Publications by authors named "Charles R Long"

29 Publications

  • Page 1 of 1

Maternal nutrient restriction in late pregnancy programs postnatal metabolism and pituitary development in beef heifers.

PLoS One 2021 8;16(4):e0249924. Epub 2021 Apr 8.

Department of Animal Science, Texas A&M University, College Station, Texas, United States of America.

Maternal undernutrition during pregnancy followed by ad libitum access to nutrients during postnatal life induces postnatal metabolic disruptions in multiple species. Therefore, an experiment was conducted to evaluate postnatal growth, metabolism, and development of beef heifers exposed to late gestation maternal nutrient restriction. Pregnancies were generated via transfer of in vitro embryos produced using X-bearing sperm from a single Angus sire. Pregnant dams were randomly assigned to receive either 100% (control; n = 9) or 70% (restricted; n = 9) of their total energy requirements from gestational day 158 to parturition. From post-natal day (PND) 301 until slaughter (PND485), heifers were individually fed ad libitum in a Calan gate facility. Calves from restricted dams were lighter than controls at birth (P<0.05) through PND70 (P<0.05) with no difference in body weight from PND105 through PND485 (P>0.10). To assess pancreatic function, glucose tolerance tests were performed on PND315 and PND482 and a diet effect was seen with glucose area under the curve being greater (P<0.05) in calves born to restricted dams compared to controls. At slaughter, total internal fat was greater (P<0.05) in heifers born to restricted dams, while whole pituitary weight was lighter (P<0.05). Heifers from restricted dams had fewer growth hormone-positive cells (somatotrophs) compared to controls (P<0.05). Results demonstrate an impaired ability to clear peripheral glucose in heifers born to restricted dams leading to increased deposition of internal fat. A reduction in the number of somatotrophs may contribute to the adipogenic phenotype of heifers born to restricted dams due to growth hormone's known anabolic roles in growth, lipolysis, and pancreatic islet function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249924PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8031383PMC
April 2021

Modification of the summative equation to estimate daily total digestible nutrients for bermudagrass pasture.

J Anim Sci 2020 Nov;98(11)

LSU AgCenter Iberia/Dean Lee Research Station, Alexandria, LA.

In forage-animal nutrition modeling, diet energy is estimated mainly from the forage total digestible nutrients (TDN). As digestibility trials are expensive, TDN is usually estimated using summative equations. Early summative equations assumed a fixed coefficient to compute digestible fiber using the lignin-to-neutral detergent fiber (NDF) ratio. Subsequently, a structural coefficient (φ) was added to the summative equations to reflect an association between lignin and cell wall components. Additional modifications to the summative equations assumed a constant φ value, and they have been used as a standard method by many commercial laboratories and scientists. For feeds with nutritive values that do not change much over time, a constant φ value may suffice. However, for forages with nutritive values that keep changing during the grazing season owing to changes in weather and plant maturity, a constant φ value may add a systematic bias to prediction because it is associated with the variable lignin-to-NDF ratio. In this study, we developed a model to estimate φ as a function of the day of the year by using the daily TDN values of bermudagrass [Cynodon dactylon (L.) Pers.], a popular warm-season perennial grass in the southern United States. The variable φ model was evaluated by using it in the TDN equation and comparing the estimated values with the observed ones obtained from several locations. Values of the various measures of fit used-the Willmott index (WI), the modeling efficiency (ME), R2, root mean square error (RMSE), and percent error (PE)-showed that using the variable φ vis-à-vis the constant φ improved the TDN equation significantly. The WI, ME, R2, RMSE, and PE values of 0.94, 0.80, 0.80, 2.5, and 4.7, respectively, indicated that the TDN equation with the variable φ model was able to mimic the observed values of TDN satisfactorily. Unlike the constant φ, the variable φ predicted more closely the forage nutritive value throughout the grazing season. The variable φ model may be useful to forage-beef modeling in accurately reflecting the impacts of plant maturity and weather on daily forage nutritive value and animal performance.
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http://dx.doi.org/10.1093/jas/skaa354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751130PMC
November 2020

Temperament influences mitochondrial capacity in skeletal muscle from 8 through 18 mo of age in Brahman heifers.

J Anim Sci 2020 Oct;98(10)

Department of Animal Science, Texas A&M University, College Station, TX.

Temperamental cattle tend to yield carcasses of poorer quality, and Brahman cattle are reportedly more temperamental than non-indicus cattle breeds. A potential link between temperament and product quality may be mitochondrial activity. We hypothesized that mitochondrial measures would be greater in temperamental compared with calm heifers and that the relationships between temperament and mitochondria would persist as heifers age. Serum cortisol and skeletal muscle (longissimus thoracis [LT] and trapezius [TRAP]) mitochondrial profiles and antioxidant activities were quantified from the same calm (n = 6) and temperamental (n = 6) Brahman heifers at 8, 12, and 18 mo of age. Data were analyzed using a mixed model ANOVA in SAS (9.4) with repeated measures. Serum cortisol was greater in temperamental compared with calm heifers throughout the study (P = 0.02). Mitochondrial volume density (citrate synthase [CS] activity) increased over time (P < 0.0001) but was similar between temperament and muscle groups. Mitochondrial function (cytochrome c oxidase activity) was greatest in the temperamental LT at 8 mo of age (P ≤ 0.0006), greatest in the temperamental TRAP at 18 mo of age (P ≤ 0.003), and did not differ by temperament at 12 mo of age. Integrative (relative to tissue wet weight) mitochondrial oxidative phosphorylation capacity with complex I substrates (PCI), PCI plus complex II substrate (PCI+II), noncoupled electron transfer system capacity (ECI+II), and E with functional complex II only (ECII) were greater in the TRAP than LT for calm heifers at all ages (P ≤ 0.002), but were similar between muscle groups in temperamental heifers. Overall, calm heifers tended to have greater intrinsic (relative to CS activity) PCI and flux control of PCI+II (P ≤ 0.1) than temperamental heifers, indicating greater utilization of complex I paired with greater coupling efficiency in calm heifers. Within the LT, integrative PCI+II was greater (P = 0.05) and ECI+II tended to be greater (P = 0.06) in temperamental compared with calm heifers. From 8- to 18-mo old, glutathione peroxidase (GPx) activity decreased (P < 0.0001) and superoxide dismutase activity increased (P = 0.02), and both were similar between muscle groups. The activity of GPx was greater in temperamental compared with calm heifers at 8 (P = 0.004) but not at 12 or 18 mo of age. These results detail divergent skeletal muscle mitochondrial characteristics of live Brahman heifers according to temperament, which should be further investigated as a potential link between temperament and product quality.
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http://dx.doi.org/10.1093/jas/skaa291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751149PMC
October 2020

Genome-wide DNA methylation alteration in prenatally stressed Brahman heifer calves with the advancement of age.

Epigenetics 2021 May 20;16(5):519-536. Epub 2020 Aug 20.

Department of Animal Science, Texas A&M AgriLife Research, USA.

Possible phenotypic impairments associated with maternal stress during gestation in beef cattle may be explained by epigenetic effects. This study examined the impact of prenatal transportation stress on DNA methylation of lymphocytes of Brahman cows over the first 5 years of life. Methylation analysis through reduced representation bisulphite sequencing was conducted on DNA from lymphocytes from 28 paired samples from 6 prenatally stressed (PNS) and 8 control (Control) females obtained initially when they were 28 days of age and 5 years of age. There were 14,386 CpG (C = cytosine; p = phosphate; G = guanine) sites differentially methylated ( < 0.01) in 5-yr-old Control cows compared to their lymphocyte DNA at 28 days of age, this number was slightly decreased in 5-yr-old PNS with 13,378 CpG sites. Only 2,749 age-related differentially methylated CpG sites were seen within PNS females. There were 2,637 CpG sites differentially methylated ( < 0.01) in PNS cows relative to Controls at 5 years of age. There were differentially methylated genes in 5-yr-old cows that contributed similarly to altered gene pathways in both treatment groups. Canonical pathways altered in PNS compared to Control cows at 5 years of age were mostly related to development and growth, nervous system development and function, and immune response. Prenatal stress appeared to alter the epigenome in Brahman cows compared to Control at 5 years of age, which implies a persistent intervention in DNA methylation in lymphocytes, and may confer long-lasting effects on gene expression, and consequently relevant phenotypic changes.
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http://dx.doi.org/10.1080/15592294.2020.1805694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078662PMC
May 2021

l-Carnitine Supplementation during In Vitro Maturation and In Vitro Culture Does not Affect the Survival Rates after Vitrification and Warming but Alters and Gene Expression.

Int J Mol Sci 2020 Aug 5;21(16). Epub 2020 Aug 5.

One Health and Veterinary Innovative Research and Development (OHVRI) Group, College of Veterinary Medicine, University of Antioquia, Medellin 050034, Colombia.

l-carnitine is a potent antioxidant used for in vitro culture systems. Controversial results have been reported using l-carnitine in culture medium at different stages of in vitro bovine embryo production. Cumulus-oocyte complexes ( = 843) were in vitro-fertilized and cultured and added (treatment group) or not added (control group) with l-carnitine. At day three of culture, each group was subdivided into two subgroups receiving no l-carnitine (group 1), 3.8 mM l-carnitine added during in vitro maturation (group 2), 1.5 mM added during the in vitro culture (group 3), and 3.8 mM and 1.5 mM added during the maturation and culture, respectively (group 4). At day 8, blastocyst embryos were examined for mitochondrial activity, the presence of lipid droplets, total cell number, gene expression, and cryotolerance by vitrification. The data were analyzed with a one-way analysis of variance. l-carnitine added in the late in vitro culture significantly reduced mitochondrial activity and lipid content, and upregulated and gene expression compared to controls ( < 0.05). l-carnitine supplementation did not significantly affect the embryo rate production or survival rate after vitrification and warming ( > 0.05). l-carnitine supplementation significantly improved embryo potential to develop viable pregnancies in agreement with a study reporting improved pregnancy rates.
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http://dx.doi.org/10.3390/ijms21165601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460650PMC
August 2020

Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.

Sci Rep 2020 05 4;10(1):7411. Epub 2020 May 4.

Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas, USA.

Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1 mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations.
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http://dx.doi.org/10.1038/s41598-020-62723-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198616PMC
May 2020

Influence of prenatal transportation stress-induced differential DNA methylation on the physiological control of behavior and stress response in suckling Brahman bull calves.

J Anim Sci 2020 Jan;98(1)

Department of Animal Science, Texas A&M University, and Texas A&M AgriLife Research, College Station, TX.

The objective of this experiment was to examine potential differential methylation of DNA as a mechanism for altered behavioral and stress responses in prenatally stressed (PNS) compared with nonprenatally stressed (Control) young bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation (Transported group) or maintained as nontransported Controls (n = 48). From the offspring born to Transported and Control cows, a subset of 28-d-old intact bulls (n = 7 PNS; n = 7 Control) were evaluated for methylation of DNA of behavior and stress response-associated genes. Methylation of DNA from white blood cells was assessed via reduced representation bisulfite sequencing methods. Because increased methylation of DNA within gene promoter regions has been associated with decreased transcriptional activity of the corresponding gene, differentially methylated (P ≤ 0.05) CG sites (cytosine followed by a guanine nucleotide) located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. Among differentially methylated genes (P ≤ 0.05) related to behavior and the stress response were OPRK1, OPRM1, PENK, POMC, NR3C2, TH, DRD1, DRD5, COMT, HTR6, HTR5A, GABRA4, GABRQ, and GAD2. Among altered (P < 0.05) signaling pathways related to behavior and the stress response were Opioid Signaling, Corticotropin-Releasing Hormone Signaling, Dopamine Receptor Signaling, Dopamine-DARPP32 Feedback in cAMP Signaling, Serotonin Receptor Signaling, and GABA Receptor Signaling. Alterations to behavior and stress response-related genes and canonical pathways supported previously observed elevations in temperament score and serum cortisol through weaning in the larger population of PNS calves from which bulls in this study were derived. Differential methylation of DNA and predicted alterations to behavior and stress response-related pathways in PNS compared with Control bull calves suggest epigenetic programming of behavior and the stress response in utero.
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http://dx.doi.org/10.1093/jas/skz368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986441PMC
January 2020

Engineering bone phenotypes in domestic animals: Unique resources for enhancing musculoskeletal research.

Bone 2020 01 8;130:115119. Epub 2019 Nov 8.

Department of Veterinary Integrative Biosciences Texas A&M University, College Station, TX 77843, United States.

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http://dx.doi.org/10.1016/j.bone.2019.115119DOI Listing
January 2020

Genetic engineering a large animal model of human hypophosphatasia in sheep.

Sci Rep 2018 11 16;8(1):16945. Epub 2018 Nov 16.

Division of Biosciences, College of Dentistry, The Ohio State University, Columbus, OH, 43210, USA.

The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.
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http://dx.doi.org/10.1038/s41598-018-35079-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240114PMC
November 2018

Prenatal transportation stress alters genome-wide DNA methylation in suckling Brahman bull calves.

J Anim Sci 2018 Dec;96(12):5075-5099

Texas A&M AgriLife Research & Extension Center, Overton, TX.

The objective of this experiment was to identify genome-wide differential methylation of DNA in young prenatally stressed (PNS) bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation or maintained as nontransported Controls (n = 48). Methylation of DNA from white blood cells from a subset of 28-d-old intact male offspring (n = 7 PNS; n = 7 Control) was assessed via reduced representation bisulfite sequencing. Samples from PNS bulls contained 16,128 CG, 226 CHG, and 391 CHH (C = cytosine; G = guanine; H = either adenine, thymine, or cytosine) sites that were differentially methylated compared to samples from Controls. Of the CG sites, 7,407 were hypermethylated (at least 10% more methylated than Controls; P ≤ 0.05) and 8,721 were hypomethylated (at least 10% less methylated than Controls; P ≤ 0.05). Increased DNA methylation in gene promoter regions typically results in decreased transcriptional activity of the region. Therefore, differentially methylated CG sites located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. In PNS bull calves, 113 pathways were altered (P ≤ 0.05) compared to Controls. Among these were pathways related to behavior, stress response, metabolism, immune function, and cell signaling. Genome-wide differential DNA methylation and predicted alterations to pathways in PNS compared with Control bull calves suggest epigenetic programming of biological systems in utero.
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http://dx.doi.org/10.1093/jas/sky350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276578PMC
December 2018

Oxygen-induced alterations in the expression of chromatin modifying enzymes and the transcriptional regulation of imprinted genes.

Gene Expr Patterns 2018 06 12;28:1-11. Epub 2018 Jan 12.

Department of Veterinary Physiology & Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, 77843-4466, USA.

Embryo culture and assisted reproductive technologies have been associated with a disproportionately high number of epigenetic abnormalities in the resulting offspring. However, the mechanisms by which these techniques influence the epigenome remain poorly defined. In this study, we evaluated the capacity of oxygen concentration to influence the transcriptional control of a selection of key enzymes regulating chromatin structure. In mouse embryonic stem cells, oxygen concentrations modulated the transcriptional regulation of the TET family of enzymes, as well as the de novo methyltransferase Dnmt3a. These transcriptional changes were associated with alterations in the control of multiple imprinted genes, including H19, Igf2, Igf2r, and Peg3. Similarly, exposure of in vitro produced bovine embryos to atmospheric oxygen concentrations was associated with disruptions in the transcriptional regulation of TET1, TET3, and DNMT3a, along with the DNA methyltransferase co-factor HELLS. In addition, exposure to high oxygen was associated with alterations in the abundance of transcripts encoding members of the Polycomb repressor complex (EED and EZH2), the histone methyltransferase SETDB1 and multiple histone demethylases (KDM1A, KDM4B, and KDM4C). These disruptions were accompanied by a reduction in embryo viability and suppression of the pluripotency genes NANOG and SOX2. These experiments demonstrate that oxygen has the capacity to modulate the transcriptional control of chromatin modifying genes involved in the establishment and maintenance of both pluripotency and genomic imprinting.
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http://dx.doi.org/10.1016/j.gep.2018.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094953PMC
June 2018

DNA methylation-independent growth restriction and altered developmental programming in a mouse model of preconception male alcohol exposure.

Epigenetics 2017 7;12(10):841-853. Epub 2017 Dec 7.

a Department of Veterinary Physiology & Pharmacology, College of Veterinary Medicine and Biomedical Sciences , Texas A&M University , College Station , Texas , USA.

The preconception environment is a significant modifier of dysgenesis and the development of environmentally-induced disease. To date, fetal alcohol spectrum disorders (FASDs) have been exclusively associated with maternal exposures, yet emerging evidence suggests male-inherited alterations in the developmental program of sperm may be relevant to the growth-restriction phenotypes of this condition. Using a mouse model of voluntary consumption, we find chronic preconception male ethanol exposure associates with fetal growth restriction, decreased placental efficiency, abnormalities in cholesterol trafficking, sex-specific alterations in the genetic pathways regulating hepatic fibrosis, and disruptions in the regulation of imprinted genes. Alterations in the DNA methylation profiles of imprinted loci have been identified in clinical studies of alcoholic sperm, suggesting the legacy of paternal drinking may transmit via heritable disruptions in the regulation of imprinted genes. However, the capacity of sperm-inherited changes in DNA methylation to broadly transmit environmentally-induced phenotypes remains unconfirmed. Using bisulphite mutagenesis and second-generation deep sequencing, we find no evidence to suggest that these phenotypes or any of the associated transcriptional changes are linked to alterations in the sperm-inherited DNA methylation profile. These observations are consistent with recent studies examining the male transmission of diet-induced phenotypes and emphasize the importance of epigenetic mechanisms of paternal inheritance beyond DNA methylation. This study challenges the singular importance of maternal alcohol exposures and suggests paternal alcohol abuse is a significant, yet overlooked epidemiological factor complicit in the genesis of alcohol-induced growth defects, and may provide mechanistic insight into the failure of FASD children to thrive postnatally.
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http://dx.doi.org/10.1080/15592294.2017.1363952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788439PMC
October 2018

Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

Antiviral Res 2016 Jan 22;125:8-13. Epub 2015 Nov 22.

College of Veterinary Medicine and Biomedical Science, Texas A & M University, College Station, TX, USA.

Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.
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http://dx.doi.org/10.1016/j.antiviral.2015.11.002DOI Listing
January 2016

Inhibition of EHMT2 Induces a Robust Antiviral Response Against Foot-and-Mouth Disease and Vesicular Stomatitis Virus Infections in Bovine Cells.

J Interferon Cytokine Res 2016 Jan 29;36(1):37-47. Epub 2015 Sep 29.

1 Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University , College Station, Texas.

The genetic regulatory network controlling the innate immune system is well understood in many species. However, the role of the epigenetic mechanisms underlying the expression of immunoregulatory genes is less clear, especially in livestock species. Histone H3 lysine 9 dimethylation (H3K9me2) is an epigenetic modification associated with transcriptional silencing within the euchromatin regions. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) is a crucial enzyme responsible for regulating the dynamics of this epigenetic modification. It has been shown that histone modifications play a role in regulating type I interferon (IFN) response. In the present study, we investigated the role of EHMT2 in the epigenetic regulation of bovine antiviral innate immunity and explored its therapeutic potential against viral infections. We evaluated the effects of pharmacological and RNAi-mediated inhibition of EHMT2 on the transcription of IFN-β and other IFN-inducible antiviral genes, as well as its effect on foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV) replication in bovine cells. We show that treatment of primary bovine cells with the synthetic EHMT2 inhibitor (UNC0638) either before or shortly after virus infection resulted in a significant increase in transcript levels of bovine IFN-β (boIFN-β; 300-fold) and other IFN-inducible genes, including IFN-stimulated gene 15 (ISG-15), myxovirus resistance 1 (Mx-1), Mx-2, RIG-I, 2',5'-oligoadenylate synthetase 1 (OAS-1), and protein kinase R (PKR). Expression of these factors correlated with a significant decrease in VSV and FMDV viral titers. Our data confirm the involvement of EHMT2 in the epigenetic regulation of boIFN-β and demonstrate the activation of a general antiviral state after EHMT2 inhibition.
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http://dx.doi.org/10.1089/jir.2015.0006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722570PMC
January 2016

Histone-lysine N-methyltransferase SETDB1 is required for development of the bovine blastocyst.

Theriogenology 2015 Nov 29;84(8):1411-22. Epub 2015 Jul 29.

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA.

Transcripts derived from select clades of transposable elements are among the first to appear in early mouse and human embryos, indicating transposable elements and the mechanisms that regulate their activity are fundamental to the establishment of the founding mammalian lineages. However, the mechanisms by which these parasitic sequences are involved in directing the developmental program are still poorly characterized. Transposable elements are regulated through epigenetic means, where combinatorial patterns of DNA methylation and histone 3 lysine 9 trimethylation (H3K9me3) suppress their transcription. From studies in rodents, SET domain bifurcated 1 (SETDB1) has emerged as the core methyltransferase responsible for marking transposable elements with H3K9me3 and temporally regulating their transcriptional activity. SETDB1 loss of function studies in mice reveal that although extraembryonic tissues do not require this methyltransferase, establishment of the embryo proper fails without it. As the bovine embryo initiates the processes of epigenetic programming earlier in the preimplantation phase, we sought to determine whether suppressing SETDB1 would block the formation of the inner cell mass. We report here that bovine SETDB1 transcripts are present throughout preimplantation development, and RNA interference-based depletion blocks embryo growth at the morula stage of development. Although we did not observe alterations in global histone methylation or transposable element transcription, we did observe increased global levels of H3K27 acetylation, an epigenetic mark associated with active enhancers. Our observations suggest that SETDB1 might interact with the epigenetic machinery controlling enhancer function and that suppression of this methyltransferase may disrupt the bovine developmental program.
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http://dx.doi.org/10.1016/j.theriogenology.2015.07.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591208PMC
November 2015

Molecular and preclinical basis to inhibit PGE2 receptors EP2 and EP4 as a novel nonsteroidal therapy for endometriosis.

Proc Natl Acad Sci U S A 2015 Aug 21;112(31):9716-21. Epub 2015 Jul 21.

Department of Integrative Biosciences, Texas A&M University, College Station, TX 77843;

Endometriosis is a debilitating, estrogen-dependent, progesterone-resistant, inflammatory gynecological disease of reproductive age women. Two major clinical symptoms of endometriosis are chronic intolerable pelvic pain and subfertility or infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent recurrence of disease. There is a fundamental need to identify nonestrogen or nonsteroidal targets for the treatment of endometriosis. Peritoneal fluid concentrations of prostaglandin E2 (PGE2) are higher in women with endometriosis, and this increased PGE2 plays important role in survival and growth of endometriosis lesions. The objective of the present study was to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on molecular and cellular aspects of the pathogenesis of endometriosis and associated clinical symptoms. Using human fluorescent endometriotic cell lines and chimeric mouse model as preclinical testing platform, our results, to our knowledge for the first time, indicate that selective inhibition of EP2/EP4: (i) decreases growth and survival of endometriosis lesions; (ii) decreases angiogenesis and innervation of endometriosis lesions; (iii) suppresses proinflammatory state of dorsal root ganglia neurons to decrease pelvic pain; (iv) decreases proinflammatory, estrogen-dominant, and progesterone-resistant molecular environment of the endometrium and endometriosis lesions; and (v) restores endometrial functional receptivity through multiple mechanisms. Our novel findings provide a molecular and preclinical basis to formulate long-term nonestrogen or nonsteroidal therapy for endometriosis.
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http://dx.doi.org/10.1073/pnas.1507931112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534219PMC
August 2015

Genome edited sheep and cattle.

Transgenic Res 2015 Feb 10;24(1):147-53. Epub 2014 Sep 10.

The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush Campus, Edinburgh, EH25 9RG, UK.

Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.
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http://dx.doi.org/10.1007/s11248-014-9832-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274373PMC
February 2015

Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication.

J Virol 2014 Oct 16;88(19):11140-53. Epub 2014 Jul 16.

Plum Island Animal Disease Center, ARS, USDA, Greenport, New York, USA

Unlabelled: Several studies have demonstrated that the delivery of type I, II, or III interferons (IFNs) by inoculation of a replication-defective human adenovirus 5 (Ad5) vector expressing IFNs can effectively control foot-and-mouth disease (FMD) in cattle and swine during experimental infections. However, relatively high doses are required to achieve protection. In this study, we identified the functional properties of a porcine fusion protein, poIRF7/3(5D), as a biotherapeutic and enhancer of IFN activity against FMD virus (FMDV). We showed that poIRF7/3(5D) is a potent inducer of type I IFNs, including alpha IFN (IFN-α), IFN-β, and IFN-ω but not type III IFN (interleukin-28B), without inducing cytotoxicity. Expression of poIRF7/3(5D) significantly and steadily reduced FMDV titers by up to 6 log10 units in swine and bovine cell lines. Treatment with an IFN receptor inhibitor (B18R) combined with an anti-IFN-α antibody neutralized the antiviral activity in the supernatants of cells transduced with an Ad5 vector expressing poIRF7/3(5D) [Ad5-poIRF7/3(5D)]. However, several transcripts with known antiviral function, including type I IFNs, were still highly upregulated (range of increase, 8-fold to over 500-fold) by poIRF7/3(5D) in the presence of B18R. Furthermore, the sera of mice treated with Ad5-poIRF7/3(5D) showed antiviral activity that was associated with the induction of high levels of IFN-α and resulted in complete protection against FMDV challenge at 6, 24, or 48 h posttreatment. This study highlights for the first time the antiviral potential of Ad5-poIRF7/3(5D) in vitro and in vivo against FMDV.

Importance: FMD remains one of the most devastating diseases that affect livestock worldwide. Effective vaccine formulations are available but are serotype specific and require approximately 7 days before they are able to elicit protective immunity. We have shown that vector-delivered IFN is an option to protect animals against many FMDV serotypes as soon as 24 h and for about 4 days postadministration. Here we demonstrate that delivery of a constitutively active transcription factor that induces the production of endogenous IFNs and potentially other antiviral genes is a viable strategy to protect against FMD.
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http://dx.doi.org/10.1128/JVI.00372-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178809PMC
October 2014

Sustained expression of insulin by a genetically engineered sertoli cell line after allotransplantation in diabetic BALB/c mice.

Biol Reprod 2014 May 2;90(5):109. Epub 2014 Apr 2.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas

Immune-privileged Sertoli cells (SCs) exhibit long-term survival after allotransplantation or xenotransplantation, suggesting they can be used as a vehicle for cell-based gene therapy. Previously, we demonstrated that SCs engineered to secrete insulin by using an adenoviral vector normalized blood glucose levels in diabetic mice. However, the expression of insulin was transient, and the use of immunocompromised mice did not address the question of whether SCs can stably express insulin in immunocompetent animals. Thus, the objective of the current study was to use a lentiviral vector to achieve stable expression of insulin in SCs and test the ability of these cells to survive after allotransplantation. A mouse SC line transduced with a recombinant lentiviral vector containing furin-modified human proinsulin cDNA (MSC-EhI-Zs) maintained stable insulin expression in vitro. Allotransplantation of MSC-EhI-Zs cells into diabetic BALB/c mice demonstrated 88% and 75% graft survival rates at 20 and 50 days post-transplantation, respectively. Transplanted MSC-EhI-Zs cells continued to produce insulin mRNA throughout the study (i.e., 50 days); however, insulin protein was detected only in patches of cells within the grafts. Consistent with low insulin protein detection, there was no significant change in blood glucose levels in the transplant recipients. Nevertheless, MSC-EhI-Zs cells isolated from the grafts continued to express insulin protein in culture. Collectively, this demonstrates that MSC-EhI-Zs cells stably expressed insulin and survived allotransplantation without immunosuppression. This further strengthens the use of SCs as targets for cell-based gene therapy for the treatment of numerous chronic diseases, especially those that require basal protein expression.
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http://dx.doi.org/10.1095/biolreprod.113.115600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076370PMC
May 2014

Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer.

Mol Reprod Dev 2014 Feb 13;81(2):183-93. Epub 2013 Dec 13.

Department of Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas.

Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection.
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http://dx.doi.org/10.1002/mrd.22271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3953569PMC
February 2014

Efficient TALEN-mediated gene knockout in livestock.

Proc Natl Acad Sci U S A 2012 Oct 1;109(43):17382-7. Epub 2012 Oct 1.

Center for Genome Engineering and Department of Animal Science, University of Minnesota, St Paul, MN 55108, USA.

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.
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http://dx.doi.org/10.1073/pnas.1211446109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491456PMC
October 2012

Down-regulation of viral replication by lentiviral-mediated expression of short-hairpin RNAs against vesicular stomatitis virus ribonuclear complex genes.

Antiviral Res 2012 Aug 26;95(2):150-8. Epub 2012 May 26.

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466, United States.

Vesicular stomatitis virus (VSV) causes great economic impact to livestock industry and is a prototype for studying non-segmented negative-stranded RNA (NSNR) viruses. In this study, we evaluated the antiviral potential of unique short-hairpin RNA (shRNA) targeting genes that form the ribonuclear protein (RNP) complex of VSV serotype Indiana (VSIV). We used lentiviral vectors to construct cell lines that stably expressed one of seven shRNAs targeting the RNP genes of VSIV, namely nucleocapsid (N), phosphoprotein (P), or polymerase (L). We reported two N-shRNA sequences targeting the 5' or 3' end of N that significantly reduced N, P, and L viral transcripts (p<0.001), reduced viral protein expression, and reduced the viral particles shed in Vero cells (p<0.01). When we analyzed the sequence diversity in the target region of this N-shRNA from two field isolates, we detected a single base substitution outside the seed region. We also reported five other shRNA sequences targeting components of the viral RNA that significantly reduce N, P, and L viral transcripts (p<0.001) but failed to efficiently impair viral replication. The differences in the efficiency of the shRNAs tested were not due to mismatches within the target region in the genome of VSIV. Although partial silencing of viral transcripts by single shRNAs impaired but did not block VSIV replication, the combination of the shRNAs identified here into a multiple shRNA vector may result in inhibition of viral replication. These data contribute to ongoing development of RNAi-based technologies to combat viral diseases.
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http://dx.doi.org/10.1016/j.antiviral.2012.05.007DOI Listing
August 2012

Genetically engineered immune privileged Sertoli cells: A new road to cell based gene therapy.

Spermatogenesis 2012 Jan;2(1):23-31

Sertoli cells are immune privileged cells, important for controlling the immune response to male germ cells as well as maintaining the tolerogenic environment in the testis. Additionally, ectopic Sertoli cells have been shown to survive and protect co-grafted cells when transplanted across immunological barriers. The survival of ectopic Sertoli cells has led to the idea that they could be used in cell based gene therapy. In this review, we provide a brief overview of testis immune privilege and Sertoli cell transplantation, factors contributing to Sertoli cell immune privilege, the challenges faced by viral vector gene therapy, the use of immune privileged cells in cell based gene therapy and describe several recent studies on the use of genetically engineered Sertoli cells to provide continuous delivery of therapeutic proteins.
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http://dx.doi.org/10.4161/spmg.19119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3341243PMC
January 2012

Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development.

Mol Reprod Dev 2011 May 8;78(5):306-17. Epub 2011 Apr 8.

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466, USA.

In studies of somatic cell nuclear transfer (SCNT), the ability of factors within the oocyte to epigenetically reprogram transferred nuclei is essential for embryonic development of the clone to proceed. However, irregular patterns of X-chromosome inactivation, abnormal expression of imprinted genes, and genomic DNA hypermethylation are frequently observed in reconstructed embryos, suggesting abnormalities in this process. To better understand the epigenetic events underlying SCNT reprogramming, we sought to determine if the abnormal DNA methylation levels observed in cloned embryos result from a failure of the oocyte to properly reprogram transcription versus differential biochemical regulation of the DNA methyltransferase family of enzymes (DNMTs) between embryonic and somatic nuclei. To address this question, we conducted real-time quantitation of Dnmt transcripts in bovine preimplantation embryos generated though in vitro fertilization (IVF), parthenogenic activation, and SCNT. By the 8-cell stage, transcripts encoding Dnmt1 become significantly down-regulated in cloned embryos, likely in response to the state of genomic hypermethylation, while the de novo methyltransferases maintain an expression pattern indistinguishable from their IVF and parthenote counterparts. Depletion of embryonic/maternal Dnmt1 transcripts within IVF embryos using short-interfering RNAs, while able to lower genomic DNA methylation levels, resulted in developmental arrest at the 8/16-cell stage. In contrast, SCNT embryos derived from a stable, Dnmt1-depleted donor cell line develop to blastocyst stage, but failed to carry to term. Our results indicate an essential role for Dnmt1 during bovine preimplantation development, and suggest proper transcriptional reprogramming of this gene family in SCNT embryos.
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http://dx.doi.org/10.1002/mrd.21306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3095725PMC
May 2011

Viral particles of endogenous betaretroviruses are released in the sheep uterus and infect the conceptus trophectoderm in a transspecies embryo transfer model.

J Virol 2010 Sep 7;84(18):9078-85. Epub 2010 Jul 7.

Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471, USA.

The sheep genome contains multiple copies of endogenous betaretroviruses highly related to the exogenous and oncogenic jaagsiekte sheep retrovirus (JSRV). The endogenous JSRVs (enJSRVs) are abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus trophectoderm and are essential for conceptus elongation and trophectoderm growth and development. Of note, enJSRVs are present in sheep and goats but not cattle. At least 5 of the 27 enJSRV loci cloned to date possess an intact genomic organization and are able to produce viral particles in vitro. In this study, we found that enJSRVs form viral particles that are released into the uterine lumen of sheep. In order to test the infectious potential of enJSRV particles in the uterus, we transferred bovine blastocysts into synchronized ovine recipients and allowed them to develop for 13 days. Analysis of microdissected trophectoderm of the bovine conceptuses revealed the presence of enJSRV RNA and, in some cases, DNA. Interestingly, we found that RNAs belonging to only the most recently integrated enJSRV loci were packaged into viral particles and transmitted to the trophectoderm. Collectively, these results support the hypothesis that intact enJSRV loci expressed in the uterine endometrial epithelia are shed into the uterine lumen and could potentially transduce the conceptus trophectoderm. The essential role played by enJSRVs in sheep reproductive biology could also be played by endometrium-derived viral particles that influence development and differentiation of the trophectoderm.
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http://dx.doi.org/10.1128/JVI.00950-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937645PMC
September 2010

Applications of RNA interference-based gene silencing in animal agriculture.

Reprod Fertil Dev 2010 ;22(1):47-58

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biological Sciences, Texas A&M University, College Station, TX 77843-4466, USA.

Classical genetic selection, recently aided by genomic selection tools, has been successful in achieving remarkable progress in livestock improvement. However, genetic selection has led to decreased genetic diversity and, in some cases, acquisition of undesirable traits. In order to meet the increased demands of our expanding population, new technologies and practices must be developed that contend with zoonotic and animal disease, environmental impacts of large farming operations and the increased food and fibre production needed to feed and clothe our society. Future increases in productivity may be dependent upon the acquisition of genetic traits not currently encoded by the genomes of animals used in standard agricultural practice, thus making classical genetic selection impossible. Genetic engineering of livestock is commonly used to produce pharmaceuticals or to impart enhanced production characteristics to animals, but has also demonstrated its usefulness in producing animals with disease resistance. However, significant challenges remain because it has been more difficult to produce animals in which specific genes have been removed. It is now possible to modify livestock genomes to block expression of endogenous and exogenous genes (such as those expressed following virus infection). In the present review, we discuss mechanisms of silencing gene expression via the biology of RNA interference (RNAi), the technology of activating the RNAi pathway and the application of this technology to enhance livestock production through increased production efficiency and prevention of disease. An increased demand for sustainable food production is at the forefront of scientific challenges and RNAi technology will undoubtedly play a key role.
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http://dx.doi.org/10.1071/RD09211DOI Listing
May 2010

Synchronisation of canine germinal vesicle stage oocytes prior to in vitro maturation alters the kinetics of nuclear progression during subsequent resumption of meiosis.

Reprod Fertil Dev 2008 ;20(5):606-14

Texas A and M University, College of Veterinary Medicine, Department of Physiology and Pharmacology, College Station, TX 77843-4466, USA.

Inhibition of meiosis before in vitro maturation (IVM) can improve meiotic competence in immature mammalian oocytes. Therefore, meiosis-inhibiting agents were evaluated singularly for the ability to arrest and synchronise germinal vesicle (GV) stage canine oocytes, and the most effective treatments were combined to improve meiotic resumption rates. Oocytes cultured in 2 ng mL(-1) oestradiol (E2), 10 IU mL(-1) eCG, or both (EG) for 72 h resulted in significantly fewer oocytes resuming meiosis in EG than the control, E2, or with eCG. Oocytes cultured in 50 or 100 micromol L(-1) of butyrolactone 1 or roscovitine (ROS) for up to 48 h did not resume meiosis nor increase subsequent meiotic resumption rates following IVM. A combination of 50 micromol L(-1) ROS and EG treatment for 48 h significantly increased the proportion of canine oocytes in meiotic arrest. More importantly, following 48 h of IVM, ROS+EG-treated oocytes demonstrated a dramatic increase in the ability to resume meiosis compared with the non-treated controls (51.3 +/- 8.2% and 10.8 +/- 4.5%, respectively; P < 0.05). These data indicate that chemical and biological meiotic inhibitors are effective at inducing GV arrest in canine oocytes. Furthermore, these inhibitors are reversible and beneficial to subsequent meiotic resumption in vitro.
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http://dx.doi.org/10.1071/rd07227DOI Listing
September 2008

Identification, amplification and characterization of miR-17-92 from canine tissue.

Gene 2007 Dec 30;404(1-2):25-30. Epub 2007 Aug 30.

Department of Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.

Recently, a novel group of genes encoding small RNA molecules, termed microRNAs (miRNAs), has been discovered to play a vital role in eukaryotic gene expression. Known to act in a post-transcriptional fashion, miRNAs can inhibit translation by binding to messenger RNA (mRNA) or by targeting mRNA for degradation. A search of genetic databases revealed significant conservation of miRNA genes between the domestic dog and the human. This finding suggests that expression patterns may also be conserved. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan Real-time PCR techniques were used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver) using the delta-delta Ct (critical threshold) method.
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http://dx.doi.org/10.1016/j.gene.2007.08.015DOI Listing
December 2007

Suppression of prion protein in livestock by RNA interference.

Proc Natl Acad Sci U S A 2006 Apr 27;103(14):5285-90. Epub 2006 Mar 27.

Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Howard Hughes Medical Institute, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.

Given the difficulty of applying gene knockout technology to species other than mice, we decided to explore the utility of RNA interference (RNAi) in silencing the expression of genes in livestock. Short hairpin RNAs (shRNAs) were designed and screened for their ability to suppress the expression of caprine and bovine prion protein (PrP). Lentiviral vectors were used to deliver a transgene expressing GFP and an shRNA targeting PrP into goat fibroblasts. These cells were then used for nuclear transplantation to produce a cloned goat fetus, which was surgically recovered at 81 days of gestation and compared with an age-matched control derived by natural mating. All tissues examined in the cloned fetus expressed GFP, and PCR analysis confirmed the presence of the transgene encoding the PrP shRNA. Most relevant, Western blot analysis performed on brain tissues comparing the transgenic fetus with control demonstrated a significant (>90%) decrease in PrP expression levels. To confirm that similar methodologies could be applied to the bovine, recombinant virus was injected into the perivitelline space of bovine ova. After in vitro fertilization and culture, 76% of the blastocysts exhibited GFP expression, indicative that they expressed shRNAs targeting PrP. Our results provide strong evidence that the approach described here will be useful in producing transgenic livestock conferring potential disease resistance and provide an effective strategy for suppressing gene expression in a variety of large-animal models.
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http://dx.doi.org/10.1073/pnas.0600813103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459347PMC
April 2006
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