Publications by authors named "Jorge A Piedrahita"

51 Publications

Validation of Transgene Expression in Gene-Edited Pigs Using CRISPR Transcriptional Activators.

CRISPR J 2020 10;3(5):409-418

Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, USA.

The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are occasionally not expressed and/or translated as expected. Therefore, there is a need to validate the expression outcomes gene modifications before investing in the costly generation of a gene-edited animal. Unfortunately, many gene targets are tissue specific and/or not expressed in cultured primary cells, making validation difficult without generating an animal. In this study, using pigs as a proof of concept, we show that CRISPR-dCas9 transcriptional activators can be used to validate functional transgene insertion in nonexpressing easily cultured cells such as fibroblasts. This is a tool that can be used across disciplines and animal species to save time and resources by verifying expected outcomes of gene edits before generating live animals.
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http://dx.doi.org/10.1089/crispr.2020.0037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580606PMC
October 2020

Clinical xenotransplantation of the heart: At the watershed.

J Heart Lung Transplant 2020 08 20;39(8):758-760. Epub 2020 Jun 20.

Departments of Surgery and; Microbiology & Immunology, and; Transplantation Biology Program, University of Michigan, Ann Arbor, Michigan.

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http://dx.doi.org/10.1016/j.healun.2020.06.002DOI Listing
August 2020

Joint laxity varies in response to partial and complete anterior cruciate ligament injuries throughout skeletal growth.

J Biomech 2020 03 16;101:109636. Epub 2020 Jan 16.

Joint Department of Biomedical Engineering, North Carolina State University and the University of North Carolina - Chapel Hill, Raleigh, NC 27695, United States; Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27695, United States; Department of Orthopaedics, University of North Carolina - Chapel Hill, Chapel Hill, NC 27599, United States. Electronic address:

Anterior cruciate ligament (ACL) injuries are increasingly common in the skeletally immature population. As such there is a need to increase our understanding of the biomechanical function of the joint following partial and complete ACL injury during skeletal growth. In this work, we aimed to assess changes in knee kinematics and loading of the remaining soft tissues following both partial and complete ACL injury in a porcine model. To do so, we applied anterior-posterior tibial loads and varus-valgus moments to stifle joints of female pigs ranging from early juvenile to late adolescent ages and assessed both kinematics and in-situ loads carried in the bundles of the ACL and other soft tissues including the collateral ligaments and the menisci. Partial ACL injury led to increased anterior tibial translation only in late adolescence and small increases in varus-valgus rotation at all ages. Complete ACL injury led to substantial increases in translation and rotation at all ages. At all ages, the medial collateral ligament and the medial meniscus combined to resist the majority of applied anterior tibial load following complete ACL transection. Across all ages and flexion angles, the contribution of the MCL ranged from 45 to 90% of the anterior load and the contribution of the medial meniscus ranged from 14 to 35% of the anterior load. These findings add to our current understanding of age-specific functional properties of both healthy and injured knees during skeletal growth.
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http://dx.doi.org/10.1016/j.jbiomech.2020.109636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410249PMC
March 2020

Tissue-specific changes in size and shape of the ligaments and tendons of the porcine knee during post-natal growth.

PLoS One 2019 23;14(10):e0219637. Epub 2019 Oct 23.

Department of Biomedical Engineering, North Carolina State University and the University of North Carolina at Chapel Hill, Raleigh, North Carolina, United States of America.

Prior studies have analyzed growth of musculoskeletal tissues between species or across body segments; however, little research has assessed the differences in similar tissues within a single joint. Here we studied changes in the length and cross-sectional area of four ligaments and tendons, (anterior cruciate ligament, patellar tendon, medial collateral ligament, lateral collateral ligament) in the tibiofemoral joint of female Yorkshire pigs through high-field magnetic resonance imaging throughout growth. Tissue lengths increased by 4- to 5-fold from birth to late adolescence across the tissues while tissue cross-sectional area increased by 10-20-fold. The anterior cruciate ligament and lateral collateral ligament showed allometric growth favoring change in length over change in cross-sectional area while the patellar tendon and medial collateral ligament grow in an isometric manner. Additionally, changes in the length and cross-sectional area of the anterior cruciate ligament did not increase as much as in the other ligaments and tendon of interest. Overall, these findings suggest that musculoskeletal soft tissue morphometry can vary within tissues of similar structure and within a single joint during post-natal growth.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219637PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6808441PMC
March 2020

In Situ Joint Stiffness Increases During Skeletal Growth but Decreases Following Partial and Complete Anterior Cruciate Ligament Injury.

J Biomech Eng 2019 Aug 1. Epub 2019 Aug 1.

4130 Engineering Building III CB7115 Raleigh, NC 27695.

Partial and complete anterior cruciate ligament (ACL) injuries occur in both pediatric and adult populations and can result in loss of joint stability and function. The sigmoidal shape of knee joint function (load-translation curve) under applied loads, includes a low-load region (described by slack length) followed by a high-load region (described by stiffness). However, the impact of age and injury on these parameters is not fully understood. The current objective was to measure the effects of age and injury on the shape of joint function in a porcine model. In response to an applied anterior-posterior tibial load, in situ slack did not change (p>0.05), despite 7-fold increases in joint size. Joint stiffness increased from an average of 10 N/mm in early youth to 47 N/mm in late adolescence (p<0.05). In situ ACL stiffness increased similarly, and changes in in situ joint stiffness and ACL stiffness were highly correlated across ages. With complete ACL injury, in situ slack length increased by 2-fold to 4-fold and in situ stiffness decreased 3-fold to 4-fold across ages (p<0.05). Partial ACL injury resulted in less dramatic, but statistically significant, increases in joint slack and significant decreases in in situ joint stiffness in the adolescent age groups (p<0.05). This work furthers our understanding of the interaction between joint biomechanics and ACL function throughout growth and the impact of ACL injury in the skeletally immature joint.
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http://dx.doi.org/10.1115/1.4044582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105148PMC
August 2019

Biomechanical Function and Size of the Anteromedial and Posterolateral Bundles of the ACL Change Differently with Skeletal Growth in the Pig Model.

Clin Orthop Relat Res 2019 Sep;477(9):2161-2174

S. G. Cone, E. P. Lambeth, M. B. Fisher, Department of Biomedical Engineering, North Carolina State University and the University of North Carolina - Chapel Hill, Raleigh, NC, USA S. G. Cone, J. A. Piedrahita, M. B. Fisher, Comparative Medicine Institute, North Carolina State University, Raleigh, NC, USA H. Ru, Department of Computational Biology and Bioinformatics, North Carolina State University, Raleigh, NC, USA L. A. Fordham, Department of Radiology, University of North Carolina - Chapel Hill, Chapel Hill, NC, USA J. A. Piedrahita, Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC, USA J. T. Spang, M. B. Fisher, Department of Orthopaedics, University of North Carolina - Chapel Hill, Chapel Hill, NC, USA.

Background: ACL injuries are becoming increasingly common in children and adolescents, but little is known regarding age-specific ACL function in these patients. To improve our understanding of changes in musculoskeletal tissues during growth and given the limited availability of pediatric human cadaveric specimens, tissue structure and function can be assessed in large animal models, such as the pig.

Questions/purposes: Using cadaveric porcine specimens ranging throughout skeletal growth, we aimed to assess age-dependent changes in (1) joint kinematics under applied AP loads and varus-valgus moments, (2) biomechanical function of the ACL under the same loads, (3) the relative biomechanical function of the anteromedial and posterolateral bundles of the ACL; and (4) size and orientation of the anteromedial and posterolateral bundles.

Methods: Stifle joints (analogous to the human knee) were collected from female Yorkshire crossbreed pigs at five ages ranging from early youth to late adolescence (1.5, 3, 4.5, 6, and 18 months; n = 6 pigs per age group, 30 total), and MRIs were performed. A robotic testing system was used to determine joint kinematics (AP tibial translation and varus-valgus rotation) and in situ forces in the ACL and its bundles in response to applied anterior tibial loads and varus-valgus moments. To see if morphological changes to the ACL compared with biomechanical changes, ACL and bundle cross-sectional area, length, and orientation were calculated from MR images.

Results: Joint kinematics decreased with increasing age. Normalized AP tibial translation decreased by 44% from 1.5 months (0.34 ± 0.08) to 18 months (0.19 ± 0.02) at 60° of flexion (p < 0.001) and varus-valgus rotation decreased from 25° ± 2° at 1.5 months to 6° ± 2° at 18 months (p < 0.001). The ACL provided the majority of the resistance to anterior tibial loading at all age groups (75% to 111% of the applied anterior force; p = 0.630 between ages). Anteromedial and posterolateral bundle function in response to anterior loading and varus torque were similar in pigs of young ages. During adolescence (4.5 to 18 months), the in situ force carried by the anteromedial bundle increased relative to that carried by the posterolateral bundle, shifting from 59% ± 22% at 4.5 months to 92% ± 12% at 18 months (data for 60° of flexion, p < 0.001 between 4.5 and 18 months). The cross-sectional area of the anteromedial bundle increased by 30 mm throughout growth from 1.5 months (5 ± 2 mm) through 18 months (35 ± 8 mm; p < 0.001 between 1.5 and 18 months), while the cross-sectional area of the posterolateral bundle increased by 12 mm from 1.5 months (7 ± 2 mm) to 4.5 months (19 ± 5 mm; p = 0.004 between 1.5 and 4.5 months), with no further growth (17 ± 7 mm at 18 months; p = 0.999 between 4.5 and 18 months). However, changes in length and orientation were similar between the bundles.

Conclusion: We showed that the stifle joint (knee equivalent) in the pig has greater translational and rotational laxity in early youth (1.5 to 3 months) compared with adolescence (4.5 to 18 months), that the ACL functions as a primary stabilizer throughout growth, and that the relative biomechanical function and size of the anteromedial and posterolateral bundles change differently with growth.

Clinical Relevance: Given the large effects observed here, the age- and bundle-specific function, size, and orientation of the ACL may need to be considered regarding surgical timing, graft selection, and graft placement. In addition, the findings of this study will be used to motivate pre-clinical studies on the impact of partial and complete ACL injuries during skeletal growth.
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http://dx.doi.org/10.1097/CORR.0000000000000884.DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000103PMC
September 2019

Xenotransplantation: Progress Along Paths Uncertain from Models to Application.

ILAR J 2018 12;59(3):286-308

Translational Medicine and The Comparative Medicine Institute, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina.

For more than a century, transplantation of tissues and organs from animals into man, xenotransplantation, has been viewed as a potential way to treat disease. Ironically, interest in xenotransplantation was fueled especially by successful application of allotransplantation, that is, transplantation of human tissue and organs, as a treatment for a variety of diseases, especially organ failure because scarcity of human tissues limited allotransplantation to a fraction of those who could benefit. In principle, use of animals such as pigs as a source of transplants would allow transplantation to exert a vastly greater impact than allotransplantation on medicine and public health. However, biological barriers to xenotransplantation, including immunity of the recipient, incompatibility of biological systems, and transmission of novel infectious agents, are believed to exceed the barriers to allotransplantation and presently to hinder clinical applications. One way potentially to address the barriers to xenotransplantation is by genetic engineering animal sources. The last 2 decades have brought progressive advances in approaches that can be applied to genetic modification of large animals. Application of these approaches to genetic engineering of pigs has contributed to dramatic improvement in the outcome of experimental xenografts in nonhuman primates and have encouraged the development of a new type of xenograft, a reverse xenograft, in which human stem cells are introduced into pigs under conditions that support differentiation and expansion into functional tissues and potentially organs. These advances make it appropriate to consider the potential limitation of genetic engineering and of current models for advancing the clinical applications of xenotransplantation and reverse xenotransplantation.
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http://dx.doi.org/10.1093/ilar/ily015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6808066PMC
December 2018

High mobility group A2 (HMGA2) deficiency in pigs leads to dwarfism, abnormal fetal resource allocation, and cryptorchidism.

Proc Natl Acad Sci U S A 2018 05 7;115(21):5420-5425. Epub 2018 May 7.

Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27607;

Expression of is strongly associated with body size and growth in mice and humans. In mice, inactivation of one or both alleles of results in body-size reductions of 20% and 60%, respectively. In humans, microdeletions involving the locus result in short stature, suggesting the function of the HMGA2 protein is conserved among mammals. To test this hypothesis, we generated HMGA2-deficient pigs via gene editing and somatic cell nuclear transfer (SCNT). Examination of growth parameters revealed that male and female pigs were on average 20% lighter and smaller than matched controls ( < 0.05). boars showed significant size reduction ranging from 35 to 85% of controls depending on age ( < 0.05), and organ weights were also affected ( < 0.05). 2 gilts and boars exhibited normal reproductive development and fertility, while boars were sterile due to undescended testes (cryptorchidism). Crossbreeding boars and gilts produced litters lacking the genotype. However, analysis of day (D) D40 and D78 pregnancies indicated that fetuses were present at the expected Mendelian ratio, but placental abnormalities were seen in the D78 concepti. Additionally, embryos generated by gene editing and SCNT produced multiple pregnancies and viable offspring, indicating that lack of HMGA2 is not lethal per se. Overall, our results show that the effect of with respect to growth regulation is highly conserved among mammals and opens up the possibility of regulating body and organ size in a variety of mammalian species including food and companion animals.
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http://dx.doi.org/10.1073/pnas.1721630115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003518PMC
May 2018

Animal Models in Tissue Engineering. Part II.

Tissue Eng Part C Methods 2017 12;23(12):827-828

2 Wake Forest Institute for Regenerative Medicine , Wake Forest Baptist Medical Center, Winston-Salem, North Carolina.

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http://dx.doi.org/10.1089/ten.TEC.2017.0458DOI Listing
December 2017

Animal Models in Tissue Engineering. Part I.

Tissue Eng Part C Methods 2017 11;23(11):641-642

2 Wake Forest Institute for Regenerative Medicine, Wake Forest Baptist Medical Center , Winston-Salem, North Carolina.

Animal models play a central and pivotal role in tissue engineering. Although advances in areas such as 3D printing and bioreactor technologies now permit the in vitro development and testing of complex scaffold/cell composites, in vivo testing remains critical not only for refining methods being developed but also for the critical efficacy and safety testing required for regulatory approval. Yet, choosing the appropriate model for a particular application remains a challenge, as each model has its own strengths and weaknesses. In some cases, there are size issues to contend with as scale-up of a 3D structure brings with it considerable challenges with regard to diffusion, infiltration, and structural forces. In others, physiological differences between species make selection of the appropriate animal model that best represents the human disease or injury critical.
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http://dx.doi.org/10.1089/ten.TEC.2017.0402DOI Listing
November 2017

Improved Chondrogenic Potential and Proteomic Phenotype of Porcine Chondrocytes Grown in Optimized Culture Conditions.

Cell Reprogram 2017 08;19(4):232-244

1 Genomics Program, North Carolina State University , Raleigh, North Carolina.

For successful cartilage tissue engineering, the ability to generate a high number of chondrocytes in vitro while avoiding terminal differentiation or de-differentiation is critical. The ability to accomplish this by using the abundant and easily sampled costal cartilage could provide a practical alternative to the use of articular cartilage and mesenchymal stem cells. Chondrocytes isolated from pig costal cartilage were expanded in either serum-free medium with FGF2 (SFM) or fetal bovine serum-containing medium (SCM), under either high (21%) or low (5%) oxygen conditions. Overall, chondrocytes cultured in SFM and low oxygen (Low-SFM) demonstrated the highest cell growth rate (p < 0.05). The effect of passage number on the differentiation status of the chondrocytes was analyzed by alkaline phosphatase (AP) staining and real-time PCR for known chondrocyte quality markers. AP staining indicated that chondrocytes grown in SCM had a higher proportion of terminally differentiated (hypertrophic) chondrocytes (p < 0.05). At the mRNA level, expression ratios of ACAN/VCAN and COL2/COL1 were significantly higher (p < 0.05) in cells expanded in Low-SFM, indicating reduced de-differentiation. In vitro re-differentiation capacity was assessed after a 6-week induction, and chondrocytes grown in Low-SFM showed similar expression ratios of COL2/COL1 and ACAN/VCAN to native cartilage. Proteomic analysis of in vitro produced cartilage indicated that the Low-SFM condition most closely matched the proteomic profile of native costal and articular cartilage. In conclusion, Low-SFM culture conditions resulted in improved cell growth rates, reduced levels of de-differentiation during expansion, greater ability to re-differentiate into cartilage on induction, and an improved proteomic profile that resembles that of in vivo cartilage.
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http://dx.doi.org/10.1089/cell.2017.0005DOI Listing
August 2017

Orientation changes in the cruciate ligaments of the knee during skeletal growth: A porcine model.

J Orthop Res 2017 12 23;35(12):2725-2732. Epub 2017 May 23.

Department of Biomedical Engineering, North Carolina State University, Raleigh, 208C Engineering Building III, North Carolina, 27695.

Musculoskeletal injuries in pediatric patients are on the rise, including significant increases in anterior cruciate ligament (ACL) injuries. Previous studies have found major anatomical changes during skeletal growth in the soft tissues of the knee. Specifically, the ACL and the posterior cruciate ligament (PCL) change in their relative orientation to the tibial plateau throughout growth. In order to develop age-specific treatments for ACL injuries, the purpose of this study was to characterize orientation changes in the cruciate ligaments of the Yorkshire pig, a common pre-clinical model, during skeletal growth in order to verify the applicability of this model for pediatric musculoskeletal studies. Hind limbs were isolated from female Yorkshire pigs ranging in age from newborn to late adolescence and were then imaged using high field strength magnetic resonance imaging. Orientation changes were quantified from the magnetic resonance images using image segmentation software. Statistically significant increases were found in the coronal and sagittal angles of the ACL relative to the tibial plateau during pre-adolescent growth. Additional changes were observed in the PCL angle, Blumensaat angle, intercondylar roof angle, and the aspect ratio of the intercondylar notch. Only the sagittal angle of the ACL relative to the tibial plateau experienced statistically significant changes through late adolescence. The age-dependent properties of the ACL and PCL in the female pig mirrored results found in female human patients, suggesting that the porcine model may provide a pre-clinical platform to study the cruciate ligaments during skeletal growth. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2725-2732, 2017.
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http://dx.doi.org/10.1002/jor.23594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5671372PMC
December 2017

Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies.

PLoS One 2017 12;12(1):e0169242. Epub 2017 Jan 12.

Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.

Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3-5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169242PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5230777PMC
August 2017

Generation of Induced Pluripotent Stem Cells (iPSCs) from Adult Canine Fibroblasts.

Methods Mol Biol 2015 ;1330:69-78

Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC, 27607, USA.

Induced pluripotent stem cells hold great potential in regenerative medicine as it enables to generate pluripotent stem cells from any available cell types. Ectopic expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) can reprogram fibroblasts directly to pluripotency as shown in multiple species. Here, we describe detailed protocols for generation of iPSCs from adult canine fibroblasts. Robust canine iPSCs will provide powerful tools not only to study human diseases, but also for the development of therapeutic approaches.
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http://dx.doi.org/10.1007/978-1-4939-2848-4_7DOI Listing
September 2016

Intravenous Cardiac Stem Cell-Derived Exosomes Ameliorate Cardiac Dysfunction in Doxorubicin Induced Dilated Cardiomyopathy.

Stem Cells Int 2015 17;2015:960926. Epub 2015 Aug 17.

Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695, USA ; Joint Department of Biomedical Engineering, UNC-Chapel Hill and NC State University, Chapel Hill and Raleigh, NC, USA ; The Cyrus Tang Hematology Center, Soochow University, Suzhou 215123, China.

Despite the efficacy of cardiac stem cells (CSCs) for treatment of cardiomyopathies, there are many limitations to stem cell therapies. CSC-derived exosomes (CSC-XOs) have been shown to be responsible for a large portion of the regenerative effects of CSCs. Using a mouse model of doxorubicin induced dilated cardiomyopathy, we study the effects of systemic delivery of human CSC-XOs in mice. Mice receiving CSC-XOs showed improved heart function via echocardiography, as well as decreased apoptosis and fibrosis. In spite of using immunocompetent mice and human CSC-XOs, mice showed no adverse immune reaction. The use of CSC-XOs holds promise for overcoming the limitations of stem cells and improving cardiac therapies.
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http://dx.doi.org/10.1155/2015/960926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553317PMC
September 2015

From "ES-like" cells to induced pluripotent stem cells: a historical perspective in domestic animals.

Theriogenology 2014 Jan;81(1):103-11

Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina, USA; Genomics Program, North Carolina State University, Raleigh, North Carolina, USA.

Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provide great potential as cell sources for gene editing to generate genetically modified animals, as well as in the field of regenerative medicine. Stable, long-term ESCs have been established in laboratory mouse and rat; however, isolation of true pluripotent ESCs in domesticated animals such as pigs and dogs have been less successful. Initially, domesticated animal pluripotent cell lines were referred to as "embryonic stem-like" cells owing to their similar morphologic characteristics to mouse ESCs, but accompanied by a limited ability to proliferate in vitro in an undifferentiated state. That is, they shared some but not all the characteristics of true ESCs. More recently, advances in reprogramming using exogenous transcription factors, combined with the utilization of small chemical inhibitors of key biochemical pathways, have led to the isolation of iPSCs. In this review, we provide a historical perspective of the isolation of various types of pluripotent stem cells in domesticated animals. In addition, we summarize the latest progress and limitations in the derivation and application of iPSCs.
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http://dx.doi.org/10.1016/j.theriogenology.2013.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3883495PMC
January 2014

Alternating current electric fields of varying frequencies: effects on proliferation and differentiation of porcine neural progenitor cells.

Cell Reprogram 2013 Oct 20;15(5):405-12. Epub 2013 Aug 20.

1 Department of Clinical Sciences, North Carolina State University , Raleigh, NC, 27607.

Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes.
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http://dx.doi.org/10.1089/cell.2013.0001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3787337PMC
October 2013

Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.

PLoS One 2013 28;8(6):e66465. Epub 2013 Jun 28.

Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina, United States of America.

Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC) driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM) and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA), Minichromosome Maintenance Complex 2 (MCM2), Bromodeoxyuridine (BrdU) and phosphorylated Histone H3 (pH3) distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/'reserve' stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2); enteroendocrine cells by Chromogranin A (CGA), Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII) and sucrase isomaltase (SIM). Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066465PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696067PMC
January 2014

Differences in X-chromosome transcriptional activity and cholesterol metabolism between placentae from swine breeds from Asian and Western origins.

PLoS One 2013 31;8(1):e55345. Epub 2013 Jan 31.

Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.

To gain insight into differences in placental physiology between two swine breeds noted for their dissimilar reproductive performance, that is, the Chinese Meishan and white composite (WC), we examined gene expression profiles of placental tissues collected at 25, 45, 65, 85, and 105 days of gestation by microarrays. Using a linear mixed model, a total of 1,595 differentially expressed genes were identified between the two pig breeds using a false-discovery rate q-value ≤0.05. Among these genes, we identified breed-specific isoforms of XIST, a long non-coding RNA responsible X-chromosome dosage compensation in females. Additionally, we explored the interaction of placental gene expression and chromosomal location by DIGMAP and identified three Sus scrofa X chromosomal bands (Xq13, Xq21, Xp11) that represent transcriptionally active clusters that differ between Meishan and WC during placental development. Also, pathway analysis identified fundamental breed differences in placental cholesterol trafficking and its synthesis. Direct measurement of cholesterol confirmed that the cholesterol content was significantly higher in the Meishan versus WC placentae. Taken together, this work identifies key metabolic pathways that differ in the placentae of two swine breeds noted for differences in reproductive prolificacy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055345PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561265PMC
July 2013

Growth requirements and chromosomal instability of induced pluripotent stem cells generated from adult canine fibroblasts.

Stem Cells Dev 2013 Mar 28;22(6):951-63. Epub 2012 Nov 28.

Genomics Program, North Carolina State University, Raleigh, NC 27607, USA.

In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing 4 transcription factors, Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Here, we report the derivation of induced pluripotent stem cells (iPSCs) from adult canine fibroblasts by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in 3 different culture media [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), or FGF2 plus LIF]. Cells cultured in both FGF2 and LIF expressed pluripotency markers [POU5F1 (OCT4), SOX2, NANOG, and LIN28] and embryonic stem cell (ESC)-specific genes (PODXL, DPPA5, FGF5, REX1, and LAMP1) and showed strong levels of alkaline phosphatase expression. In vitro differentiation by formation of embryoid bodies and by directed differentiation generated cell derivatives of all 3 germ layers as confirmed by mRNA and protein expression. In vivo, the ciPSCs created solid tumors, which failed to reach epithelial structure formation, but expressed markers for all 3 germ layers. Array comparative genomic hybridization and chromosomal fluorescence in situ hybridization analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance, there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary, we were able to derive ciPSCs from adult fibroblasts by using 4 transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF are still unknown.
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http://dx.doi.org/10.1089/scd.2012.0393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585736PMC
March 2013

Lack of genomic imprinting of DNA primase, polypeptide 2 (PRIM2) in human term placenta and white blood cells.

Epigenetics 2012 May 1;7(5):429-31. Epub 2012 May 1.

Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC, USA.

PRIM2, encoding a subunit of primase involved in DNA replication and transcription, is expressed in the placenta and is crucial for mammalian development and growth. Its role in placental function is not well understood. Recently, PRIM2 was reported as imprinted in human white blood cells (WBC). We report here our failure to confirm imprinting of the PRIM2 locus in human placenta or WBC. The discordance between our results and those of others are likely due to an incorrectly annotated PRIM2 pseudogene found in the human genome database.
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http://dx.doi.org/10.4161/epi.19777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368808PMC
May 2012

Production of ELOVL4 transgenic pigs: a large animal model for Stargardt-like macular degeneration.

Br J Ophthalmol 2011 Dec 26;95(12):1749-54. Epub 2011 Aug 26.

Department of Animal Science, North Carolina State University, Raleigh, North Carolina, USA.

Background: Truncation mutations in the elongation of very long chain fatty acids-4 (AF277094, MIM #605512) (ELOVL4) gene cause Stargardt-like macular dystrophy type 3 (STGD3). Mice expressing truncated ELOVL4 develop rapid retinal degeneration, but are poor STGD3 models since mice lack a macula. Photoreceptor topography in the pig retina is more similar to that in humans as it includes the cone rich, macula-like area centralis. The authors generated transgenic pigs expressing human disease-causing ELOVL4 mutations to better model the pathobiology of this macular disease.

Methods: Pronuclear DNA microinjection and somatic cell nuclear transfer were used to produce transgenic pigs for two different ELOVL4 mutations: the 5 base pair deletion (5 bpdel) and the 270 stop mutation (Y270terEYFP). Retinal transgene expression, morphology and electrophysiology were examined.

Results: The authors obtained four lines of Y270terEYFP and one line of 5 bpdel transgenic animals. Direct fluorescence microscopy indicated that the Y270terEYFP protein is expressed in photoreceptors and mislocalised within the cell. Immunohistochemical examination of transgenic pigs showed photoreceptor loss and disorganised inner and outer segments. Electroretinography demonstrated diminished responses in both transgenic models.

Conclusions: These transgenic pigs provide unique animal models for examining macular degeneration and STGD3 pathogenesis.
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http://dx.doi.org/10.1136/bjophthalmol-2011-300417DOI Listing
December 2011

Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies.

Transgenic Res 2012 Apr 9;21(2):383-92. Epub 2011 Aug 9.

Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621, USA.

The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.
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http://dx.doi.org/10.1007/s11248-011-9542-6DOI Listing
April 2012

The role of imprinted genes in fetal growth abnormalities.

Birth Defects Res A Clin Mol Teratol 2011 Aug 6;91(8):682-92. Epub 2011 Jun 6.

Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA.

Epigenetics, and in particular imprinted genes, have a critical role in the development and function of the placenta, which in turn has a central role in the regulation of fetal growth and development. A unique characteristic of imprinted genes is their expression from only one allele, maternal or paternal and dependent on parent of origin. This unique expression pattern may have arisen as a mechanism to control the flow of nutrients from the mother to the fetus, with maternally expressed imprinted genes reducing the flow of resources and paternally expressed genes increasing resources to the fetus. As a result, any epigenetic deregulation affecting this balance can result in fetal growth abnormalities. Imprinting-associated disorders in humans, such as Beckwith-Wiedemann and Angelman syndrome, support the role of imprinted genes in fetal growth. Similarly, assisted reproductive technologies in animals have been shown to affect the epigenome of the early embryo and the expression of imprinted genes. Their role in disorders such as intrauterine growth restriction appears to be more complex, in that imprinted gene expression can be seen as both causative and protective of fetal growth restriction. This protective or compensatory effect needs to be explored more fully.
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http://dx.doi.org/10.1002/bdra.20795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189628PMC
August 2011

Perspectives on transgenic livestock in agriculture and biomedicine: an update.

Reprod Fertil Dev 2011 ;23(1):56-63

Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC 27606, USA.

It has been 30 years since the first transgenic mouse was generated and 26 years since the first example of transferring the technology to livestock was published. While there was tremendous optimism in those initial years, with most convinced that genetically modified animals would play a significant role in agricultural production, that has not come to be. So at first sight one could conclude that this technology has, to a large extent, failed. On the contrary, it is believed that it has succeeded beyond our original expectations, and we are now at what is perhaps the most exciting time in the development and implementation of these technologies. The original goals, however, have drastically changed and it is now biomedical applications that are playing a central role in pushing both technical and scientific developments. The combination of advances in somatic cell nuclear transfer, the development of induced pluripotent stem cells and the completion of the sequencing of most livestock genomes ensures a bright and exciting future for this field, not only in livestock but also in companion animal species.
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http://dx.doi.org/10.1071/RD10246DOI Listing
April 2011

Development of a model of sacrocaudal spinal cord injury in cloned Yucatan minipigs for cellular transplantation research.

Cell Reprogram 2010 Dec;12(6):689-97

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.

Research into transplantation strategies to treat spinal cord injury (SCI) is frequently performed in rodents, but translation of results to clinical patients can be poor and a large mammalian model of severe SCI is needed. The pig has been considered an optimal model species in which to perform preclinical testing, and the Yucatan minipig can be cloned successfully utilizing somatic cell nuclear transfer (SCNT). However, induction of paralysis in pigs poses significant welfare and nursing challenges. The present study was conducted to determine whether Yucatan SCNT clones could be used to develop an SCI animal model for cellular transplantation research. First, we demonstrated that transection of the sacrocaudal spinal cord in Yucatan SCNT clones produces profound, quantifiable neurological deficits restricted to the tail. We then established that neurospheres could be isolated from brain tissue of green fluorescence protein (GFP) transfected SCNT clones. Finally, we confirmed survival of transplanted GFP-expressing neural stem cells in the SCI lesion and their differentiation into glial and neuronal lineages for up to 4 weeks without immunosuppression. We conclude that this model of sacrocaudal SCI in Yucatan SCNT clones represents a powerful research tool to investigate the effect of cellular transplantation on axonal regeneration and functional recovery.
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http://dx.doi.org/10.1089/cell.2010.0039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004133PMC
December 2010

Generation and characterization of neurospheres from canine adipose tissue-derived stromal cells.

Cell Reprogram 2010 Aug;12(4):417-25

North Carolina State University, College of Veterinary Medicine, Raleigh, North Carolina 27606, USA.

Adipose tissue-derived stromal cells (ADSCs) have been identified as a powerful stem cell source for cellular transplantation therapy. The dog is increasingly used as a model of human neurological disease; however, few studies have reported induction of canine ADSCs to neural lineages. We characterized canine ADSCs and investigated whether they could be induced to differentiate into neural lineages. Subcutaneous adipose tissue collected from the dorsal epaxial region of adult dogs aged from 1 to 6 years was cultured to produce ADSCs that were then induced to neural lineages. RT-PCR, flow cytometry, and immunocytochemistry were performed to characterize these cell populations. Morphologically fibroblast-like ADSCs were isolated and had similar characteristics to mesenchymal stem cells. Under neurogenic conditions containing basic fibroblast growth factor and epidermal growth factor, ADSCs formed spherical cellular aggregates that resembled neurospheres. RT-PCR confirmed expression of Sox2 and CD90 by these aggregates. Expression of neural stem/progenitor markers (Nestin, Sox2, Vimentin) and neural lineage markers (A2B5, GFAP, Tuj1) was shown on immunocytochemistry. After differentiation, 60% of the cells were Tuj1 positive. In conclusion, we isolated and generated neural progenitor cells from canine ADSCs. ADSCs have potential for future autologous cell transplantation therapy for neurological disorders.
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http://dx.doi.org/10.1089/cell.2009.0093DOI Listing
August 2010

Varying phenotypes in swine versus murine transgenic models constitutively expressing the same human Sonic hedgehog transcriptional activator, K5-HGLI2 Delta N.

Transgenic Res 2010 Oct 23;19(5):869-87. Epub 2010 Jan 23.

Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA.

This study was undertaken to characterize the effects of constitutive expression of the hedgehog transcriptional activator, Gli2, in porcine skin. The keratinocyte-specific human transgene, K5-hGli2 Delta N, was used to produce transgenic porcine lines via somatic cell nuclear transfer techniques. In mice, K5-hGli2 Delta N induces epithelial downgrowths resembling basal cell carcinomas. Our porcine model also developed these basal cell carcinoma-like lesions, however gross tumor development was not appreciated. In contrast to the murine model, diffuse epidermal changes as well as susceptibility to cutaneous infections were seen in the swine model. Histologic analysis of transgenic piglets revealed generalized epidermal changes including: epidermal hyperplasia (acanthosis), elongated rete ridges, parakeratotic hyperkeratosis, epidermal neutrophilic infiltration, capillary loop dilation and hypogranulosis. By 2 weeks of age, the transgenic piglets developed erythematic and edematous lesions at high contact epidermal areas and extensor surfaces of distal limb joints. Despite antibiotic treatment, these lesions progressed to a deep bacterial pyoderma and pigs died or were euthanized within weeks of birth. Non-transgenic littermates were phenotypically normal by gross and histological analysis. In summary, constitutive expression of the human hGli2 Delta N in keratinocytes, results in cutaneous changes that have not been reported in the K5-hGli2 Delta N murine model. These findings indicate a need for a multiple species animal model approach in order to better understand the role of Gli2 in mammalian skin.
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http://dx.doi.org/10.1007/s11248-010-9362-0DOI Listing
October 2010

Characterization of conserved and nonconserved imprinted genes in swine.

Biol Reprod 2009 Nov 1;81(5):906-20. Epub 2009 Jul 1.

Department of Molecular Biomedical Sciences, College of Veterinary Medicine, Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina 27606, USA.

To increase our understanding of imprinted genes in swine, we carried out a comprehensive analysis of this gene family using two complementary approaches: expression and phenotypic profiling of parthenogenetic fetuses, and analysis of imprinting by pyrosequencing. The parthenote placenta and fetus were smaller than those of controls but had no obvious morphological differences at Day 28 of gestation. By Day 30, however, the parthenote placentas had decreased chorioallantoic folding, decreased chorionic ruggae, and reduction of fetal-maternal interface surface in comparison with stage-matched control fetuses. Using Affymetrix Porcine GeneChip microarrays and/or semiquantitative PCR, brain, fibroblast, liver, and placenta of Day 30 fetuses were profiled, and 25 imprinted genes were identified as differentially expressed in at least one of the four tissue types: AMPD3, CDKN1C, COPG2, DHCR7, DIRAS3, IGF2 (isoform specific), IGF2AS, IGF2R, MEG3, MEST, NAP1L5, NDN, NNAT, OSBPL1A, PEG3, APEG3, PEG10, PLAGL1, PON2, PPP1R9A, SGCE, SLC38A4, SNORD107, SNRPN, and TFPI2. For DIRAS3, PLAGL1, SGCE, and SLC38A4, tissue-specific differences were detected. In addition, we examined the imprinting status of candidate genes by quantitative allelic pyrosequencing. Samples were collected from Day 30 pregnancies generated from reciprocal crosses of Meishan and White Composite breeds, and single-nucleotide polymorphisms were identified in candidate genes. Imprinting was confirmed for DIRAS3, DLK1, H19, IGF2AS, NNAT, MEST, PEG10, PHLDA2, PLAGL1, SGCE, and SNORD107. We also found no evidence of imprinting in ASB4, ASCL2, CD81, COMMD1, DCN, DLX5, and H13. Combined, these results represent the most comprehensive survey of imprinted genes in swine to date.
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http://dx.doi.org/10.1095/biolreprod.109.078139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770020PMC
November 2009

Identification of SNPs and INDELS in swine transcribed sequences using short oligonucleotide microarrays.

BMC Genomics 2008 May 29;9:252. Epub 2008 May 29.

Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA.

Background: Genome-wide detection of single feature polymorphisms (SFP) in swine using transcriptome profiling of day 25 placental RNA by contrasting probe intensities from either Meishan or an occidental composite breed with Affymetrix porcine microarrays is presented. A linear mixed model analysis was used to identify significant breed-by-probe interactions.

Results: Gene specific linear mixed models were fit to each of the log2 transformed probe intensities on these arrays, using fixed effects for breed, probe, breed-by-probe interaction, and a random effect for array. After surveying the day 25 placental transcriptome, 857 probes with a q-value < or = 0.05 and |fold change| > or = 2 for the breed-by-probe interaction were identified as candidates containing SFP. To address the quality of the bioinformatics approach, universal pyrosequencing assays were designed from Affymetrix exemplar sequences to independently assess polymorphisms within a subset of probes for validation. Additionally probes were randomly selected for sequencing to determine an unbiased confirmation rate. In most cases, the 25-mer probe sequence printed on the microarray diverged from Meishan, not occidental crosses. This analysis was used to define a set of highly reliable predicted SFPs according to their probability scores.

Conclusion: By applying a SFP detection method to two mammalian breeds for the first time, we detected transition and transversion single nucleotide polymorphisms, as well as insertions/deletions which can be used to rapidly develop markers for genetic mapping and association analysis in species where high density genotyping platforms are otherwise unavailable.SNPs and INDELS discovered by this approach have been publicly deposited in NCBI's SNP repository dbSNP. This method is an attractive bioinformatics tool for uncovering breed-by-probe interactions, for rapidly identifying expressed SNPs, for investigating potential functional correlations between gene expression and breed polymorphisms, and is robust enough to be used on any Affymetrix gene expression platform.
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http://dx.doi.org/10.1186/1471-2164-9-252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2442091PMC
May 2008