Topic:Cloning
Cloning in horses involves the process of creating a genetically identical copy of an original horse through somatic cell nuclear transfer (SCNT). This technique involves transferring the nucleus of a somatic cell from the donor horse into an enucleated oocyte, which is then stimulated to develop into an embryo and implanted into a surrogate mare. Cloning has been utilized for various purposes, including the preservation of valuable genetics, reproduction of geldings, and research into genetic diseases. The practice raises discussions on genetic diversity, animal welfare, and ethical considerations. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and implications of cloning in equine science.
Cloning and functional characterization of recombinant equine P-selectin. The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Further...
Equine cloning. Equine cloning is now in use as a clinical technique. It is available commercially, and its efficiency seems to be increasing. The foals produced by cloning may differ in some phenotypic and behavioral traits from the original animal but should produce offspring that reflect those that the original donor animal would have produced. This is especially true in the case of male animals, where the mitochondrial DNA is not passed to the progeny. Results of pregnancies due in 2006 should add significantly to our understanding of the factors affecting production of viable cloned foals and of the simi...
Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep. Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and sign...
Molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene on horse chromosome 2p16–>p15. The mammalian collagen, type IX, alpha 2 gene (COL9A2) encodes the alpha-2 chain of type IX collagen and is located on horse chromosome 2p16-->p14 harbouring a quantitative trait locus for osteochondrosis. We isolated a bacterial artificial chromosome (BAC) clone containing the equine COL9A2 gene and determined the complete genomic sequence of this gene. Cloning and characterization of equine COL9A2 revealed that the equine gene consists of 32 exons spanning approximately 15 kb. The COL9A2 transcript encodes a single protein of 688 amino acids. Thirty two single nucleotide polymorphisms (SNPs)...
Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer. Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo devel...
Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane. We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency ...
cDNA cloning and substrate specificity of equine tryptase, a possible mediator in equine heaves. Mast cell mediators are believed to play a central role in inflammatory lung disorders such as human allergic and occupational asthma. Equine heaves is characterized by reversible neutrophilic airway inflammation and airway obstruction, primarily due to bronchospasm and mucus hypersecretion, following exposure of susceptible horses to organic stable dusts. As such, heaves shares many similarities with human occupational dust-induced asthma and therefore it is proposed that mast cells may also be implicated in the pathogenesis of heaves. Tryptase, a mast cell-specific proteinase, can be used as...
Distribution of CCR3 mRNA expression in horse tissues. CCL11 (also known as eotaxin) is a very potent and selective mediator of eosinophil migration which exerts its effects through its receptor, CCR3. In this study we report the cloning of an equine CCR3 cDNA sequence and investigation of the localization of CCR3 mRNA expression in horse tissues. Equine CCR3 displayed high levels of sequence identity with CCR3 sequences in other species. RT-PCR analysis revealed the expression of CCR3 in colon, lung and spleen of normal horses. In situ hybridisation experiments indicated that expression of CCR3 mRNA in colon was predominantly in eosinophils and t...
[Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay]. According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was c...
Developments in European horse breeding and consequences for veterinarians in equine reproduction. The liberalization of European animal breeding legislation and an increasing diversity of equestrian sports have led to a constant rise in the number of horse breeds and breed registries. In addition to the trend towards more and smaller breed registries, there is another trend towards an international expansion of the bigger established sport horse breeds. Regional breeds, at least in smaller countries, may no longer be able to run an independent breeding programme. The typical horse breeder, in the future, will be a female and qualified in equestrian sports. Artificial insemination (AI) main...
Derivation, maintenance, and induction of the differentiation in vitro of equine embryonic stem cells. We describe here the isolation and maintenance of pluripotent embryonic stem (ES) cells from equine blastocysts that have been frozen and thawed. Equine ES cells appear to maintain a normal diploid karyotype in culture. These cells express markers that are characteristic of mouse ES cells, namely, alkaline phosphatase, stage-specific-embryonic antigen 1, STAT3, and Oct4. We also describe protocols for the induction of differentiation in vitro to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor, and platelet-derived growth factor and to he...
Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia. The aim of the current study was to clone the equine adenosine A(2A) receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA(2A)-R expression construct was generated by ligation of the eA(2A) cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA(2A)-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K(D)), and receptor densities (B(max)) ...
Cloning and pharmacological characterization of the equine adenosine A3 receptor. The aim of this study was to establish a heterologous expression system for the equine adenosine A(3) receptor (eA(3)-R) in an effort to ascertain its pharmacologic profile. Initially, radioligand binding assays identified clones expressing the eA(3)-R in human embryonic kidney cells (HEK) based on the specific binding of [(125)I]AB-MECA. Subsequently, adenylate cyclase assays were utilized to demonstrate functional coupling of the eA(3)-R to the G-protein/adenylate cyclase system. Equilibrium competition binding assays were then performed using selective and non-selective A(3) agonists and an...
Molecular cloning and gonadotropin-dependent regulation of equine prostaglandin F2alpha receptor in ovarian follicles during the ovulatory process in vivo. The progressive rise in gonadotropins prior to ovulation triggers a marked increase in intrafollicular levels of prostaglandin F(2alpha)(PGF(2alpha)), which is known to interact with PGF(2alpha) receptor (FP). Little is known about the regulation of FP during ovulation. This study was undertaken to characterize the equine FP and its gonadotropin-dependent regulation in preovulatory follicles prior to ovulation. The full-length equine FP encodes a 366-amino acid protein that is 82-93% homologous to other species. Using semi-quantitative RT-PCR/Southern blot, we showed that FP mRNA expression wa...
Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract. We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect bl...
Cloning and expression of 51-kDa antigenic protein of Neorickettsia risticii NR-JA1. Neorickettsia (Ehrlichia) risticii is a causative agent of acute diarrheal syndrome in horses, commonly known as Potomac horse fever. Korean isolate of N. risticii NR-JA1 was cultivated in mouse macrophage cell line P388D1. A complete ORF of p51 antigenic protein gene was amplified and cloned into pQE32 and pcDNA3.1 vectors and the resultant clones were named as pQE32/Nr-51 and pcDNA3.1/Nr-51, respectively. Recombinant p51 (rp51) protein antigen was expressed in E. coli (pQE32/Nr-51) and cos-7 cell line (pcDNA3.1/Nr-51). The rp51 protein showed immunoreactivity with anti- mouse p51 antibodies....
Equine cloning: applications and outcomes. Cloning is one of several new assisted reproductive techniques being developed for clinical use in the equine industry. Potential uses of equine cloning include: (1) the preservation of genetics from individual animals that would otherwise not be able to reproduce, such as geldings; (2) the preservation of genetic material of endangered and/or exotic species, such as the Mongolian wild horse (Przewalski's horse); and (3) because of the companion animal role that horses fill for some individuals, it is likely that some horse owners will have individual animals cloned for emotional fulfillment. ...
Studies of fibronectin-binding proteins of Streptococcus equi. Streptococcus equi subsp. equi is the causative agent of strangles, a disease of the upper respiratory tract in horses. The initiation of S. equi subsp. equi infection is likely to involve cell surface-anchored molecules mediating bacterial adhesion to the epithelium of the host. The present study describes the cloning and characterization of FNEB, a fibronectin-binding protein with cell wall-anchoring motifs. FNEB can thus be predicted as cell surface located, contrary to the two previously characterized fibronectin-binding proteins in S. equi subsp. equi, FNE and SFS. Assays of antibody tite...
Cloning and expression of the extra-cellular part of the alpha chain of the equine high-affinity IgE receptor and its use in the detection of IgE. The high-affinity receptor for IgE (FcepsilonRI) plays a central role in IgE-mediated allergic reactions. Cross-linking of FcepsilonRI by IgE-antigen complexes results in the activation of mast cells and basophils and is thought to contribute to the immunopathology of Heaves, a chronic obstructive pulmonary disease of horses. Recombinant protein corresponding to the extra-cellular portion of the FcepsilonRI alpha subunit, cloned and sequenced previously, was expressed using both mammalian cells and insect cells. The yield of expressed protein was considerably greater using insect cells and the...
Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type. The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear...
Molecular characterization and functional expression of equine interleukin-1 type I and type II receptor cDNAs. cDNA generated from lipopolysaccharide-stimulated equine peripheral blood mononuclear cells was used to amplify and clone type I and type II equine interleukin-1 receptors (IL-1RI and IL-1RII) using primers derived from semi-conserved regions between human and mouse IL-1RI and IL-1RII sequences, respectively. 5' and 3' terminal sequences of equine IL-1RI and IL-1RII were amplified by 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of equine IL-1RI demonstrated 77, 64 and 63% similarity with human, mouse and rat sequences, respectively. The predicted amino acid seque...
Molecular cloning and characterization of markers and cytokines for equid myeloid cells. The myeloid cell system comprises of monocytes, macrophages (MPhi), dendritic cells (DC), Kupffer cells, osteoclasts or microglia and is also known as the mononuclear phagocytic system (MPS). Essential cytokines to differentiate or activate these cells include GM-CSF or IL-4. Important markers for characterization include CD1, CD14, CD68, CD163 and CD206. All these markers, however, were not cloned or further characterized in equids by use of monoclonal antibodies earlier. To overcome this problem with the present study, two approaches were used. First, we cloned equine cytokines and markers, ...
Establishment of cloned Anaplasma phagocytophilum and analysis of p44 gene conversion within an infected horse and infected SCID mice. Diverse p44 alleles at the p44 expression locus (p44Es) encoding surface-exposed major membrane proteins, P44s, of Anaplasma phagocytophilum were hypothesized to be garnered by recombination to enact antigenic variation. However, this hypothesis has not been proven so far, due to inability to clone this obligate intragranulocytic rickettsia. To define the p44E recombination, we developed a novel method to clone A. phagocytophilum. This isogenic cloned population containing a defined p44E was used to infect a naive horse and severe combined immunodeficiency (SCID) mice. During a 58-day infectio...
Sequence analysis of canine and equine ferritin H and L subunit cDNAs. Canine and equine ferritin H and L subunit cDNA clones were obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) and TA cloning from various tissues. Canine liver and spleen ferritin H subunit cDNA clones contained an open reading frame for the same 182-amino acid protein as that reported in canine brain ferritin H subunit cDNA although there were substitutions in the 3'-noncoding regions. Ferritin L subunit cDNA clones from canine liver, spleen, and kidney showed identical coding sequences encoding the 174-amino acid protein except for a single nucleotide substitution in ki...
The development and application of the modern reproductive technologies to horse breeding. Although the horse was probably the first animal to experience and benefit from artificial insemination, it trailed the field somewhat with regard to the application of embryo transfer and other oocyte and embryo-related modern breeding technologies. But with a late run it is now back in mid-field and gaining fast on the other large domestic species in the application of the many technological advances of the past 20 years to sound breeding practice. Improvements in extenders and cryoprotectants have resulted in a veritable upsurge in the transport and insemination of cooled and frozen stallio...
Update on equine ICSI and cloning. Intracytoplasmic sperm injection (ICSI) has recently become efficient enough to be considered for clinical use. With ICSI, one spermatozoa is injected into a mature oocyte. Harvesting of an oocyte ex vivo, followed by ICSI and transfer of the fertilized oocyte to the oviduct, may be applicable when semen quality is insufficient for standard insemination. Sperm injection, followed by in vitro embryo culture to the blastocyst stage, may be used in cases where multiple oocytes are to be fertilized (e.g. when oocytes are collected post-mortem). Nuclear transfer (cloning) of horses is possible but ...
A tumor necrosis factor receptor family protein serves as a cellular receptor for the macrophage-tropic equine lentivirus. Characterization of cellular receptors for human, simian, and feline immunodeficiency viruses that are tropic for lymphocytes and macrophages have revealed a common theme of a sequential binding of viral envelope proteins with two coreceptors to mediate virus infection of target cells. In contrast to these dual tropic immunodeficiency viruses, the ungulate lentiviruses, including equine infectious anemia virus (EIAV), exclusively infect cells of the monocyte-macrophage lineage to cause progressive degenerative diseases without clinical immunodeficiency. EIAV causes a uniquely dynamic disease t...
Expression of equine glucose transporter type 4 in skeletal muscle after glycogen-depleting exercise. To clone and sequence cDNA for equine insulin-responsive glucose transporter (glucose transporter type 4 [GLUT-4]) and determine effects of glycogen-depleting exercise and meal type after exercise on GLUT-4 gene expression in skeletal muscle of horses. Methods: Muscle biopsy specimens from 7 healthy adult horses. Methods: Total RNA was extracted from specimens, and GLUT-4 cDNA was synthesized and sequenced. Horses were exercised on 3 consecutive days. On the third day of exercise, for 8 hours after exercise, horses were either not fed, fed half of daily energy requirements as hay, or fed an is...