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, comparative sequence analysis and mRNA expression of calcium-transporting genes in horses. Epithelial calcium transport occurs by paracellular and transcellular mechanisms. Transcellular transport in intestinal and renal epithelia involves several transport proteins, including transient receptor potential vanilloid member 5 (TRPV5), member 6 (TRPV6), calbindin D9k (CB9), calbindin D28k (CB28), sodium calcium exchanger 1 (NCX1), plasma membrane calcium ATPase 1 (PMCA1), and the vitamin D receptor (VDR). We are interested in the horse because of its unique calcium physiology (high blood calcium, high intestinal calcium absorption, high renal excretion of calcium, low vitamin D concent...
Contribution of each of four Superantigens to Streptococcus equi-induced mitogenicity, gamma interferon synthesis, and immunity. Streptococcus equi is the causative agent of strangles, the most frequently diagnosed infectious disease of horses worldwide. The disease is characterized by abscessation and swelling of the lymph nodes of the head and neck, which can literally strangle the horse to death. S. equi produces four recently acquired phage-associated bacterial superantigens (sAgs; SeeH, SeeI, SeeL, and SeeM) that share homology with the mitogenic toxins of Streptococcus pyogenes. The aim of this study was to characterize the contribution of each of these S. equi sAgs to mitogenic activity in vitro and quantify the ...
Detection of treponemes in canker lesions of horses by 16S rRNA clonal sequencing analysis. Equine canker is a chronic pododermatitis of the hoof in horses. Although spirochetes are detectable histopathologically in the lesions, the precise etiology remains unclear. This study reports the 16S rRNA gene sequencing of randomly selected clones based on PCR with Treponema-specific primers, using the canker lesions from two horses and healthy frog and sole from a horse. A total of 114 clones were obtained from the lesions, but no clones were detected in the healthy hoof tissues. The clones from the canker lesions examined were grouped into 19 operational taxonomic units, such as treponema...
Cloning, sequencing and expression analysis of the equine hepcidin gene by real-time PCR. Equine serum or plasma iron concentration drops quickly during inflammation. Accumulation of iron inside macrophages and reduction of the intestinal absorption of this element cause hypoferremia during systemic inflammatory processes. These mechanisms are mediated by hepcidin, a 25 amino acids peptide synthesized mainly in the liver in response to iron stores and inflammation. Hepcidin is an important peptide for systemic iron homeostasis and also has antibacterial and antifungal activities. Hepcidin up-regulation is particularly useful during acute inflammation, especially before adaptive imm...
Cloning of IgE-binding proteins from Simulium vittatum and their potential significance as allergens for equine insect bite hypersensitivity. Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides and sometimes Simulium spp. The aim of this investigation was to identify Simulium allergens associated with IBH. A phage surface display cDNA library expressing recombinant Simulium vittatum salivary gland proteins was screened using sera of IBH-affected horses sensitized to S. vittatum salivary gland proteins as shown in immunoblot, resulting in the identification of seven cDNAs encoding IgE-binding proteins. The deduced amino acid sequences of these proteins showed sequence similarities to a...
[Construction and in vitro evaluation of an infectious clone of the equine infectious anemia virus vaccine strain EIAV(FDDV) with four reverse-mutated vaccine-specific sites in the S2 gene]. To elucidate the function of the S2 gene in equine infectious anemia virus (EIAV) and its role in the attenuation of the Chinese attenuated EIAV vaccine strains, the S2 in the EIAV vaccine strain EIAV (FDDV) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. Based on the sequence variation of the S2 gene between the EIAV virulent strains and vaccine strains, all the four vaccine-specific sites in the S2 of an EIAV(FDDV) infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAV(DV115). The reverse-mutated molecula...
Expressed gene sequences of the equine cytokines interleukin-17 and interleukin-23. This report describes the initial cloning and characterization of the equine interleukin-17 (IL-17) expressed gene sequence from mRNA obtained from equine intestinal tissue and interleukin-23 (IL-23) expressed gene sequence from mRNA obtained from equine peripheral blood mononuclear cells. Equine IL-17 has 462 nucleotides in the translated region, determined by homology with known human and mouse sequences, and shares 84% and 75% identity, respectively. For the deduced amino acid sequences, the identity with human and mouse is 76% and 70%. Equine IL-23 has 579 nucleotides in the translated reg...
Expression and purification of active recombinant equine lysozyme in Escherichia coli. Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purificati...
Immunogenicity and antigenicity of the recombinant EMA-1 protein of Theileria equi expressed in the yeast Pichia pastoris. The equine piroplasmosis caused by Theileria equi is one of the most important parasitic diseases of the equine, causing damage to animal health and economic losses. In T. equi, 2 merozoite surface proteins, equi merozoite antigen EMA-1 and EMA-2, have been identified as the most immunodominant antigens. This suggests that these antigens might be used as immunobiological tools. The EMA-1 of Theileria equi was cloned and expressed in the yeast Pichia pastoris. The transformed yeast was grown at high cell density, expressing up to 389 mg x L(-1) of recombinant protein. The protein was concentrat...
Further Development of an Equine Cell Line that can be Propagated over 100 Times. Cell lines originating from horses are necessary for isolation and propagation of equine herpesviruses (EHV). Although we established an equine-derived cell line, FHK-Tcl3, propagation ceased after fewer than 40 passages. In this study, FHK-Tcl3 cell propagation continued beyond 40 passages, achieving over 100 passages. FHK-Tcl3 cells were then cloned by limiting dilution at the 100th passage. Cloned cells were termed FHK-Tcl3.1. FHK-Tcl3.1 cells grew well and were propagated every 3 to 4 days by splitting 1:5. In addition, EHV-1, -2 and -4 showed a clear cytopathic effect (CPE) in FHK-Tcl3.1 ...
Identification and isolation of cDNA clones encoding the abundant secreted proteins in the saliva proteome of Culicoides nubeculosus. Culicoides spp. are vectors of several infectious diseases of veterinary importance and a major cause of allergy in horses and other livestock. Their saliva contains a number of proteins which enable blood feeding, enhance disease transmission and act as allergens. We report the construction of a novel cDNA library from Culicoides nubeculosus linked to the analysis of abundant salivary gland proteins by mass spectrometry. Fifty-four novel proteins sequences are described including those of the enzymes maltase, hyaluronidase and two serine proteases demonstrated to be present in Culicoides sali...
Effect of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line on efficiency of equine nuclear transfer. We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase ...
Risk of equine infectious anemia virus disease transmission through in vitro embryo production using somatic cell nuclear transfer. Prevention and regulation of equine infectious anemia virus (EIAV) disease transmission solely depend on identification, isolation, and elimination of infected animals because of lack of an effective vaccine. Embryo production via the somatic cell nuclear transfer (SCNT) technology uses oocytes collected mainly from untested animals, which creates a potential risk of EIAV transmission through infected embryos. The current review examines the risk of EIAV disease transmission through SCNT embryo production and transfer. Equine infectious anemia virus is a lentivirus from the family Retroviridae...
Restriction of equine infectious anemia virus by equine APOBEC3 cytidine deaminases. The mammalian APOBEC3 (A3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. The A3 locus on the chromosome 28 of the horse genome contains multiple A3 genes: two copies of A3Z1, five copies of A3Z2, and a single copy of A3Z3, indicating a complex evolution of multiple gene duplications. We have cloned and analyzed for expression the different equine A3 genes and examined as well the subcellular distribution of the corresponding proteins. Additionally, we have tested the functional antiretroviral activity of the equi...
Synthesis and characterization of biologically active recombinant elk and horse FSH. The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in...
Equine thrombospondin II and secreted protein acidic and cysteine-rich in a model of normal and pathological wound repair. Wound healing in horses is complicated, particularly when wounds are on the limb. The objectives of this study were to clone equine thrombospondin II (THBS2) and secreted protein acidic and cysteine-rich (SPARC) cDNAs and to compare the spatiotemporal expression of mRNAs and proteins during repair of body and limb wounds. These molecules were targeted in view of their potential biological contribution to angiogenesis, which is exacerbated during the repair of limb wounds in horses. Cloning was achieved by screening size-selected cDNA libraries previously derived from 7-day-old wounds. Expressi...
Expression of biologically active recombinant equine interferon-gamma in Escherichia coli. Interferon gamma (IFN-gamma) is a pleiotropic cytokine that is recognized as an important modulator of the immune response. To date, there is no report that prokaryocyte-derived recombinant equine IFN-gamma has antiviral activity. In this report, the gene coding equine IFN-gamma (EIFN-gamma) mature protein was cloned into pET-28a (+) and the recombinant EIFN-gamma was expressed in Escherichia coli (E. coli). The antiviral activity of expressed recombinant EIFN-gamma was evaluated by using a recombinant Vesicular Stomatitis Virus expressing green fluorescence protein (rVSV-GFP) system in the eq...
Establishment and characterization of a fibroblast cell line from the Mongolian horse. A fibroblast line was successfully established from Mongolian horse ear marginal tissue by using a primary explant technique and cell cryogenic preservation technology. Biological analysis showed the following: The cells were adherent and exhibited density-dependent inhibition of proliferation; assays of microbial contamination from bacteria, fungi, and mycoplasma were negative; the population doubling time of the cells was 33.9 h; and a 2n chromosome number of 64 at a frequency higher than 80%. A lack of cross-contamination of this cell line with other species was confirmed by isoenzyme analy...
Equine cytochrome P450 2C92: cDNA cloning, expression and initial characterization. Substantial gaps exist in our knowledge of the metabolic clearance of therapeutic agents in horses. Accordingly, a cytochrome P450 monooxygenase in the 2C family was cloned from an equine liver, sequenced and expressed in a baculovirus expression system. Catalytic activities of the recombinant protein were measured with a number of substrates. The protein, assigned CYP2C92, displayed optimal catalytic activity with diclofenac using molar ratios of CYP2C92 to NADPH CYP450 reductase of 1:18. Addition of cytochrome b(5) to diclofenac incubations had no significant effect on metabolic turnover. CY...
OB-cadherin cloning and expression in a model of wound repair in horses. Horses suffer from a debilitating impediment in repairing wounds located on the lower limb that leads to the development of a fibroproliferative disorder (exuberant granulation tissue). This condition is a source of wastage since it often forces retirement from competition. Treatments that resolve or prevent this condition are still lacking, maybe due to deficient knowledge of the underlying molecular mechanisms. Fibroblast-to-myofibroblast conversion is an essential step allowing contraction during wound repair and is accompanied by an increase in OB-cadherin expression. Objective: To clone e...
Nucleotide structure and expression of equine pigment epithelium-derived factor during repair of experimentally induced wounds in horses. To clone full-length equine pigment epithelium-derived factor (PEDF) complementary DNA (cDNA) and to evaluate its temporal expression during repair of wounds in horses. Methods: 4 clinically normal 2-to 3-year-old Standardbred mares. Methods: Full-length equine PEDF cDNA was cloned by screening size-selected cDNA libraries derived from biopsy specimens obtained from the wound edge 7 days after experimental creation of a 6.25-cm(2) full-thickness wound in the skin of the lateral thoracic wall. Expression was evaluated in normal skin and in biopsy specimens obtained weekly from experimentally in...
Hypoxic/ischemic encephalopathy associated with placental insufficiency in a cloned foal. Hypoxic/ischemic encephalopathy in a cloned American Quarter horse foal was initially associated with placental insufficiency and exacerbated by protracted hypotension during anesthesia for a surgical procedure. The foal, born at the Texas A&M Veterinary Medical Center, was diagnosed at birth with neonatal maladjustment syndrome that was accompanied by dysmaturity, muscle contracture of the front limbs, and a blood clot within the lumen of the urinary bladder. Seizures that developed after anesthesia were attributed to hypoxia/ischemia during anesthesia and culminated in death. Macroscopically...
[Applications of SSCP and HMA for polymorphic analysis of horse MHC-I alleles]. In this article, we report the analysis of genetic polymorphisms of horse MHC-I molecules by SSCP and HMA, which are methods based on the technique of polyacrylamide gel electrophoresis (PAGE). Our results showed that SSCP was not a suitable method for the analysis of genetic polymorphisms of horse MHC-I molecules due to the failure in generating satisfied separation of DNA fragments, even if experimental conditions were optimized. However, the HMA method produced clearly separated DNA fragments of horse MHC-I molecules, after the experimental conditions, such as the running temperature and th...
Laminin receptor 1 is differentially expressed in thoracic and limb wounds in the horse. Healing of wounds located on the distal limbs of horses is often complicated by retarded epithelialization and the development of exuberant granulation tissue (proud flesh). Treatments that definitively resolve this pathological process are still unavailable. Molecular studies of the repair mechanism might contribute to the development of new therapeutic strategies. The study presented herein aimed to clone the full length cDNA and to study the spatio-temporal expression profile of mRNA and protein for LAMR1, previously attributed a role in wound epithelialization, during the repair of body an...
Novel interferon delta genes in mammals: cloning of one gene from the sheep, two genes expressed by the horse conceptus and discovery of related sequences in several taxa by genomic database screening. Interferons (IFNs) are cytokines of vertebrates with many biological effects including antiviral, immunoregulatory and antiproliferative activities. Among them, mammalian type I IFNs represents a large family of related proteins, mainly virus-inducible, divided in 10 distinct subfamilies named alpha, beta, omega, delta, epsilon, alphaomega, nu, tau, kappa and zeta (or Limitin). Some type I IFN subfamilies are physiologically expressed by the conceptus during early pregnancy in ungulates. This is the case in ruminants with IFN-tau (which triggers the maintenance of the maternal corpus luteum du...
A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species. Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated ...
Somatic cell nuclear transfer in horses. The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids an...
Complementary DNA cloning, functional expression and characterization of a novel cytochrome P450, CYP2D50, from equine liver. Members of the CYP2D family constitute only about 2-4% of total hepatic CYP450s, however, they are responsible for the metabolism of 20-25% of commonly prescribed therapeutic compounds. CYP2D enzymes have been identified in a number of different species. However, vast differences in the metabolic activity of these enzymes have been well documented. In the horse, the presence of a member of the CYP2D family has been suggested from studies with equine liver microsomes, however its presence has not been definitively proven. In this study a cDNA encoding a novel CYP2D enzyme (CYP2D50) was cloned f...
Gonadotropin-dependent regulation of the prostaglandin E2 receptor in equine preovulatory follicles during the ovulatory process in mares. The objectives of the study were to clone the primary structure of the prostaglandin E2 receptor subtype 2 (PTGER2) cDNA and to characterize its regulation in equine follicles during gonadotropin-induced ovulation. Results from DNA isolation indicated that the equine PTGER2 cDNA encodes a predicted 353-amino acid protein, which is highly similar (76-85%) to known mammalian homologues. The regulation of PTGER2 was studied by semi-quantitative RT-PCR/Southern blot using preparations of theca interna and mural granulosa cells isolated from equine follicles 0-39 hr post-treatment with human chorio...
The cloned equine thyrotropin receptor is hypersensitive to human chorionic gonadotropin; identification of three residues in the extracellular domain involved in ligand specificity. The receptors for TSH, LH/chorionic gonadotropin (CG), and FSH belong to the same subfamily of G protein-coupled receptors. The specificity of recognition of their cognate hormone involves a limited number of residues in the leucine-rich repeats present in the N-terminal ectodomain of the receptor. It is admitted that receptors of this subfamily coevoluted with their respective ligands. The secretion of CG is restricted to gestation of primates and Equidae. We hypothesized that, facing the challenge of a new hormone, the glycoprotein hormone receptors would have evolved differently in Equidae ...