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.
[Construction of an infectious clone of equine infectious anemia virus by N-glycosylation reverse-mutations]. To elucidate the role of N-glycosylation in fetal donkey dermal cell (FDD)-attenuated equine infectious anemia virus (EIAV), we constructed an N-glycosylation reverse-mutation molecular clone, pLGN191N236N246. This viral molecular clone was derived from the infectious clone pLGFD3-8 by site-directed mutagenesis. This clone was used to transfect fetal donkey dermal (FDD) cells. Infectious characteristics of transfectants were monitored by RT-PCR, indirect immune fluorescence and reverse transcriptase activity assay. After three passages in FDD cells, viral replications in the supernatant of cel...
Equine ANXA2 and MMP1 expression analyses in an experimental model of normal and pathological wound repair. Wounds on horse limbs can develop exuberant granulation tissue which resembles the human keloid. Clues gained from the study of over-scarring in horses might help control fibro-proliferative disorders. Objective: The aim of the present study was to clone full-length equine ANXA2 cDNA then to study spatio-temporal expression of ANXA2 and MMP1 mRNA and protein, potential contributors to remodeling, during repair of body (normal) and limb (fibro-proliferative) wounds in an established horse wound model. Methods: Cloning of ANXA2 was achieved by screening size-selected cDNA libraries. Expression w...
Molecular cloning and characterization of equine Toll-like receptor 9. Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three l...
Experimental embryology of mammals at the Jastrzebiec Institute of Genetics and Animal Breeding. Our Department of Experimental Embryology originated from The Laboratory of Embryo Biotechnology, which was organized and directed by Dr. Maria Czlonkowska until her premature death in 1991. Proving successful international transfer of frozen equine embryos and generation of an embryonic sheep-goat chimaera surviving ten years were outstanding achievements of her term. In the 1990s, we produced advanced fetuses of mice after reconstructing enucleated oocytes with embryonic stem (ES) cells, as well as mice originating entirely from ES cells by substitution of the inner cell mass with ES cells. ...
Characterization of a novel, testis-specific equine serine/threonine kinase. Testis-specific protein kinases are important because of their potential role in spermiogenesis, sperm maturation, and sperm function. In the present study, a novel serine-threonine kinase with high identity to human serine-threonine kinase 31 (STK31) was cloned from equine testis and expression of the protein was characterized in equine testis and ejaculated spermatozoa. Five over-lapping independent clones were plaque purified after screening of a lambda ZAP cDNA expression library constructed from equine testis. Sequence analysis and alignment of all five clones showed high identity with hu...
Equine CTNNB1 and PECAM1 nucleotide structure and expression analyses in an experimental model of normal and pathological wound repair. Wound healing in horses is fraught with complications. Specifically, wounds on horse limbs often develop exuberant granulation tissue which behaves clinically like a benign tumor and resembles the human keloid in that the evolving scar is trapped in the proliferative phase of repair, leading to fibrosis. Clues gained from the study of over-scarring in horses should eventually lead to new insights into how to prevent unwanted scar formation in humans. cDNA fragments corresponding to CTNNB1 (coding for beta-catenin) and PECAM1, genes potentially contributing to the proliferative phase of repair,...
Equine laminitis: membrane type matrix metalloproteinase-1 (MMP-14) is involved in acute phase onset. Enzymatic separation at the hoof lamellar dermal-epidermal interface may play a role in the development of laminitis and characterising and locating matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMPs or TIMPs) in lamellar tissues may further understanding of pathogenesis. Objective: To clone and sequence the cDNA encoding lamellar MMP-14 and TIMP-2, and quantify their transcription in normal and laminitic tissue; and to develop antibody to locate MMP-14 in lamellar tissues. Methods: Tissue samples were obtained from an oligofructose induced model of laminitis. Tot...
Molecular cloning and characterization of the alphaX subunit from CD11c/CD18 horse integrin. This work reports the cloning and sequence determination of the horse alpha subunit of the integrin CD11c/CD18, a marker of dendritic cells. A cDNA clone of 4582 base pairs was obtained. It encodes a protein segment of 1086 amino acid residues of the extracellular domain with 10 potential sites of glycosylation, a transmembrane domain of 32 residues and a C-terminal cytoplasmic tail of 24 residues. A phylogenetic analysis of this integrin shows close similarity (83%) with that of Canis familiaris.
Cloning, expression and purification of envelope proteins E1 and E2 of western equine encephalitis virus and potential use of them as antigens in immunoassays. The genes encoding envelope proteins E1 and E2 of western equine encephalitis virus (WEEV) were respectively cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. The recombinant C-terminal 6xHis-tagged WEEV E1 and E2 were expressed in bacteria as inclusion bodies that were subsequently solubilized with 8M urea, purified by immobilized metal ion affinity chromatography and finally refolded using an arginine system. The purified 6xHis-tagged proteins showed 50kDa bands as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, consistent with the expected size...
Cloning and structural analysis of equine platelet endothelial cell adhesion molecule (PECAM, CD31) and vascular cell adhesion molecule-1 (VCAM-1, CD106). Platelet endothelial cell adhesion molecule (PECAM, CD31) and vascular cell adhesion molecule-1 (VCAM-1, CD106) are essential for leukocyte emigration and diapedesis. PECAM is an essential histologic marker of endothelial cells; VCAM-1 is a prototype marker for endothelial cell activation. In this study, equine PECAM and VCAM mRNA were cloned and sequenced. Both genes are highly conserved amongst several species. This study also revealed conserved structural and regulatory motifs, emphasizing the importance of these genes' physiological roles in immunological responses.
Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test. The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia col...
Establishment of a novel equine cell line for isolation and propagation of equine herpesviruses. In the present study, an equine-derived cell line was established by transfecting primary fetal horse kidney (FHK) cells with expression plasmid encoding simian virus 40 (SV40) large T antigen and then cloning them by limiting dilution. The cloned cell line, named FHK-Tcl3, grew well and could be propagated over 30 times by splitting them 1:3. Equine herpesvirus (EHV)-1 and EHV-4 replicated well in FHK-Tcl3. EHV-2 and EHV-4 were isolated from samples collected from horses in the field using FHK-Tcl3, and EHV-3 also propagated in FHK-Tcl3. These results indicated that this novel cell line, FHK-...
Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin. We evaluated the effect of different activation treatments on the production of blastocysts and foals by nuclear transfer. Donor cells were prepared using roscovitine treatment, which has previously been associated with increased production of viable offspring. All activation treatments were followed by culture in 6-dimethylaminopurine (6-DMAP) for 4 h. In experiment 1, blastocyst production after activation by injection of sperm extract followed by treatment with ionomycin was significantly higher than that for activation with a serial treatment of ionomycin, 6-DMAP, and ionomycin (12.5 vs 2....
Heterospecific nuclear-transferred embryos derived from equine fibroblast cells and enucleated bovine oocytes. This study was conducted to reconstruct heterogeneous embryos using equine skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplast for investigating the reprogramming of equine somatic cell nuclear in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Adult horse skin fibroblast cells serum-starved were used as donor somatic cells. Bovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electofusion. The fused eggs were activated by inomycin ...
Development and characterization of an infectious cDNA clone of the virulent Bucyrus strain of Equine arteritis virus. Strains of Equine arteritis virus (EAV) differ in the severity of the disease that they induce in horses. Infectious cDNA clones are potentially useful for identification of genetic determinants of EAV virulence; to date, two clones have been derived from a cell culture-adapted variant of the original (Bucyrus) isolate of EAV, and it has previously been shown that recombinant virus derived from one of these (rEAV030) is attenuated in horses. A complete cDNA copy of the genome of the virulent Bucyrus strain of EAV has now been assembled into a plasmid vector. In contrast to rEAV030, recombinant...
Immune selection of equine infectious anemia virus env variants during the long-term inapparent stage of disease. The principal neutralizing domain (PND) of equine infectious anemia virus (EIAV) is located in the V3 region of SU. Genetic variation in the PND is considered to play an important role in immune escape and EIAV persistence; however, few studies have characterized genetic variation in SU during the inapparent stage of disease. To better understand the mechanisms of virus persistence, we undertook a longitudinal study of SU variation in a pony experimentally inoculated with the virulent EIAV(Wyo). Viral RNA isolated from the inoculum and from sequential sera samples was amplified by RT-PCR, clon...
Cloning of equine prostaglandin dehydrogenase and its gonadotropin-dependent regulation in theca and mural granulosa cells of equine preovulatory follicles during the ovulatory process. The mammalian ovulatory process is accompanied by a gonadotropin-dependent increase in follicular levels of prostaglandin E2 (PGE2) and PGF2alpha, which are metabolized by 15-hydroxy prostaglandin dehydrogenase (PGDH). Little is known about ovarian PGDH regulation in non-primate species. The objectives of this study were to characterize the structure of equine PGDH and its regulation in follicles during human chorionic gonadotropin (hCG)-induced ovulation. The full-length equine PGDH was obtained by RT-PCR, 5'- and 3'-rapid amplification of cDNA ends (RACE). Its open reading frame encodes a 26...
Molecular characterization of tumor necrosis alpha-induced protein 6 and its human chorionic gonadotropin-dependent induction in theca and mural granulosa cells of equine preovulatory follicles. The preovulatory rise in gonadotropins causes an expansion of the cumulus-oocyte complex, a process requiring the induction of several genes. The objectives of this study were to clone the equine tumor necrosis factor alpha-induced protein 6 (TNFAIP6), and investigate its regulation in equine follicles during human chorionic gonadotropin (hCG)-induced ovulation. The isolation of the equine TNFAIP6 cDNA revealed that it contains an open reading frame of 834 bp (including the stop codon), encoding a predicted 277 amino acid protein that is highly similar (91-93% identity) to known mammalian homo...
Molecular cloning of equine 17beta-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo. The type 1 form of 17beta-hydroxysteroid dehydrogenase (17betaHSD1) was the first isoform to be identified and is capable of converting estrone to 17beta-estradiol. This study was aimed at characterizing the molecular structure of the equine 17betaHSD1 gene and cDNA, as well as its molecular regulation during human chorionic gonadotropin (hCG)-induced follicular luteinization/ovulation in vivo. The equine 17betaHSD1 gene was cloned from an equine genomic library and shown to have a conserved genomic structure composed of six exons. Its cDNA sequence was also identified and coded for a 308 amin...
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...