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.
Molecular cloning and sequencing of the low-affinity IgE receptor (CD23) for horse and cattle. Expression of the low-affinity IgE receptor (CD23) on the surface of mononuclear cells is a critical event in the development of IgE-mediated immunologic responses. Using PCR and cDNA library screening, a 2.7kb cDNA encoding equine CD23 and a 627bp PCR fragment of cattle CD23 were sequenced and characterized. The equine CD23 sequence encodes a complete and continuous open reading frame of 327 amino acids, while the shorter cattle fragment encodes 209 amino acids corresponding to nucleotides 325-1098 of the equine CD23 transcript. In addition to high identities in their nucleotides and translat...
Isolation and characterization of an arterivirus defective interfering RNA genome. Equine arteritis virus (EAV), the type member of the family Arteriviridae, is a single-stranded RNA virus with a positive-stranded genome of approximately 13 kb. EAV uses a discontinuous transcription mechanism to produce a nested set of six subgenomic mRNAs from which its structural genes are expressed. We have generated the first documented arterivirus defective interfering (DI) RNAs by serial undiluted passaging of a wild-type EAV stock in BHK-21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was cloned. Upon transfection into EAV-infected BHK-21 cells, transcripts derived from this clo...
A DNA fragment of Leptospira interrogans encodes a protein which shares epitopes with equine cornea. Horses infected with Leptospira interrogans present several clinical disorders, one of them being recurrent uveitis. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. With the aim to make progress on defining the molecular basis and pathogenesis of equine recurrent uveitis, here we describe the cloning of one DNA fragment from a Leptospira interrogans serovar pomona genomic lambda gt11 library. Although there are references of transcription of leptospiral genes in E. coli from their own leptospiral promoters, in this recombinant construc...
Cloning, expression, sequence analysis, and characterization of streptokinases secreted by porcine and equine isolates of Streptococcus equisimilis. Streptokinases secreted by nonhuman isolates of group C streptococci (Streptococcus equi, S. equisimilis, and S. zooepidemicus) have been shown to bind to different mammalian plasminogens but exhibit preferential plasminogen activity. The streptokinase genes from S. equisimilis strains which activated either equine or porcine plasminogen were cloned, sequenced, and expressed in Escherichia coli. The streptokinase secreted by the equine isolate had little similarity to any known streptokinases secreted by either human or porcine isolates. The streptokinase secreted by the porcine isolate had li...
Sequence and characterization of cDNA encoding the motilin precursor from chicken, dog, cow and horse. Evidence of mosaic evolution in prepromotilin. Motilin is involved in the regulation of the fasting motility pattern in man and in dog, but may have a different role in other species. Immunoreactive motilin has been demonstrated in several species, but the sequence is mostly unknown. The aim of this study was to isolate and sequence the cDNA encoding the motilin precursor from several mammalian species and from chicken. Total RNA was isolated from the duodenal mucosa of the chicken, dog, cow and horse. In each case single stranded cDNA was synthesized. Motilin cDNA fragments were amplified by PCR, ligated into a plasmid and cloned. Clones ...
The equine herpesvirus 2 E1 open reading frame encodes a functional chemokine receptor. Several herpesviruses contain open reading frames (ORFs) that encode potential homologs of eucaryotic genes. Equine herpesvirus 2 (EHV-2) is a gammaherpesvirus related to other lymphotropic herpesviruses such as herpesvirus saimiri and Epstein-Barr virus. The E1 ORF of EHV-2, a G protein-coupled receptor homolog, shows 31 to 47% amino acid identity with known CC chemokine receptors. To investigate whether E1 may encode a functional receptor, we cloned the E1 ORF and expressed it in stably transfected cell lines. We report here the identification of the CC chemokine eotaxin as a functional liga...
Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay. A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. ...
The molecular genetics of red and green color vision in mammals. To elucidate the molecular mechanisms of red-green color vision in mammals, we have cloned and sequenced the red and green opsin cDNAs of cat (Felis catus), horse (Equus caballus), gray squirrel (Sciurus carolinensis), white-tailed deer (Odocoileus virginianus), and guinea pig (Cavia porcellus). These opsins were expressed in COS1 cells and reconstituted with 11-cis-retinal. The purified visual pigments of the cat, horse, squirrel, deer, and guinea pig have lambdamax values at 553, 545, 532, 531, and 516 nm, respectively, which are precise to within +/-1 nm. We also regenerated the "true" red ...
Novel cathelicidins in horse leukocytes(1). Cathelicidins are precursors of defense peptides of the innate immunity and are widespread in mammals. Their structure comprises a conserved prepropiece and an antimicrobial domain that is structurally varied both intra- and inter-species. We investigated the complexity of the cathelicidin family in horse by a reverse transcription-PCR-based cloning strategy of myeloid mRNA and by Southern and Western analyses. Three novel cathelicidin sequences were deduced from bone marrow mRNA and designated equine cathelicidins eCATH-1, eCATH-2 and eCATH-3. Putative antimicrobial domains of 26, 27 and 40 r...
Dual regulation of promoter II- and promoter 1f-derived cytochrome P450 aromatase transcripts in equine granulosa cells during human chorionic gonadotropin-induced ovulation: a novel model for the study of aromatase promoter switching. Estradiol biosynthesis is a key biochemical trait of developing follicles. To study its regulation in equine follicles, the objectives of this study were to clone and determine the structure of equine cytochrome P450 aromatase (P450AROM), and characterize the regulation of P450AROM and P450 17alpha-hydroxylase/C17-20 lyase (P45017alpha) messenger RNAs (mRNAs) in vivo in equine preovulatory follicles isolated during hCG-induced ovulation. Two distinct P450AROM complementary DNAs (cDNAs) were isolated from an equine preovulatory follicle cDNA library. One clone was 2682 bp in length and included...
Equine arteritis virus derived from an infectious cDNA clone is attenuated and genetically stable in infected stallions. Virus derived from an infectious cDNA clone of equine arteritis virus (EAV030H) was intranasally inoculated into two stallions, neither of which subsequently developed clinical manifestations of equine viral arteritis (EVA). Virus was isolated from nasal swabs and mononuclear cells collected from both stallions
Cloning, sequencing and functional expression of zebra (Equus burchelli) LH. Although donkey luteinizing hormone exhibits a very high degree of amino acid sequence identity with horse LH, its FSH activity in non-equine species is tenfold lower. The coding regions of the common zebra (Equus burchelli) glycoprotein hormone alpha-subunit and LH beta-subunit transcripts were cloned by reverse transcription-PCR from pituitary gland RNA to investigate more precisely the structure-function relationships of this gonadotrophin family. Zebra LH was then expressed in COS-7 cells and its LH and FSH activities were assessed in a rat Leydig cell bioassay (for LH) and in a cell line ...
Molecular cloning of cDNA for equine follistatin and its gene expression in the reproductive tissues of the mare. A cDNA clone encoding equine follistatin was isolated from an equine ovarian cDNA library. Out of 1.2 x 10(5) independent clones screened, one positive clone was isolated and its cDNA sequence determined. The isolated clone, named EQ-FS-1, contained a complete open reading frame encoding 344 amino acid residues. The similarity of its deduced amino acid sequence to these of other mammalian species was greater than 95%. Although its expression level varied among the tissues examined, follistatin mRNA was detected in the equine uteroplacental tissues, follicles and corpora lutea by Northern blot ...
Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus. African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a ...
Identification of a new aspartic proteinase expressed by the outer chorionic cell layer of the equine placenta. The pregnancy-associated glycoproteins (PAGs) are placental antigens that were initially characterized as pregnancy markers in the maternal circulation of domestic ruminant species. They are members of the aspartic proteinase gene family, having greatest sequence identity with pepsinogens. However, some are not capable of functioning as enzymes. The PAGs are associated with a large gene family within the Artiodactyla order (cattle, camels, pigs). So far, no members of this family have been characterized in species outside this order. This report describes the cloning and initial characterizati...
Human chorionic gonadotropin induces an inverse regulation of steroidogenic acute regulatory protein messenger ribonucleic acid in theca interna and granulosa cells of equine preovulatory follicles. The time- and gonadotropin-dependent regulation of steroidogenic acute regulatory protein (StAR) has not been characterized in vivo in preovulatory follicles of large monoovulatory species or sexually mature animals. The objectives of this study were to clone equine StAR and describe the regulation of its messenger RNA (mRNA) in equine follicles after the administration of an ovulatory dose of hCG. The screening of an equine follicle complementary DNA (cDNA) library with a mouse StAR cDNA probe revealed two forms of equine StAR that differ only in the length of their 3'-untranslated region (3'...
Molecular characteristics of equine stromelysin and the tissue inhibitor of metalloproteinase 1. To clone the entire coding sequence of equine matrix metalloproteinase-3 (MMP-3, stromelysin) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and compare their nucleotide and amino acid sequences with those of MMP-3 and TIMP-1 from other species. Methods: Articular cartilage harvested from the joints of 4 foals, 2 yearlings, and 3 adult horses. Methods: A cDNA library was constructed from mRNA extracted from equine chondrocytes. The library was screened and clones selected that contained the cDNA for MMP-3 and TIMP-1. The cDNA was sequenced and the nucleotide and deduced amino acid sequen...
Cloning and characterization of the equine F18 gene, which has a novel exon. A genomic clone isolated from an equine genomic library probed with an oligonucleotide (CAG)10 showed high sequence similarity to the human F18 gene and was tentatively named equine F18 gene. Because the human F18 gene is expressed in many tissues, we examined whether this equine clone was also expressed in equine tissues. The cDNA encoding equine F18 was obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR) from equine thymus. The nucleotide sequence of the equine F18 cDNA (1940 bp) was determined and contained both the ATG initiation codon and a poly(A) sequence. The cDNA ...
Construction of a horse BAC library and cytogenetical assignment of 20 type I and type II markers. A horse BAC library was constructed with about 40,000 clones and mean insert size of 110 kb representing a 1.5 genome equivalent coverage and a probability of finding a single sequence of 0.75. It was characterized by PCR screening of about 130 sequences of horse microsatellites and exonic gene sequences retrieved from databases. BACs containing 8 microsatellites and 12 genes were subsequently localized by fluorescent in situ hybridization (FISH) on chromosomes. Two linkage groups were newly assigned to chromosomes: LG2 to ECA3 and LG5 to ECA24, and five linkage groups were also oriented--LG3,...
Molecular basis for antigenic variation of a protective strain-specific antigen of Ehrlichia risticii. Ehrlichia risticii, the causative agent of Potomac horse fever, has recently been isolated from many vaccinated horses with typical clinical signs of the disease. The heterogeneity of the E. risticii isolates obtained from the vaccinated horses necessitates the identification of the molecular basis of strain variations to elucidate the vaccine failure and to aid in the development of an efficient vaccine against this disease. As an attempt, two major cross-reacting surface antigen genes of 50- and 85-kDa antigens, present separately in strains 25-D (isolated in 1984) and 90-12 (isolated in 199...
Report of the Second Equine Leucocyte Antigen Workshop, Squaw valley, California, July 1995. The final assignment of antibody clusters for leucocyte antigens and immunoglobulins, as described in detail in Sections 3 and 4, is summarized in Table 4. Together with other mAbs developed outside of ELAW II (Table 9) this pool of reagents represent a powerful array of tools for the study of equine immunity. The Second Equine Leucocyte Antigen Workshop made considerable advances in pursuing the objectives of establishing the specificities of mAbs and achieving consensus on the nomenclature for equine leucocyte and immunoglobulin molecules. Of equal importance, several productive collaboratio...
Cloning of equine interleukin 1 receptor antagonist and determination of its full-length cDNA sequence. To clone equine interleukin 1 receptor antagonist (IL-1ra) and determine its full-length cDNA sequence. Methods: A cDNA library derived from lipopolysaccharide-stimulated equine monocytes was screened by means of plaque hybridization to radiolabeled equine IL-1ra DNA probes generated by means of the polymerase chain reaction. The cDNA nucleotide sequence for equine IL-1ra was determined by use of the dideoxy chain termination technique, analyzed by use of computer software for sequence characteristics, and compared with sequences reported for IL-1ra of other species. Results: The cDNA of equin...
CLoning of equine interleukin 1 alpha and equine interleukin 1 beta and determination of their full-length cDNA sequences. To clone equine interleukin 1 alpha (IL-1 alpha) and equine interleukin 1 beta (IL-1 beta) and determine their full-length cDNA sequences. Methods: The mRNA isolated from lipopolysaccharide-stimulated cultured equine monocytes was reverse transcribed, and a cDNA library was constructed in a lambda phage. The cDNA library was screened by means of plaque hybridization with radiolabeled human IL-1 alpha and IL-1 beta cDNA probes. The cDNA nucleotide sequences for equine IL-1 alpha and equine IL-1 beta were determined by use of the dideoxy chain termination technique. The cDNA sequences were analy...
A conserved structural element in horse and mouse IGF2 genes binds a methylation sensitive factor. The equine IGF2 gene has been cloned and characterised. It spans a 9 kb region, which is substantially less than the corresponding human gene. Three coding exons and three untranslated leader exons, all highly homologous to those in other species, were identified. Downstream of the polyadenylation site in exon 6, a dinucleotide repeat sequence was identified. Three putative promoters (P1-P3) were localised in the 5' region of the gene. RNase protection analysis revealed two active promoters in fetal tissues, P2 and P3, whereas P3 was the only promoter active in adult tissues. This represents a...
An aspartic proteinase expressed in the equine placenta. This manuscript describes the cloning of a novel aspartic proteinase expressed in the placenta of the horse (order Perrisodactyla). Evidence for similar genes in the cat (Carnivora) and ruminants (Artiodactyla), indicates that these molecules have been conserved within widely divergent species with distinct types of placentation. Since ePAG is produced by the outer cell layer (trophoblast) of the placenta, it can tentatively be grouped with the pregnancy-associated glycoproteins (PAG) of cattle, sheep, and pig. The high sequence identity that ePAG shares with pepsinogens as well as the PAG, in...
Equus caballus gelsolin–cDNA sequence and protein structural implications. We have generated and characterized the cDNA from equine smooth muscle that encodes gelsolin, an actin-modulating protein. Overlapping cDNA clones synthesized by the reverse transcriptase/polymerase chain reaction and clones isolated from a horse genomic library provided the complete primary structure for the intracellular isoform of gelsolin, while cDNA complemented with protein sequence data produced the full-length primary transcript of the gelsolin isoform found circulating in equine plasma. The deduced amino acid sequences of the intracellular and secreted versions of equine gelsolin infe...
Development and characterization of an in vivo pathogenic molecular clone of equine infectious anemia virus. An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon of rev), and the 3' long terminal repeat (LTR). Modified 19-2-6A mol...
Molecular cloning and cartilage gene expression of equine stromelysin 1 (matrix metalloproteinase 3). To clone and determine molecular structure of equine stromelysin 1 (matrix metalloproteinase 3) and examine stromelysin expression in articular cartilage. SAMPLES AND PROCEDURE: Total RNA was harvested from equine arthritic cartilage specimens and was used for reverse transcription and polymerase chain reaction amplification to develop overlapping complementary DNA (cDNA) clones. Four cDNA sequences were ligated into plasmid (pGEM3Z) constructs and subcloned into bacterial expression vectors, and sequence was determined by automated dye terminator sequencing. Stromelysin mRNA expression was as...