scholarly journals 308 DEVELOPMENT OF TRANSGENIC LIVESTOCK WITH REDUCED MYOSTATIN EXPRESSION USING RNA INTERFERENCE

2009 ◽  
Vol 21 (1) ◽  
pp. 251
Author(s):  
K. Tessanne ◽  
T. Stroud ◽  
C. Long ◽  
G. Hannon ◽  
S. Sadeghieh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Lentiviral Vector ◽  
Muscle Growth ◽  
Negative Regulator ◽  
Large Animal ◽  
Large Animal Models ◽  
Transgenic Livestock ◽  
Fetal Fibroblasts ◽  
Wide Range

RNA interference (RNAi) is a means of regulating gene expression by targeting mRNA in a sequence-specific manner for degradation or translational inhibition. Short hairpin RNAs (shRNAs) and siRNAs have been extensively employed for manipulating gene expression in a wide range of species. However, the great majority of this work has involved in vitro studies with cells grown in culture. Our goal for this project is to produce transgenic livestock in which myostatin, a negative regulator of muscle growth, has been targeted for silencing by RNAi. In theory, livestock in which myostatin has been silenced should exhibit increased muscle growth and development. To that end, we designed shRNAs to target the bovine myostatin mRNA sequence. The shRNAs were cloned into a lentiviral vector that contains a cytomegalovirus promoter controlling green fluorescent protein and shRNA expression as well as neomycin resistance. Infective lentivirus was made in HEK293T cells through co-transfection of the lentiviral vector, a packaging plasmid, and a plasmid expressing the VSVG pseudotype. Bovine fetal fibroblasts were transduced, selected using Geneticin®, and nuclear transfer was utilized to produce cloned transgenic embryos. There were 186 fusion attempts resulting in 160 fused embryos (fusion rate = 86%). Of these, 54 reached the blastocyst stage (34%) and 10 embryos were transferred into 5 recipient females (2 embryos per recipient). At 40 days, ultrasound revealed 1 confirmed pregnancy. Current plans are to harvest this fetus at 90 days and analyze it for evidence of myostatin knockdown. The production of transgenic animals exhibiting myostatin knockdown through lentiviral-mediated RNAi will demonstrate the utility of RNAi in the study of gene function in large animal models without the need for homologous recombination techniques, which are currently inefficient in species other than mice.

scholarly journals Influence of hyperthyroid conditions on gene expression in extraocular muscles of rats

2009 ◽  
Vol 37 (3) ◽  
pp. 231-238 ◽  
Author(s):  
Thomas S. Postler ◽  
Murat T. Budak ◽  
Tejvir S. Khurana ◽  
Neal A. Rubinstein
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Protein Localization ◽  
Muscle Growth ◽  
Extraocular Muscles ◽  
Negative Regulator ◽  
Mrna Levels ◽  
Sprague Dawley ◽  
Wide Range ◽  
Canonical Pathways

Extraocular muscles (EOMs) are a highly specialized type of tissue with a wide range of unique properties, including characteristic innervation, development, and structural proteins. Even though EOMs are frequently and prominently affected by thyroid-associated diseases, little is known about the direct effects of thyroid hormone on these muscles. To create a comprehensive profile of changes in gene expression levels in EOMs induced by thyroid hormone, hyperthyroid conditions were simulated by treating adult Sprague-Dawley rats with intraperitoneal injections of the thyroid hormone 3,3′,5-triiodo-l-thyronine (T3); subsequently, microarray analysis was used to determine changes in mRNA levels in EOMs from T3-treated animals relative to untreated control animals. The expression of 468 transcripts was found to be significantly altered, with 466 of these transcripts downregulated in EOMs from T3-treated animals. The biological processes into which the affected genes could be grouped included cellular metabolism, transport, biosynthesis, protein localization, and cell homeostasis. Moreover, 15 distinct biochemical canonical pathways were represented among the genes with altered transcription levels. Strikingly, myostatin ( Gdf8), a potent negative regulator of muscle growth, was found to be strongly downregulated in EOMs from T3-treated animals. Together, these findings suggest that pathological concentrations of thyroid hormone have a unique effect on gene expression in EOMs, which is likely to play a hitherto neglected role in thyroid-associated ophthalmopathies.

scholarly journals Tyrosine-Derived Polycarbonate Nerve Guidance Tubes Elicit Pro-Regenerative Extracellular Matrix Deposition When Used to Bridge Segmental Nerve Defects in Swine

2020 ◽  
Author(s):  
JC Burrell ◽  
D Bhatnagar ◽  
DP Brown ◽  
NS Murthy ◽  
J Dutton ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Schwann Cell ◽  
Cell Infiltration ◽  
Large Animal ◽  
Matrix Deposition ◽  
Regenerative Response ◽  
Large Animal Models ◽  
Segmental Nerve ◽  
Collagen Iii ◽  
Wide Range

AbstractPromising biomaterials should be tested in appropriate large animal models that recapitulate human inflammatory and regenerative responses. Previous studies have shown tyrosine-derived polycarbonates (TyrPC) are versatile biomaterials with a wide range of applications across multiple disciplines. The library of TyrPC has been well studied and consists of thousands of polymer compositions with tunable mechanical characteristics and degradation and resorption rates that are useful for nerve guidance tubes (NGTs). NGTs made of different TyrPCs have been used in segmental nerve defect models in small animals. The current study is an extension of this work and evaluates NGTs made using two different TyrPC compositions in a 1 cm porcine peripheral nerve repair model. We first evaluated a nondegradable TyrPC formulation, demonstrating proof-of-concept chronic regenerative efficacy up to 6 months with similar nerve/muscle electrophysiology and morphometry to the autograft repair control. Next, we characterized the acute regenerative response using a degradable TyrPC formulation. After 2 weeks in vivo, TyrPC NGT promoted greater deposition of pro-regenerative extracellular matrix (ECM) constituents (in particular collagen I, collagen III, collagen IV, laminin and fibronectin) compared to commercially available collagen-based NGTs. This corresponded with dense Schwann cell infiltration and axon extension across the lumen. These findings confirmed results reported previously in a mouse model and reveal that TyrPC NGTs were well tolerated in swine and facilitated host axon regeneration and Schwann cell infiltration in the acute phase across segmental defects - likely by eliciting a favorable neurotrophic ECM milieu. This regenerative response ultimately can contribute to functional recovery.

scholarly journals Characterisation of the horse transcriptome from immunologically active tissues

2014 ◽  
Author(s):  
Joanna Moreton ◽  
Sunir Malla ◽  
Aziz Aboobaker ◽  
Rachael Tarlinton ◽  
Richard D Emes
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
Animal Models ◽  
Genome Annotation ◽  
Large Animal ◽  
Suitable Model ◽  
Rodent Models ◽  
Large Animal Models ◽  
Human Disorders ◽  
Novel Isoforms

The immune system of the horse has not been well studied, despite the fact that the horse displays several features such as sensitivity to bacterial lipopolysaccharide that make them in many ways a more suitable model of some human disorders than the current rodent models. The difficulty of working with large animal models has however limited characterisation of gene expression in the horse immune system with current annotations for the equine genome restricted to predictions from other mammals and the few described horse proteins. This paper outlines sequencing of 184 million transcriptome short reads from immunologically active tissues of three horses including the genome reference “Twilight”. In a comparison with the Ensembl horse genome annotation, we found 8,763 potentially novel isoforms.

Abstract 251: Development of a Transgenic Goat Model with Cardiac-Specific Overexpression of Transforming Growth Factor-β1 to Study the Relationship Between Atrial Fibrosis and Atrial Fibrillation

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Irina A Polejaeva ◽  
Justin Hall ◽  
Qinggang Meng ◽  
Xinchang Zhou ◽  
Benjamin R Sessions ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Large Animal ◽  
Atrial Pacing ◽  
Atrial Fibrosis ◽  
Transgenic Goat ◽  
Large Animal Models ◽  
Fetal Fibroblasts ◽  
Tgf Β1

Studies on patients, large animal models and transgenic mouse models have shown a strong association of atrial fibrosis with atrial fibrillation (AF). However, it is unclear whether there is a causal relationship between atrial fibrosis and AF or whether these events appear as a result of independent pathological changes in the heart. We are testing the hypothesis that goats that overexpress TGF-β1 (transforming growth factor beta1) specifically in cardiac myocytes will develop atrial fibrosis that in turn will lead to AF. Many aspects of AF-related remodeling have been studied comprehensively in goat models. However, these AF models are typically mechanically induced (eg, the rapid atrial pacing model). This unique transgenic goat model has the potential to offer insights into the role of fibrosis in AF initiation and progression without the confounding effects of mechanical AF induction. Somatic cell nuclear transfer (SCNT or cloning) was used to produce TGF-β1 transgenic pregnancies. First, pcDNA3.1DV5-MHC-TGF-β1cys33ser vector was constructed by subcloning the MHC-TGF-β1 fragment from the plasmid pUC-BM20-MHC-TGF-β1 into the pcDNA3.1D V5 vector. The NeonTM transfection system was used to electroporate primary goat fetal fibroblasts. After two weeks of G418 selection, the resulting G418 resistant colonies were screened by PCR to confirm transgene integration into goat genomic DNA. PCR positive cells were used for SCNT. Cloned embryos (n=264) were cultured for 12-60 h in vitro and then transferred into synchronized recipient females (n=15). Confirmation of pregnancy was done by ultrasonography on day 30 post-transfer. At the time of this abstract submission, 40% (6/15) of recipients were confirmed to be pregnant as determined by the presence of a heartbeat. The range for the stage of gestation is between day-60 and day-120. The first delivery date is April 28, 2012. Several reports documented no pregnancy losses after 30 days of gestation in goats. Therefore, we expect that most if not all of these pregnancies will result in delivery of live offspring. To our knowledge, this will be the first transgenic goat model generated for cardiovascular research.

20-Hydroxyecdysone activates the protective arm of the RAAS via Mas receptor

2021 ◽  
Author(s):  
René Lafont ◽  
Maria Serova ◽  
Blaise Didry-Barca ◽  
Sophie Raynal ◽  
Louis Guibout ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mechanism Of Action ◽  
Muscle Growth ◽  
Negative Regulator ◽  
Pharmacological Effects ◽  
Reporter System ◽  
Myostatin Gene ◽  
Mas Receptor ◽  
Myoblast Cell Line ◽  
Myoblast Cell

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysteroid receptors (EcR/RXR complex) and at least one membrane GPCR receptor (DopEcR). It also displays numerous pharmacological effects in mammals, where its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERβ receptor. The goal of this study was to better understand 20E mechanism of action in mammals. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) was used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with angiotensin-(1-7), the endogenous ligand of Mas. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using small interfering RNA (siRNA) or pharmacological inhibitors. 17β-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7) antagonists. A mechanism involving cooperation between the Mas receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the Mas receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and, indeed, the proposed mechanism may explain the close similarity between angiotensin-(1-7)’s and 20E’s effects. Our findings open up many possible therapeutic developments involving stimulation of the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

scholarly journals Characterisation of the horse transcriptome from immunologically active tissues

2014 ◽  
Author(s):  
Joanna Moreton ◽  
Sunir Malla ◽  
Aziz Aboobaker ◽  
Rachael Tarlinton ◽  
Richard D Emes
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
Animal Models ◽  
Genome Annotation ◽  
Large Animal ◽  
Suitable Model ◽  
Rodent Models ◽  
Large Animal Models ◽  
Human Disorders ◽  
Novel Isoforms

The immune system of the horse has not been well studied, despite the fact that the horse displays several features such as sensitivity to bacterial lipopolysaccharide that make them in many ways a more suitable model of some human disorders than the current rodent models. The difficulty of working with large animal models has however limited characterisation of gene expression in the horse immune system with current annotations for the equine genome restricted to predictions from other mammals and the few described horse proteins. This paper outlines sequencing of 184 million transcriptome short reads from immunologically active tissues of three horses including the genome reference “Twilight”. In a comparison with the Ensembl horse genome annotation, we found 8,763 potentially novel isoforms.

scholarly journals 20-Hydroxyecdysone activates the protective arm of the renin angiotensin system via Mas receptor

2020 ◽  
Author(s):  
René Lafont ◽  
Sophie Raynal ◽  
Maria Serova ◽  
Blaise Didry-Barca ◽  
Louis Guibout ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mechanism Of Action ◽  
Muscle Growth ◽  
Renin Angiotensin System ◽  
Negative Regulator ◽  
Pharmacological Effects ◽  
Reporter System ◽  
Myostatin Gene ◽  
Renin Angiotensin ◽  
Mas Receptor

ABSTRACT20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysone receptors (EcRs) and at least one membrane GPCR receptor (DopEcR) and displays numerous pharmacological effects in mammals. However, its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERβ receptor. The goal of our study was to better understand 20E mechanism of action.A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) was used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with Angiotensin-(1-7), the endogenous ligand of Mas. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using small interfering RNA (siRNA) or pharmacological inhibitors.17β-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7) antagonists. A mechanism involving cooperation between Mas receptor and a membrane-bound palmitoylated estrogen receptor is proposed.The possibility to activate the Mas receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and indeed this mechanism may explain the close similarity between angiotensin-(1-7) and 20E effects. Our findings open a lot of possible therapeutic developments by stimulating the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

A Simple and Highly Effective Method for the Stable Transduction of Uncultured Porcine Hepatocytes Using Lentiviral Vector

2005 ◽  
Vol 14 (7) ◽  
pp. 489-496 ◽  
Author(s):  
Tuan Huy Nguyen ◽  
Tatiana Khakhoulina ◽  
Andrew Simmons ◽  
Philippe Morel ◽  
Didier Trono
Keyword(s):  
Vitamin E ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Wide Spectrum ◽  
Large Animal ◽  
Uw Solution ◽  
Immunodeficient Mice ◽  
Vector Coding ◽  
Large Animal Models ◽  
Porcine Hepatocytes

Gene therapy is an attractive approach for the treatment of a wide spectrum of liver diseases. Lentiviral vectors allow the stable integration of transgenes into the genome of nondividing differentiated cells including hepatocytes and could provide long-lasting expression of a therapeutic gene. To develop such approaches, preclinical studies in large animal models such as pigs are necessary to evaluate the feasibility and safety of stable lentiviral integration and long-term vector expression. In addition, effective lentivector-mediated gene transfer onto porcine hepatocytes could advance in cell-based therapies for acute liver failure. To investigate this issue, porcine hepatocytes were transduced in suspension immediately after their isolation in University of Wisconsin (UW) solution containing vitamin E. Up to 80% of hepatocytes stably expressed a GFP transgene after a single exposure to lentiviral vector coding for GFP under the control of either liver-specific or ubiquitous promoters. Moreover, porcine hepatocytes cryopreserved in UW solution containing fetal bovine serum, dimethyl sulfoxide, and vitamin E remained highly transducible with lentiviral vector after thawing. When thawed, transduced in suspension, and immediately transplanted into the spleen of immunodeficient mice, ex vivo lentivirally transgene marked xenogeneic hepatocytes were detected in murine liver. We demonstrated that porcine hepatocytes are highly susceptible to lentiviral vector and describe an easy methodology to efficiently, rapidly, and stably introduce transgenes into uncultured porcine hepatocytes.

scholarly journals CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine miR-17-92 Cluster

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 113 ◽  
Author(s):  
Chao Lu ◽  
Daxin Pang ◽  
Mengjing Li ◽  
Hongming Yuan ◽  
Tingting Yu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Rna Interference ◽  
Integration Site ◽  
Classical Swine Fever ◽  
Mirna Cluster ◽  
Transgenic Pigs ◽  
Blastocyst Development ◽  
Endogenous Gene ◽  
Fetal Fibroblasts

Successful RNAi applications depend on strategies allowing stable and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. In this study, we proposed an endogenous microRNA (miRNA) cluster as a novel integration site for small hairpin RNAs (shRNAs). We successfully integrated exogenous shRNAs at the porcine miRNA-17-92 (pmiR-17-92) cluster via a CRISPR/Cas9-mediated knock-in strategy. The anti-EGFP or anti-CSFV shRNAs could be stably and effectively expressed at the control of the endogenous promoter of the pmiR-17-92 cluster. Importantly, we confirmed that hitchhike expression of anti- classical swine fever (CSFV) shRNA had no effect on cell growth, blastocyst development and endogenous pmiR-17-92 expression in selected transgene (TG) porcine fetal fibroblasts (PFFs) clones. Moreover, these TG PFFs could inhibit the replication of CSFV by half and could be further used for generation of transgenic pigs. Taken together, these results show that our RNA interference (RNAi) expression strategy benefits numerous applications, from miRNA, genome and transgenic research, to gene therapy.

Bringing RNA Interference (RNAi) into the High School Classroom

2013 ◽  
Vol 75 (9) ◽  
pp. 698-703
Author(s):  
Sibani Sengupta
Keyword(s):  
Gene Expression ◽  
High School ◽  
Protein Synthesis ◽  
High School Students ◽  
Rna Interference ◽  
Negative Regulator ◽  
Transcriptional Level ◽  
School Students ◽  
Transcriptional Regulatory Mechanism ◽  
High School Classroom

RNA interference (abbreviated RNAi) is a relatively new discovery in the field of mechanisms that serve to regulate gene expression (a.k.a. protein synthesis). Gene expression can be regulated at the transcriptional level (mRNA production, processing, or stability) and at the translational level (protein synthesis). RNAi acts in a gene-specific manner and degrades the specific message (mRNA) to lower mRNA stability and, in the process, decreases protein production. The RNAi mechanism thus acts as a negative regulator of gene expression and undoubtedly has been one of the most significant developments in genetics and molecular biology in recent years. I present a teaching module that can help high school students experience this unique post-transcriptional regulatory mechanism.

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