ABSTRACT
Genetic engineering of non-human primates, which are most closely related to humans, has been expected to generate ideal animal models for human genetic diseases. The common marmoset (Callithrix jacchus) is a non-human primate species adequate for the production of genetically modified animals because of their small body size and high reproductive capacity. Autologous embryo transfer (AET) is routinely utilized in assisted reproductive technologies for humans but not for experimental animals. This study has developed a novel method for efficiently producing mutant marmosets using AET and CRISPR/Cas9 systems. The embryos were recovered from oviducts of naturally mated females, injected with Cas9/guide RNA, and transferred into the oviducts of the donors. This AET method can reduce the time for in vitro culture of embryos to less than 30 min. This method uses an embryo donor as the recipient, thus reducing the number of animals and allowing for "Reduction" in the 3R principles of humane experimental technique. Furthermore, this method can utilize nulliparous females as well as parous females. We applied our novel method and generated the 6 marmosets carrying mutations in the fragile X mental retardation 1 (FMR1) gene using only 18 females including 14 nulliparous females.
Subject(s)
Callithrix/genetics , Embryo Transfer/methods , Fragile X Mental Retardation Protein/genetics , Genetic Engineering/methods , Animals , Autografts , CRISPR-Cas Systems , Embryo Culture Techniques , Female , Models, Animal , MutationABSTRACT
A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1-ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.
Subject(s)
Norovirus/isolation & purification , Organic Chemicals/chemistry , Real-Time Polymerase Chain Reaction/methods , Animals , Base Sequence , Benzothiazoles , Cell Line , DNA Primers , Diamines , Feces/virology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Norovirus/genetics , Open Reading Frames , Plasmids , Quinolines , RNA, Viral/genetics , Reproducibility of Results , Temperature , Time FactorsABSTRACT
Murine norovirus (MNV) has considerable genetical and biological diversity and is recognized worldwide as the most common contaminant in laboratory mouse colonies. This study developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method with the potential to detect a broad range of MNV. RT-LAMP, using a set of five primers containing mixed bases, obtained results under isothermal conditions at 62°C for 90min. Sensitivity of RT-LAMP was 50-fold less than that of two-step TaqMan real-time reverse transcription-polymerase chain reaction (TaqMan RT-PCR). Diagnostic performance of RT-LAMP on RNA extracted from mouse fecal specimens was compared with TaqMan RT-PCR and nested RT-PCR. MNV was detected in 54 of 120 mouse fecal specimens by RT-LAMP, and RT-LAMP had an estimated sensitivity and specificity of 96.4% and 100% compared with TaqMan RT-PCR, and 94.7% and 100% compared with nested RT-PCR. RT-LAMP, which does not require expensive instruments, might be useful for the screening of mice actively or persistently infected with MNV.
Subject(s)
Caliciviridae Infections/veterinary , Norovirus/isolation & purification , Nucleic Acid Amplification Techniques/methods , Reverse Transcription , Rodent Diseases/diagnosis , Rodent Diseases/virology , Animals , Caliciviridae Infections/virology , DNA Primers/genetics , Feces/virology , Mice , Norovirus/genetics , Sensitivity and Specificity , Temperature , Time FactorsABSTRACT
We examined the number of ribosomal gene (rDNA) loci in the metaphase spreads of 54 dogs by FISH method. We found that in 16 dogs (30%) one or two loci were missing. The total number of rDNA loci was varied from 5 to 7 in males and from 4 to 6 in females. As the male dog consistently bears the rDNA on the Y chromosome, the polymorphism of the rDNA locus was ascribed to the absence of autosomal rDNA loci. Indeed, the frequency of polymorphism is almost equivalent in both sexes in the beagle. In one female beagle dog, remarkable intense fluorescence signals were observed at the four autosomal loci, indicating the in situ amplification of rDNA.
Subject(s)
DNA, Ribosomal/genetics , Dogs/genetics , Animals , Chromosomes/genetics , Female , In Situ Hybridization, Fluorescence , Karyotyping , Male , Metaphase , Polymorphism, GeneticABSTRACT
A colorimetric loop-mediated isothermal amplification (LAMP) assay with hydroxy naphthol blue was designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa. The LAMP assay showed 100% specificity for the serogroup and other bacteria, and the sensitivity was 10-fold higher than that of the PCR assays. The LAMP assay could detect P. aeruginosa inoculated in mouse feces at 130 colony-forming units (CFU)/0.1g feces (3.25 CFU/reaction). The assay was completed within 2h from DNA extraction. In a field trial, the LAMP assay revealed that none of the 27 samples was obtained from 2 specific pathogen-free (SPF) mouse facilities that were monitoring infection with P. aeruginosa; 1 out of 12 samples from an SPF mouse facility that was not monitoring infection with P. aeruginosa and 2 out of 7 samples from a conventional mouse facility were positive for P. aeruginosa. In contrast, P. aeruginosa was not detected in any of the samples by a conventional culture assay. Thus, this colorimetric LAMP assay is a simple and rapid method for P. aeruginosa detection.
Subject(s)
Bacteriological Techniques/methods , Feces/microbiology , Nucleic Acid Amplification Techniques/methods , Pseudomonas aeruginosa/isolation & purification , Animals , Bacterial Outer Membrane Proteins/genetics , Colorimetry/methods , Mice , Naphthalenesulfonates , Pseudomonas Infections/veterinary , Sensitivity and Specificity , Staining and Labeling/methodsABSTRACT
The AT-tailing method is a labelling technique that utilises oligo(dA-dT)-dependent signal amplification. In this study, a new immunohistochemical application of the immunoAT method was developed. This method uses an oligo(dA-dT)-conjugated primary antibody (direct immunoAT method) or an oligo(dA-dT)-conjugated secondary antibody (indirect immunoAT method). Fifteen-base oligo(dA-dT)-conjugated antibodies (IgG-ATs) were prepared in advance by conjugating maleimide-activated oligo(dA-dT) to IgG via free sulfhydryl residues that had been introduced on the surface of IgG using Traut's reagent. Following the reaction with the target antigen and the IgG-AT, oligo(dA-dT) was elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP and biotinylated dUTP, consequently labelling the antigen-antibody complex with a large amount of biotin. To initially evaluate the immunoAT method, the presence or absence of prion protein (PrP(sc)) was determined in formalin-fixed and paraffin-embedded sections of the medulla oblongata of cattle which had been under active surveillance for bovine spongiform encephalopathy. Sections were examined using direct and indirect immunoAT methods and the EnVision+ system (Dako) under conditions that were identical except for the differing IgG-AT and AT-tailing methods. PrP(sc) detection was consistent using all three methods. The clearest signals were obtained using the indirect immunoAT method, suggesting significant potential for this method.
Subject(s)
Encephalopathy, Bovine Spongiform/diagnosis , Immunohistochemistry/methods , Oligodeoxyribonucleotides/chemistry , Poly dA-dT/chemistry , PrPSc Proteins/isolation & purification , Animals , Antibodies/metabolism , Cattle , Formaldehyde , In Situ Hybridization , Medulla Oblongata/chemistry , Medulla Oblongata/pathology , Paraffin Embedding , PrPSc Proteins/immunology , Tissue FixationABSTRACT
Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct--namely, magnesium pyrophosphate--without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 microM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.
