Transgenes are segments of DNA that are introduced into an organism's genome using recombinant DNA technology, and their application in horses is an area of genetic research. This process involves the stable integration of foreign genes into the horse's genetic material, potentially altering traits or introducing new characteristics. Research in this field explores the methodologies for successful gene transfer, the expression and regulation of transgenes in equine cells, and the implications for horse health and performance. Transgenic techniques can be used to study genetic diseases, improve disease resistance, or enhance specific traits in horses. This page compiles peer-reviewed research studies and scholarly articles that investigate the methods, outcomes, and ethical considerations of transgene technology in equine science.
Goodrich LR, McIlwraith CW, Grieger J, Kraus VB, Stabler T, Werpy N, Phillips J, Samulski RJ, Frisbie D.To evaluate the effects of a gene transfer approach to IL-1β inhibition in an equine osteochondral chip fragment model of joint injury using a self-complementary adeno-associated virus with interleukin receptor antagonist transgene cassette (scAAVIL-1ra), as posttraumatic osteoarthritis in horses, similar to people, is a significant clinical problem. Methods: 16 horses were utilized for the study. Methods: All horses had an osteochondral chip fragment induced arthroscopically in one middle carpal joint while the contralateral joint was sham operated. Eight horses received either scAAVIL-1ra o...
Yuen BP, Wong KS, So YM, Kwok WH, Cheung HW, Wan TSM, Ho EN, Wong WT.Gene doping involves the misuse of genetic materials to alter an athlete's performance, which is banned at all times in both human and equine sports. Quantitative polymerase chain reaction (qPCR) assays have been used to control the misuse of transgenes in equine sports. Our laboratory recently developed and implemented duplex as well as multiplex qPCR assays for transgenes detection. To further advance gene doping control, we have developed for the first time a sensitive and definitive PCR-liquid chromatography high-resolution tandem mass spectrometry (PCR-LC-HRMS/MS) method for transgene det...
Wilkin T, Hamilton NA, Cawley AT, Bhat S, Baoutina A.The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: , , , and . The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplific...
Thampi P, Seabaugh KA, Pezzanite LM, Chu CR, Phillips JN, Grieger JC, McIlwraith CW, Samulski RJ, Goodrich LR.Gene therapy approaches using adeno-associated viral vectors have been successfully tested in the equine post-traumatic osteoarthritis (PTOA) model. Owing to differences in the levels of transgene expression and adverse tissue reactions observed in published studies, we sought to identify a safe therapeutic dose of scAAVIL-1ra in an inflamed and injured joint that would result in improved functional outcomes without any adverse events. scAAVIL-1ra was delivered intra-articularly over a 100-fold range, and horses were evaluated throughout and at the end of the 10-week study. A dose-related incr...
Ohnuma A, Tozaki T, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI.To ensure fair competition and sports integrity, gene doping is prohibited in horseracing and equine sports. One gene doping method is by administering exogenous genes, called transgenes, to postnatal animals. Although several transgene detection methods have been developed for horses, many are unsuitable for multiplex detection. In this proof-of-concept study, we developed a highly sensitive and multiplex transgene detection method using multiple πCode with identification patterns printed on the surface. The following steps were employed: (1) multiplex polymerase chain reaction amplification...
Tozaki T, Ohnuma A, Hamilton NA, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time ...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan prob...
Tozaki T, Ohnuma A, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI.Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin () transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the transgene was cloned into a plasmid for use as a model. We extracted the spiked transgene f...
Minato E, Aoshima K, Kobayashi A, Ohnishi N, Sasaki N, Kimura T.Equine herpesvirus 1 (EHV-1) uses equine major histocompatibility complex class I (MHC class I) as an entry receptor. Exogenous expression of equine MHC class I genes in murine cell lines confers susceptibility to EHV-1 infection. To examine the in vivo role of equine MHC class I as an entry receptor for EHV-1, we generated transgenic (Tg) mice expressing equine MHC class I under the control of the CAG promoter. Equine MHC class I protein was expressed in the liver, spleen, lung, and brain of Tg mice, which was confirmed by Western blot. However, equine MHC class I antigen was only detected in...
Perry S, Larhammar M, Vieillard J, Nagaraja C, Hilscher MM, Tafreshiha A, Rofo F, Caixeta FV, Kullander K.Neuronal networks within the spinal cord, collectively known as the central pattern generator (CPG), coordinate rhythmic movements underlying locomotion. The transcription factor doublesex and mab-3-related transcription factor 3 (DMRT3) is involved in the differentiation of the dorsal interneuron 6 class of spinal cord interneurons. In horses, a non-sense mutation in the gene has major effects on gaiting ability, whereas mice lacking the gene display impaired locomotor activity. Although the gene is necessary for normal spinal network formation and function in mice, a direct role for -deri...
Sanchez-Garcia J, Fernandez-Funez P.Prion diseases are fatal neurodegenerative diseases caused by misfolding of the prion protein (PrP). These conditions affect humans and animals, including endemic forms in sheep and deer. Bovine, rodents, and many zoo mammals also developed prion diseases during the "mad-cow" epidemic in the 1980's. Interestingly, rabbits, horses, and dogs show unusual resistance to prion diseases, suggesting that specific sequence changes in the corresponding endogenous PrP prevents the accumulation of pathogenic conformations. In vitro misfolding assays and structural studies have identified S174, S167, and ...
Jonsdottir S, Svansson V, Stefansdottir SB, Mäntylä E, Marti E, Torsteinsdottir S.Insect bite hypersensitivity is an immunoglobulin (Ig)E-mediated dermatitis of horses initiated by bites of midges of the genus Culicoides. Culicoides spp. are not indigenous to Iceland and the prevalence of insect bite hypersensitivity is much higher in horses born in Iceland and exported as compared to Icelandic horses born in a Culicoides rich environment. Immunotherapy is therefore needed. Objective: The aim of the study was to express an allergen from Culicoides in barley grain and investigate whether an immune response could be obtained in healthy Icelandic horses by oral treatment with ...
Braid LR, Hu WG, Davies JE, Nagata LP.: Mesenchymal stromal cells (MSCs) are being exploited as gene delivery vectors for various disease and injury therapies. We provide proof-of-concept that engineered MSCs can provide a useful, effective platform for protection against infectious disease. Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne pathogen affecting humans and equines and can be used in bio-warfare. No licensed vaccine or antiviral agent currently exists to combat VEEV infection in humans. Direct antibody administration (passive immunity) is an effective, but short-lived, method of providing immediate prote...
O'Rourke JP, Olsen JC, Bunnell BA.Gene transfer into hematopoietic cells may allow correction of a variety of hematopoietic and metabolic disorders. Optimized HIV-1 based lentiviral vectors have been developed for improved gene transfer and transgene expression into hematopoietic cells. However, the use of HIV-1 based vectors for human gene therapy may be limited due to ethical and biosafety issues. We report that vectors based on the non-primate equine infectious anemia virus (EIAV) transduce a variety of human hematopoietic cell lines and primary blood cells. To investigate optimization of gene expression in hematopoietic ce...
O'Rourke JP, Newbound GC, Kohn DB, Olsen JC, Bunnell BA.This report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) lentiviral vectors in a variety of human cell types in vitro. EIAV and HIV vectors transduced equivalent numbers of proliferating and G1/S- and G2/M-arrested cells, and both had very low efficiencies of transduction into G0-arrested cells. Analysis of the levels of both the enhanced green fluorescent protein (EGFP) and mRNA demonstrated that the HIV-transduced cells expressed greater levels of EGFP protein and RNA th...
Farmerie TA, Abbud RA, Budworth PR, Clay CM, Keri RA, McDowell KJ, Wolfe MW, Nilson JH.The equine glycoprotein hormone alpha-subunit gene is expressed in both pituitary and placenta, unlike that of all other nonprimate mammals studied, in which expression is limited to pituitary. Previous studies of the 5'-flanking region of the equine alpha-subunit promoter have revealed unique characteristics as well as similarities with the human alpha-subunit promoter, which demonstrates a similar pattern of tissue-specific expression. We have cloned and sequenced the equine alpha-subunit gene and have used tissue culture systems and transgenic mice to characterize its expression. Unlike the...
Sanchez-Garcia J, Fernandez-Funez P.Prion diseases are fatal neurodegenerative diseases caused by misfolding of the prion protein (PrP). These conditions affect humans and animals, including endemic forms in sheep and deer. Bovine, rodents, and many zoo mammals also developed prion diseases during the "mad-cow" epidemic in the 1980's. Interestingly, rabbits, horses, and dogs show unusual resistance to prion diseases, suggesting that specific sequence changes in the corresponding endogenous PrP prevents the accumulation of pathogenic conformations. In vitro misfolding assays and structural studies have identified S174, S167, and ...
Perry S, Larhammar M, Vieillard J, Nagaraja C, Hilscher MM, Tafreshiha A, Rofo F, Caixeta FV, Kullander K.Neuronal networks within the spinal cord, collectively known as the central pattern generator (CPG), coordinate rhythmic movements underlying locomotion. The transcription factor doublesex and mab-3-related transcription factor 3 (DMRT3) is involved in the differentiation of the dorsal interneuron 6 class of spinal cord interneurons. In horses, a non-sense mutation in the gene has major effects on gaiting ability, whereas mice lacking the gene display impaired locomotor activity. Although the gene is necessary for normal spinal network formation and function in mice, a direct role for -deri...
O'Rourke JP, Newbound GC, Kohn DB, Olsen JC, Bunnell BA.This report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) lentiviral vectors in a variety of human cell types in vitro. EIAV and HIV vectors transduced equivalent numbers of proliferating and G1/S- and G2/M-arrested cells, and both had very low efficiencies of transduction into G0-arrested cells. Analysis of the levels of both the enhanced green fluorescent protein (EGFP) and mRNA demonstrated that the HIV-transduced cells expressed greater levels of EGFP protein and RNA th...
Tozaki T, Ohnuma A, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI.Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin () transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the transgene was cloned into a plasmid for use as a model. We extracted the spiked transgene f...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan prob...
Braid LR, Hu WG, Davies JE, Nagata LP.: Mesenchymal stromal cells (MSCs) are being exploited as gene delivery vectors for various disease and injury therapies. We provide proof-of-concept that engineered MSCs can provide a useful, effective platform for protection against infectious disease. Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne pathogen affecting humans and equines and can be used in bio-warfare. No licensed vaccine or antiviral agent currently exists to combat VEEV infection in humans. Direct antibody administration (passive immunity) is an effective, but short-lived, method of providing immediate prote...
O'Rourke JP, Olsen JC, Bunnell BA.Gene transfer into hematopoietic cells may allow correction of a variety of hematopoietic and metabolic disorders. Optimized HIV-1 based lentiviral vectors have been developed for improved gene transfer and transgene expression into hematopoietic cells. However, the use of HIV-1 based vectors for human gene therapy may be limited due to ethical and biosafety issues. We report that vectors based on the non-primate equine infectious anemia virus (EIAV) transduce a variety of human hematopoietic cell lines and primary blood cells. To investigate optimization of gene expression in hematopoietic ce...
Ohnuma A, Tozaki T, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI.To ensure fair competition and sports integrity, gene doping is prohibited in horseracing and equine sports. One gene doping method is by administering exogenous genes, called transgenes, to postnatal animals. Although several transgene detection methods have been developed for horses, many are unsuitable for multiplex detection. In this proof-of-concept study, we developed a highly sensitive and multiplex transgene detection method using multiple πCode with identification patterns printed on the surface. The following steps were employed: (1) multiplex polymerase chain reaction amplification...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time ...
Tozaki T, Ohnuma A, Hamilton NA, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using...
Minato E, Aoshima K, Kobayashi A, Ohnishi N, Sasaki N, Kimura T.Equine herpesvirus 1 (EHV-1) uses equine major histocompatibility complex class I (MHC class I) as an entry receptor. Exogenous expression of equine MHC class I genes in murine cell lines confers susceptibility to EHV-1 infection. To examine the in vivo role of equine MHC class I as an entry receptor for EHV-1, we generated transgenic (Tg) mice expressing equine MHC class I under the control of the CAG promoter. Equine MHC class I protein was expressed in the liver, spleen, lung, and brain of Tg mice, which was confirmed by Western blot. However, equine MHC class I antigen was only detected in...
Farmerie TA, Abbud RA, Budworth PR, Clay CM, Keri RA, McDowell KJ, Wolfe MW, Nilson JH.The equine glycoprotein hormone alpha-subunit gene is expressed in both pituitary and placenta, unlike that of all other nonprimate mammals studied, in which expression is limited to pituitary. Previous studies of the 5'-flanking region of the equine alpha-subunit promoter have revealed unique characteristics as well as similarities with the human alpha-subunit promoter, which demonstrates a similar pattern of tissue-specific expression. We have cloned and sequenced the equine alpha-subunit gene and have used tissue culture systems and transgenic mice to characterize its expression. Unlike the...
Thampi P, Seabaugh KA, Pezzanite LM, Chu CR, Phillips JN, Grieger JC, McIlwraith CW, Samulski RJ, Goodrich LR.Gene therapy approaches using adeno-associated viral vectors have been successfully tested in the equine post-traumatic osteoarthritis (PTOA) model. Owing to differences in the levels of transgene expression and adverse tissue reactions observed in published studies, we sought to identify a safe therapeutic dose of scAAVIL-1ra in an inflamed and injured joint that would result in improved functional outcomes without any adverse events. scAAVIL-1ra was delivered intra-articularly over a 100-fold range, and horses were evaluated throughout and at the end of the 10-week study. A dose-related incr...
Jonsdottir S, Svansson V, Stefansdottir SB, Mäntylä E, Marti E, Torsteinsdottir S.Insect bite hypersensitivity is an immunoglobulin (Ig)E-mediated dermatitis of horses initiated by bites of midges of the genus Culicoides. Culicoides spp. are not indigenous to Iceland and the prevalence of insect bite hypersensitivity is much higher in horses born in Iceland and exported as compared to Icelandic horses born in a Culicoides rich environment. Immunotherapy is therefore needed. Objective: The aim of the study was to express an allergen from Culicoides in barley grain and investigate whether an immune response could be obtained in healthy Icelandic horses by oral treatment with ...
Wilkin T, Hamilton NA, Cawley AT, Bhat S, Baoutina A.The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: , , , and . The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplific...
Yuen BP, Wong KS, So YM, Kwok WH, Cheung HW, Wan TSM, Ho EN, Wong WT.Gene doping involves the misuse of genetic materials to alter an athlete's performance, which is banned at all times in both human and equine sports. Quantitative polymerase chain reaction (qPCR) assays have been used to control the misuse of transgenes in equine sports. Our laboratory recently developed and implemented duplex as well as multiplex qPCR assays for transgenes detection. To further advance gene doping control, we have developed for the first time a sensitive and definitive PCR-liquid chromatography high-resolution tandem mass spectrometry (PCR-LC-HRMS/MS) method for transgene det...
Goodrich LR, McIlwraith CW, Grieger J, Kraus VB, Stabler T, Werpy N, Phillips J, Samulski RJ, Frisbie D.To evaluate the effects of a gene transfer approach to IL-1β inhibition in an equine osteochondral chip fragment model of joint injury using a self-complementary adeno-associated virus with interleukin receptor antagonist transgene cassette (scAAVIL-1ra), as posttraumatic osteoarthritis in horses, similar to people, is a significant clinical problem. Methods: 16 horses were utilized for the study. Methods: All horses had an osteochondral chip fragment induced arthroscopically in one middle carpal joint while the contralateral joint was sham operated. Eight horses received either scAAVIL-1ra o...
