates of non-transgenic donors after either CAHE or OHE. Our results show that embryos reconstructed from APP and GFP transgenic donors have compromised in vitro developmental rates after CAHE rather than after the OHE method; however, a high efficiency with both enucleation methods was observed when using non-transgenic somatic cells. Table 1.Comparison of two enucleation methods for the production of transgenic pig embryos

scholarly journals Intratracheal aerosolization of viral vectors to newborn pig airways

BioTechniques ◽  
2020 ◽  
Vol 68 (5) ◽  
pp. 235-239
Author(s):  
Ashley L Cooney ◽  
Patrick L Sinn
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Endotracheal Tube ◽  
Viral Vectors ◽  
Small Animal ◽  
Large Animal ◽  
Large Animal Models ◽  
Airway Diseases ◽  
Efficient Delivery ◽  
Large Animals

Gene therapy for airway diseases requires efficient delivery of nucleic acids to the airways. In small animal models, gene delivery reagents are commonly delivered as a bolus dose. However, large animal models are often more relevant for the transition from preclinical studies to human trials. Aerosolizing viral vectors to the lungs of large animals can maximize anatomical distribution. Here, we describe a technique for aerosolization of viral vectors to the airways of newborn pigs. Briefly, a pig is anesthetized and intubated with an endotracheal tube, and a microsprayer is passed through the endotracheal tube. A fine mist is then sprayed into the distal trachea. Widespread and uniform distribution of transgene expression is critical for developing successful lung gene therapy treatments.

scholarly journals The Representative Porcine Model for Human Cardiovascular Disease

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Yoriyasu Suzuki ◽  
Alan C. Yeung ◽  
Fumiaki Ikeno
Keyword(s):  
Animal Models ◽  
Porcine Model ◽  
Small Animal ◽  
Large Animal ◽  
Murine Models ◽  
Practical Applications ◽  
Large Animal Models ◽  
Small Animal Models ◽  
Insight Into

To improve human health, scientific discoveries must be translated into practical applications. Inherent in the development of these technologies is the role of preclinical testing using animal models. Although significant insight into the molecular and cellular basis has come from small animal models, significant differences exist with regard to cardiovascular characteristics between these models and humans. Therefore, large animal models are essential to develop the discoveries from murine models into clinical therapies and interventions. This paper will provide an overview of the more frequently used large animal models, especially porcine models for preclinical studies.

Rabbit Anti-Thymocyte Globulin (rATG) Administrated Concomitantly with Liver Delivery of AAV2-hFIX Can Promote Inhibitor Formation In Rhesus Macaques.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3765-3765 ◽  
Author(s):  
Jonathan D. Finn ◽  
Patricia Favaro ◽  
J. Fraser Wright ◽  
Federico Mingozzi ◽  
Katherine A. High ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Neutralizing Antibodies ◽  
Rhesus Macaques ◽  
Rescue Therapy ◽  
Factor Ix ◽  
Large Animal ◽  
Solid Organ ◽  
Large Animal Models ◽  
Human Factor Ix

Abstract Abstract 3765 Adeno-associated viral (AAV) vectors are one of the most extensively studied vector platforms for gene therapy applications. Our group is currently developing AAV vectors for the therapeutic treatment of hemophilia B (HB) in humans. The first clinical trial using an AAV2 vector to express human Factor IX (hFIX) (AAV2-hFIX16) from the liver of HB patients revealed a cytotoxic T lymphocyte (CTL) response directed against AAV capsid that occurred 4–6 weeks following treatment that was associated with a decline in transgene expression. Thus, immunosuppressive (IS) therapies may be required during AAV2 vector administration at high doses to prevent or to halt the immune mediated destruction of transduced hepatocytes. Previous work in murine and non-human primate (NHP) models has shown that sustained AAV-mediated expression of transgenes can induce tolerance, and that this is in part, dependent on CD4+ CD25+ FoxP3+ regulatory T cells (Tregs). Here we investigate the safety of a Treg sparing anti-T cell IS regimen in the context of liver mediated AAV2 gene transfer. Rabbit anti-thymocyte globulin (rATG) is an immune suppressive drug that is used in solid organ transplant and autoimmune disease. rATG has been shown to dramatically deplete the majority of T-cells, however some studies have shown that rATG spares Tregs and can induce tolerance in human T cells. rATG was administered to rhesus macaques (along with an 8-week course of Mycophenolate Mofetil (MMF) and sirolimus) either at the time of AAV vector administration (AAV2-hFIX16), or 5 weeks post-vector administration (rescue therapy). The administration of ATG at week 5 had no detrimental effect on hFIX expression and was not associated with inhibitor formation (n=3) indicating that rATG might be safe to use as an IS ‘rescue' agent, after the detection of an ongoing immune response against transduced cells. Interestingly we observed that early administration of rATG prevented tolerance induction and resulted in inhibitor formation in 2 of 3 animals upon withdrawal of IS. The inhibitor formation was associated with transient elevations in circulating levels of IL-2, IL-4, IL-10 and IFN-g. These results are comparable to previous findings in NHP using an anti-CD25 IS regimen (Daclizumab) at the time of vector administration (Blood 2007, 110(7):2334-41). We conclude that the timing of IS regimens is critical, and that IS regimens that alter the numbers, frequency, and/or function of T-cells at the time of vector administration can result in neutralizing antibodies (inhibitors) to the transgene product (hFIX). These data suggest that there might be multiple mechanisms responsible for maintaining tolerance in this model, and that Tregs alone might not be sufficient. This study highlights the critical need for safety studies in large animal models of potential immune suppressive regimens in the context of gene transfer before translating to the clinic. Disclosures: High: Genzyme, Inc: Consultancy, Patents & Royalties; Third Rock Ventures: Consultancy; Novo-Nordisk: Consultancy; Shire, Inc.: Consultancy.

Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer

2005 ◽  
Vol 72 (1) ◽  
pp. 68-76 ◽  
Author(s):  
Nicole L. Webster ◽  
Monica Forni ◽  
Maria Laura Bacci ◽  
Roberto Giovannoni ◽  
Riccardo Razzini ◽  
...  
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Fluorescent Proteins ◽  
Transgenic Pigs

scholarly journals A Review of the Current Mammalian Models of Alzheimer’s Disease and Challenges That Need to Be Overcome

2021 ◽  
Vol 22 (23) ◽  
pp. 13168
Author(s):  
Natasha Elizabeth Mckean ◽  
Renee Robyn Handley ◽  
Russell Grant Snell
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Animal Models ◽  
Mouse Model ◽  
Mouse Models ◽  
Small Animal ◽  
Large Animal ◽  
Complex Disorders ◽  
Large Animal Models ◽  
The Impact

Alzheimer’s disease (AD) is one of the looming health crises of the near future. Increasing lifespans and better medical treatment for other conditions mean that the prevalence of this disease is expected to triple by 2050. The impact of AD includes both the large toll on individuals and their families as well as a large financial cost to society. So far, we have no way to prevent, slow, or cure the disease. Current medications can only alleviate some of the symptoms temporarily. Many animal models of AD have been created, with the first transgenic mouse model in 1995. Mouse models have been beset by challenges, and no mouse model fully captures the symptomatology of AD without multiple genetic mutations and/or transgenes, some of which have never been implicated in human AD. Over 25 years later, many mouse models have been given an AD-like disease and then ‘cured’ in the lab, only for the treatments to fail in clinical trials. This review argues that small animal models are insufficient for modelling complex disorders such as AD. In order to find effective treatments for AD, we need to create large animal models with brains and lifespan that are closer to humans, and underlying genetics that already predispose them to AD-like phenotypes.

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.

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