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 characterization of gene TNF alpha encoding equine tumor necrosis factor alpha. We report the molecular cloning and nucleotide sequence of the equine gene encoding tumor necrosis factor alpha. The 2610-bp genomic sequence was derived from three overlapping polymerase chain reaction products.
Three monoclonal antibodies identifying antigens on all equine T lymphocytes, and two mutually exclusive T-lymphocyte subsets. The aim of this study was to produce monoclonal antibodies (mAb) recognizing equine lymphocyte surface antigens. Fusions were conducted using BALB/c mice hyperimmunized with equine thymocytes. Hybridoma supernatants were screened by flow cytometry and positive hybridomas were cloned twice by limiting dilution. These mAb were then characterized for tissue distribution by immunohistology and flow cytometry, and by precipitation and analysis of the lymphocyte antigens which they recognized. Three mAb (CVS5, CVS4 and CVS8) are described which recognize only T lymphocytes in peripheral blood. Two-c...
Characterization of variable regions in the envelope and S3 open reading frame of equine infectious anemia virus. The polymerase chain reaction was used to amplify and clone parts of the envelope gene and overlapping S3 open reading frame, thought to encode rev, of the virulent in vivo-derived Th-1 isolate of equine infectious anemia virus (EIAV). The results indicated that EIAV consists of a heterogeneous mixture of genotypes present at the first febrile cycle after initial infection. We showed that the Th-1 isolate apparently contains nondefective genotypes as well as types which have transmembrane protein truncations or are rev deficient. Furthermore, we could confirm the presence of a hypervariable re...
DNA probes for the detection of Babesia caballi. A genomic library of Babesia caballi DNA was constructed in the plasmid vector pUC13. The specificity of the clones for B. caballi was established by the lack of hybridization to Babesia equi, Babesia bovis, Babesia bigemina and equine DNA. Two probes, pBC11 and pBC191, were isolated that could detect 0.25 ng and 0.125 ng of B. caballi DNA, corresponding to a parasitaemia of 0.12% and 0.06% respectively. pBC191 could detect B. caballi parasites in the blood of an experimentally infected horse as well as in naturally infected horses.
Cloning the cDNA for horse growth hormone and expression in Escherichia coli. A 514 bp cDNA transcript coding for 78% of horse (Equus caballus.) GH has been cloned and sequenced. The deduced amino acid sequence corresponded precisely to that previously obtained by protein sequencing and, in addition, provided new sequence information for the signal peptide. The missing 3' fragment of the cDNA was reconstructed using synthetic oligonucleotides and site-specific directed mutagenesis. The complete cDNA sequence was then inserted into an expression vector (PIN-III-lppp-5) which utilizes a bacterial signal peptide to secrete the expressed product into the periplasmic space o...
Evolution of placenta-specific gene expression: comparison of the equine and human gonadotropin alpha-subunit genes. Primate and equine species are thought to be unique among mammals in synthesizing placental gonadotropin glycoprotein hormones. Human chorionic gonadotropin (CG) and equine pregnant mare's serum gonadotropin (PMSG) are produced in placenta by the specific activation of a glycoprotein hormone alpha-subunit gene and a corresponding beta-subunit gene. The evolutionary mechanisms for the apparently independent acquisition of tissue specificity were investigated by cloning the 5' flanking region of the equine alpha-subunit gene and comparing the DNA elements and trans-acting factors involved in pla...
Equine infectious anemia virus derived from a molecular clone persistently infects horses. A full-length molecular clone of equine infectious anemia virus (EIAV) was isolated from a persistently infected canine fetal thymus cell line (Cf2Th). Upon transfection of equine dermis cells, the clone, designated CL22, yielded infectious EIAV particles (CL22-V) that replicated in vitro in both Cf2Th cells and an equine dermis cell strain. Horses infected with CL22-V developed an antibody response to viral proteins and possessed viral DNA in peripheral blood mononuclear cells, as determined by polymerase chain reaction assays. In addition, horses infected with CL22-V became persistently infe...
Molecular cloning of Ehrlichia risticii and development of a gene probe for the diagnosis of Potomac horse fever. A gene bank of Ehrlichia risticii was constructed in plasmid vector pUC13. Five clones representing discrete regions of the E. risticii genome were tested for their ability to hybridize specifically to E. risticii DNA. None of the clones cross-hybridized with Ehrlichia equi DNA, whereas four of these clones cross-hybridized with Ehrlichia canis and Ehrlichia sennetsu DNAs. However, one clone carrying a 1-kilobase HindIII fragment of E. risticii DNA failed to cross-react with the genomes of E. sennetsu, E. canis, and E. equi in dot blot hybridization assays. The sensitivity of this probe for th...
Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins. We isolated and characterized six cDNA clones from an equine infectious anemia virus-infected cell line that displays a Rev-defective phenotype. With the exception of one splice site in one of the clones, all six cDNAs exhibited the same splicing pattern and consisted of four exons. Exon 1 contained the 5' end of the genome; exon 2 contained the tat gene from mid-genome; exon 3 consisted of a small section of env, near the 5' end of the env gene; and exon 4 contained the putative rev open reading frame from the 3' end of the genome. The structures of the cDNAs predict a bicistronic message in ...
Application of cloned fragments of equine herpesvirus type-1 DNA for detection of virus-specific DNA in equine tissues. Tissue specimens obtained from equine herpesvirus-1 (EHV-1), subtype 1-infected aborted foetuses were analysed for the presence of virus DNA by means of Southern blot and dot blot hybridisations. The specificity of the methods was confirmed although the sensitivity was inferior to classical techniques such as virus isolation. However, the possibility of detecting the state of the virus DNA and the ability to distinguish between subtypes were important features, and the dot blot method was shown to have potential for a rapid diagnostic test. This report demonstrates some potential practical app...
Chromosomal localization of the major histocompatibility complex of the horse (ELA) by in situ hybridization. The first gene assignment to a horse chromosome is reported for equine leucocyte antigen (ELA), the major histocompatibility complex of the horse. A cloned DNA sequence derived from a class I gene of the porcine major histocompatibility complex was used as a probe for an in situ hybridization experiment. We present the regional localization of ELA, using this sequence, to equine chromosome 20q14-q22.
Antigenic variation of equine infectious anemia virus as detected by virus neutralization. Brief report. The antigenic structure of 16 viruses isolated from four horses which were inoculated with a clone of equine infectious anemia (EIA) virus was compared by the neutralization test. The antigenic structure of viruses isolated after development of neutralizing antibody differed from virus to virus. Back mutation of the antigenic structure was also demonstrated by serial passage of the virus in horses. These results suggest that EIA virus is subject to multidirectional antigenic variation. The possibility that the variants originated in the heterologous virus population in the inoculum seems to be...
Evidence that the recently discovered theta 1-globin gene is functional in higher primates. A new subfamily of the alpha-globin-like family has recently been identified in higher primates, rabbit, galago and possibly the horse. One member of this subfamily, theta 1, is downstream from the adult alpha 1-globin gene. In orang-utan, but not in rabbit or galago, the theta 1-gene appears to be structurally intact, suggesting that it may be functional in this species. The orang-utan theta 1-gene possesses initiation and termination codons, and the predicted polypeptide differs from the orang-utan alpha 1-globin by 55 amino acids. The upstream promoter boxes CCAAT and ATA are present, altho...
Application of recombinant DNA techniques to structure-function studies of equine protein hormones. Complementary (c)DNA libraries have been made from horse pituitary gland and endometrial cup tissues with the aim of isolating the genes for the horse gonadotrophins (FSH, LH and CG) and growth hormone (GH). Southern (DNA) and Northern (RNA) blotting techniques were used to demonstrate that several heterologous (human and ovine) cDNA probes would be adequate for isolating the horse genes. A human cDNA probe was then used to isolate the horse gonadotrophin alpha-subunit cDNA from the pituitary and endometrial cup libraries. The nucleotide sequences from both tissue sources were identical, there...
Nucleotide and deduced amino acid sequence of the influenza neuraminidase genes of two equine serotypes. Equine influenza is caused by two serotypes of type A influenza virus, EIV-A1 and EIV-A2. The complete nucleotide sequence of the neuraminidase (NA) genes of both the A1 (N7 subtype) and A2 (N8 subtype) serotype has been determined following cloning of full-length viral NA cDNAs into pBR322. Analysis of the deduced amino acid sequences reveals that the N7 and N8 genes share expected extensive homologies with the previously sequenced N1, N2, and N9 NA subtypes. These homologies include conservation of basic NA gene and protein structure, cysteine residues, potential glycosylation sites, and res...
Molecular cloning and physical characterization of integrated equine infectious anemia virus: molecular and immunologic evidence of its close relationship to ovine and caprine lentiviruses. Molecular clones of the integrated form of the genome of equine infectious anemia virus (EIAV), the etiologic agent of a naturally occurring, worldwide disease of horses, were obtained. The restriction map of a full-length genome was determined. Additional evidence for the close evolutionary relationship between EIAV and a prototype lentivirus (caprine arthritis encephalitis virus) was acquired by Southern blotting and immunological analyses. An interspecies radioimmunoassay was developed in which EIAV and ovine and caprine lentiviruses could be detected equally well. These studies make availa...
Molecular cloning and expression in Escherichia coli of equine type I interferons. Using human interferon-alpha 2 (IFN-alpha 2) and IFN-beta DNA to probe an equine genomic library we isolated recombinant phages containing genes for equine interferon-alpha (EqIFN-alpha), interferon-beta (EqIFN-beta), and interferon-omega (EqIFN-omega). Sequence and hybridization analyses of these genes reveal that the equine genome contains gene families of each of these three type I interferon classes. The mature proteins of EqIFN-alpha are 71-77% homologous to human IFN-alpha polypeptides, and, when expressed in E. coli, possess antiviral activity on both equine and human cells. By contrast...
Cloning and fine mapping the DNA of equine herpesvirus type one defective interfering particles. Equine herpesvirus type one (EHV-1) defective interfering (DI) particle DNA fragments were inserted into the XbaI site of the plasmid vector pACYC184. Five DI XbaI fragments, which ranged in molecular weight from 4.5 to 6.7 MDa, were selected for detailed analysis. Each DI DNA clone was labeled with 32P-deoxynucleotides by nick translation and hybridized to genomic digests of EHV-1 standard (STD) DNA bound to nitrocellulose. All five clones were shown to hybridize to DNA sequences derived from the left terminus (0.0-0.04 map units) of the long (L) region and from the short (S) region inverted ...
Cloning and characterization of an equine cutaneous papillomavirus. Equine papillomaviruses (EqPV) from naturally occurring cases of cutaneous papillomatosis in several ponies and one horse were isolated, cloned, and characterized. Group specific papillomavirus structural antigens were detected in sections of the papillomas by the peroxidase-antiperoxidase technique, and virions were observed in the in the nuclei of cells in the stratum granulosum and corneum. Negatively stained virions purified from papilloma homogenates by isopycnic CsCl centrifugation were 55 nm in diameter and had typical papillomavirus morphology. The entire viral genomes of two separate ...
Generation of packaging-defective DNA molecules of equine adenovirus. Equine adenovirus (EAd) DNA prepared from infected bovine kidney (MDBK) cells contained additional sequences of about 100 to 700 bp at the left-hand end of the genome. These aberrant viral genomes were produced even after the first passage of the wild type EAd in MDBK cells and their relative amounts did not change significantly during serial passage. The left terminal fragments of two defective viral DNAs were cloned into the plasmid vector pBR322 and the nucleotide sequences of their terminal regions were analyzed. The data indicate that one viral DNA contained a duplication of the inverted ...
DNA sequences from the quagga, an extinct member of the horse family. To determine whether DNA survives and can be recovered from the remains of extinct creatures, we have examined dried muscle from a museum specimen of the quagga, a zebra-like species (Equus quagga) that became extinct in 1883 (ref. 1). We report that DNA was extracted from this tissue in amounts approaching 1% of that expected from fresh muscle, and that the DNA was of relatively low molecular weight. Among the many clones obtained from the quagga DNA, two containing pieces of mitochondrial DNA (mtDNA) were sequenced. These sequences, comprising 229 nucleotide pairs, differ by 12 base substitu...
Production of monozygotic (identical) horse twins by embryo micromanipulation. The blastomeres of 192- to 8-cell embryos recovered surgically 1-3 days after ovulation from 23 Pony mares were mechanically separated and inserted, in various combinations, into evacuated pig zonae pellucidae to make 27 'half' and 17 'quarter' micromanipulated embryos. These were embedded in agar and cultured in vivo in the ligated oviducts of ewes for 3.5-5 days to allow development to the late morula/early blastocyst stage. Subsequent surgical or non-surgical transfer of 13 'half' and 17 'quarter' embryos to mares resulted in 10 established pregnancies, including 2 monozygotic pairs. Surgic...
Molecular cloning of equine herpesvirus type 1 DNA: analysis of standard and defective viral genomes and viral sequences in oncogenically transformed cells. Genomic DNA sequences of equine herpesvirus type 1 (EHV-1) have been cloned as BamHI and EcoRI restriction fragments into the plasmid pBR322 and propagated in Escherichia coli. With the exception of two EcoRI restriction fragments that reside in the S region of the viral genome, all of the cloned fragments demonstrated the same electrophoretic mobilities, restriction cleavage sites, and blot-hybridization patterns as did the parent fragments produced by BamHI or EcoRI digestion of virion DNA. The EcoRI J fragment and the BamHI E fragment of the L-region terminus were cloned after the addition ...