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1.
PLoS One ; 14(6): e0218412, 2019.
Article in English | MEDLINE | ID: mdl-31220119

ABSTRACT

The Consortium for Mouse Cell Line Authentication was formed to validate Short Tandem Repeat (STR) markers for intraspecies identification of mouse cell lines. The STR profiling method is a multiplex polymerase chain reaction (PCR) assay comprised of primers targeting 19 mouse STR markers and two human STR markers (for interspecies contamination screening). The goals of the Consortium were to perform an interlaboratory study to-(1) validate the mouse STR markers to uniquely identify mouse cell lines (intraspecies identification), (2) to provide a public database of mouse cell lines with the National Institute of Standards and Technology (NIST)-validated mouse STR profiles, and (3) to publish the results of the interlaboratory study. The interlaboratory study was an international effort that consisted of 12 participating laboratories representing institutions from academia, industry, biological resource centers, and government. The study was based on 50 of the most commonly used mouse cell lines obtained from the American Type Culture Collection (ATCC). Of the 50 mouse cell lines, 18 had unique STR profiles that were 100% concordant (match) among all Consortium laboratory members, and the remaining 32 cell lines had discordance that was resolved readily and led to improvement of the assay. The discordance was due to low signal and interpretation issues involving artifacts and genotyping errors. Although the total number of discordant STR profiles was relatively high in this study, the percent of labs agreeing on allele calls among the discordant samples was above 92%. The STR profiles, including electropherogram images, for NIST-validated mouse cell lines will be published on the NCBI BioSample Database (https://www.ncbi.nlm.nih.gov/biosample/). Overall, the interlaboratory study showed that the multiplex PCR method using 18 of the 19 mouse STR markers is capable of discriminating at the intraspecies level between mouse cell lines. Further studies are ongoing to refine the assay including (1) development of an allelic ladder for improving the accuracy of allele calling and (2) integration of stutter filters to identify true stutter.


Subject(s)
Genotype , Genotyping Techniques/methods , Microsatellite Repeats/genetics , Multiplex Polymerase Chain Reaction/methods , Alleles , Animals , Cell Line , Humans , Mice
2.
Toxicol Pathol ; 45(5): 593-603, 2017 07.
Article in English | MEDLINE | ID: mdl-28782456

ABSTRACT

We report the identification, pathogenesis, and transmission of a novel polyomavirus in severe combined immunodeficient F344 rats with null Prkdc and interleukin 2 receptor gamma genes. Infected rats experienced weight loss, decreased fecundity, and mortality. Large basophilic intranuclear inclusions were observed in epithelium of the respiratory tract, salivary and lacrimal glands, uterus, and prostate gland. Unbiased viral metagenomic sequencing of lesioned tissues identified a novel polyomavirus, provisionally named Rattus norvegicus polyomavirus 2 (RatPyV2), which clustered with Washington University (WU) polyomavirus in the Wuki clade of the Betapolyomavirus genus. In situ hybridization analyses and quantitative polymerase chain reaction (PCR) results demonstrated viral nucleic acids in epithelium of respiratory, glandular, and reproductive tissues. Polyomaviral disease was reproduced in Foxn1rnu nude rats cohoused with infected rats or experimentally inoculated with virus. After development of RatPyV2-specific diagnostic assays, a survey of immune-competent rats from North American research institutions revealed detection of RatPyV2 in 7 of 1,000 fecal samples by PCR and anti-RatPyV2 antibodies in 480 of 1,500 serum samples. These findings suggest widespread infection in laboratory rat populations, which may have profound implications for established models of respiratory injury. Additionally, RatPyV2 infection studies may provide an important system to investigate the pathogenesis of WU polyomavirus diseases of man.


Subject(s)
Polyomavirus Infections , Polyomavirus , Tumor Virus Infections , Animals , Female , Lung/virology , Male , Metagenomics , Polyomavirus/genetics , Polyomavirus/isolation & purification , Polyomavirus/pathogenicity , Polyomavirus Infections/complications , Polyomavirus Infections/genetics , Polyomavirus Infections/transmission , Polyomavirus Infections/virology , Rats , Sequence Analysis, DNA , Severe Combined Immunodeficiency/complications , Tissue Distribution , Tumor Virus Infections/complications , Tumor Virus Infections/genetics , Tumor Virus Infections/transmission , Tumor Virus Infections/virology , Viral Load/genetics
3.
J Am Assoc Lab Anim Sci ; 55(6): 782-788, 2016 11.
Article in English | MEDLINE | ID: mdl-27931317

ABSTRACT

Sampling of bedding debris within the exhaust systems of ventilated racks may be a mechanism for detecting murine pathogens in colony animals. This study examined the effectiveness of detecting pathogens by PCR analysis of exhaust debris samples collected from ventilated racks of 2 different rack designs, one with unfiltered air flow from within the cage to the air-exhaust pathway, and the other had a filter between the cage and the air-exhaust pathway. For 12 wk, racks were populated with either 1 or 5 cages of mice (3 mice per cage) infected with one of the following pathogens: mouse norovirus (MNV), mouse parvovirus (MPV), mouse hepatitis virus (MHV), Helicobacter spp., Pasteurella pneumotropica, pinworms, Entamoeba muris, Tritrichomonas muris, and fur mites. Pathogen shedding by infected mice was monitored throughout the study. In the filter-containing rack, PCR testing of exhaust plenums yielded negative results for all pathogens at all time points of the study. In the rack with open air flow, pathogens detected by PCR analysis of exhaust debris included MHV, Helicobacter spp., P. pneumotropica, pinworms, enteric protozoa, and fur mites; these pathogens were detected in racks housing either 1 or 5 cages of infected mice. Neither MPV nor MNV was detected in exhaust debris, even though prolonged viral shedding was confirmed. These results demonstrate that testing rack exhaust debris from racks with unfiltered air flow detected MHV, enteric bacteria and parasites, and fur mites. However, this method failed to reliably detect MNV or MPV infection of colony animals.


Subject(s)
Air Filters/microbiology , Air Filters/parasitology , Housing, Animal , Infections/veterinary , Rodent Diseases/microbiology , Rodent Diseases/parasitology , Animals , Bacteria/classification , Bacteria/isolation & purification , Infections/microbiology , Infections/parasitology , Infections/virology , Mice , Parasites/classification , Parasites/isolation & purification , Polymerase Chain Reaction , Rodent Diseases/virology , Viruses/classification , Viruses/isolation & purification
4.
Stem Cells Dev ; 21(14): 2606-12, 2012 Sep 20.
Article in English | MEDLINE | ID: mdl-22455749

ABSTRACT

Germline-competent rat embryonic stem (ES) cell lines are important resources for the creation of mutant rat models using ES-cell-based gene targeting technology. The ability to isolate germline-competent ES cell lines from any rat strain, including genetically modified strains, would allow for more sophisticated genetic manipulations without extensive breeding. Sprague Dawley (SD) males carrying an enhanced green fluorescent protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. A number of ES cell lines were established and subjected to rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing. Two male ES cell lines, SD-Tg.EC1/Rrrc and SD-Tg.EC8/Rrrc, were injected into blastocysts recovered from a cross of Dark Agouti (DA) males with SD females. Resulting chimeric animals were bred with wild-type SD mates to verify the germline transmissibility of the ES cell lines by identifying pups carrying the ES cell line-derived EGFP transgene. While both ES cell lines gave rise to chimeric animals, only SD-Tg.EC1 was germline competent. This confirms the feasibility of deriving germline-competent ES cell lines from transgenic rat strains and provides a novel ES cell line with a stable green fluorescent protein (GFP) reporter for future genetic manipulations to create new rat models.


Subject(s)
Cell Line , Chimera/genetics , Embryonic Stem Cells/cytology , Germ Cells/cytology , Transgenes , Animals , Cell Culture Techniques/methods , Cell Shape , Embryo Transfer/methods , Embryo, Mammalian/cytology , Female , Green Fluorescent Proteins/metabolism , Inheritance Patterns , Karyotype , Male , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Rats , Rats, Sprague-Dawley , Rats, Transgenic , Specific Pathogen-Free Organisms , Transcription Factors/metabolism
5.
Methods Mol Biol ; 597: 287-99, 2010.
Article in English | MEDLINE | ID: mdl-20013241

ABSTRACT

Currently, most genetically engineered rat strains are created by methods that involve random integration of transgenes into the genome. The ability to identify the chromosomal location of the transgene insertion site enables the development of efficient genotyping assays, allows segregation of multiple transgene integration sites to be followed while breeding, and facilitates characterization of possible positional effects on phenotype. Here we describe a method for determining the chromosomal location of transgene insertion that combines restriction endonuclease enzyme digest with subsequent rounds of PCR amplification to produce amplicons representing the chromosomal regions flanking the integrated transgene. This method provides a reliable means for determining the exact location of insertion of transgenes within the genome.


Subject(s)
Polymerase Chain Reaction/methods , Transgenes , Animals , Base Sequence , Chromosomes/genetics , DNA/isolation & purification , DNA/metabolism , DNA Primers/genetics , DNA Restriction Enzymes/metabolism , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Rats , Sequence Analysis, DNA/methods
6.
Comp Med ; 59(6): 545-52, 2009 Dec.
Article in English | MEDLINE | ID: mdl-20034429

ABSTRACT

Pronuclear injection has been a successful strategy for generating genetically engineered mouse models to better understand the functionality of genes. A characteristic of pronuclear injection is that random integration of the transgene into the genome can disturb a functional gene and result in a phenotype unrelated to the transgene itself. In this study, we have characterized a mouse model containing an insertional mutation that, in the homozygous state, severely affects spermatogenesis as characterized by lack of sperm motility and acrosomal aplasia. Whereas homozygous female mice had normal fertility, male mice homozygous for the insertional mutation were unable to produce pups by natural mating with either homozygous or wild-type female mice. No fertilized embryos were produced by matings to homozygous male mice, and no sperm were present in the reproductive tract of mated female mice. Spermatozoa isolated from homozygous male mice exhibited head and midpiece defects, but no major defects in the principal piece of these sperm. Histologic examination and immunohistochemical staining of the testes revealed vacuolar degeneration of Sertoli cells and loss of structural seminiferous tubule integrity and organization, indicating that spermatogenesis is severely affected in this mouse model. Although the males are always infertile, the severity of the histologic and sperm morphologic defects appeared to be age-related.


Subject(s)
Green Fluorescent Proteins/genetics , Infertility, Male/genetics , Mutation , Proteins/genetics , Spermatogenesis/genetics , Transgenes , Animals , Base Sequence , DNA Primers , Female , Immunohistochemistry , Male , Mice , RNA, Untranslated
7.
ILAR J ; 49(3): 347-355, 2008.
Article in English | MEDLINE | ID: mdl-18506068

ABSTRACT

High-tech biomedical advances have led to increases both in the number of mice used for research and in exchanges of mice and/or their tissues between institutions. The latter are associated with the risk of dissemination of infectious agents. Because of the lack of international standardization of health surveillance programs, health certificates for imported rodents may be informative but may not address the needs of the importing facility. Preservation of mouse germplasm is achieved by cryopreservation of spermatozoa, embryos, or ovaries, and embryonic stem cells are used for the production of genetically engineered mice. After embryo transfer, recipients and rederived pups that test negative in microbiological screening for relevant microorganisms are released into full barrier holding areas. However, current research shows that embryos may also transmit microorganisms, especially viruses, to the recipient mice. In this article, we discuss regulations and practical issues in the shipping of live mice and mouse tissues, including spermatozoa, embryos, ovaries, and embryonic stem cells, and review work on microbial contamination of these biological materials. In addition, we present ways to reduce the risk of transmission of pathogens to mice under routine conditions.


Subject(s)
Embryo, Mammalian/microbiology , Reproductive Techniques, Assisted , Animals , Embryo Culture Techniques/methods , Embryo Culture Techniques/standards , Fertilization in Vitro/methods , Fertilization in Vitro/standards , Mice , Risk Assessment , Risk Factors
8.
Comp Med ; 57(2): 193-9, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17536620

ABSTRACT

The SPRD-Pkdr1 rat model is widely used for the study of human autosomal dominant polycystic kidney disease. This rat model carries the Cy allele of the Pkdr1 gene, which results in polycystic kidney disease. Because the Cy allele is lethal in the homozygous state at weanling age, the breeding colony must be maintained in the heterozygous state. A random breeding scheme in which production of homozygous pups with enlarged kidneys indicates heterozygous breeders is commonly used. This study was performed to determine whether biochemical markers (blood urea nitrogen [BUN] or creatinine), ultrasonography, or genetic analysis could be used to select breeding animals in the SPRD-Pkdr1/Rrrc colony and thus replace the random breeding scheme with a more efficient selective breeding scheme. BUN was predictive of the Cy allele in 8- to 9-wk-old male but not female rats. Ultrasonography identified animals with polycystic kidney disease in both sexes by 9 wk of age. Microsatellite marker polymorphism analysis could not be used to determine carrier status for the Cy allele, but restriction fragment length polymorphism analysis appropriately detected the Cy allele in 100% of the animals examined. In conclusion, multiple methods can be used for detecting the Cy allele, making possible a selective breeding scheme that markedly reduces the necessary number of breeder animals and eliminates the euthanasia of offspring needed with a random test-mating scheme.


Subject(s)
Breeding/methods , Disease Models, Animal , Polycystic Kidney Diseases/genetics , Rats/genetics , Alleles , Animals , Base Sequence , Blood Urea Nitrogen , Creatinine/blood , Genetic Markers , Genotype , Heterozygote , Inbreeding , Molecular Sequence Data , Polycystic Kidney Diseases/diagnostic imaging , Polymorphism, Restriction Fragment Length , Rats, Inbred Strains , TRPP Cation Channels/chemistry , Ultrasonography
9.
Comp Med ; 57(1): 51-6, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17348291

ABSTRACT

We used primary and nested polymerase chain reaction (PCR) assays to determine the presence of mouse parvovirus (MPV) in mouse sperm, oocytes, preimplantation embryos, and ovarian tissues collected from MPV-infected mice. The primary PCR assay detected MPV in 56% of the sperm samples. MPV was not eliminated by passing sperm samples through a Percoll gradient. After Percoll treatment, MPV was still present in 50% of the samples according to primary PCR assay. Oocyte samples that did not undergo extensive washing procedures had detectable MPV in 7% of the samples based on the primary PCR assay, but nested PCR assay detected higher (28%) infection rate. However, MPV was not detected in oocytes that underwent extensive washing procedures, as assessed by either primary or nested PCR assay. Although primary PCR did not detect MPV in embryos, a nested PCR assay determined that 50% of the embryos were positive for the virus. In addition, ovarian tissues were collected from 3 different mouse colonies with enzootic MPV infection. Ovarian tissue collected from 129CT, 101/R1, and Sencar mice had high incidence (38%, 63%, and 65%, respectively) of MPV infection on the basis of nested PCR amplification. These results demonstrate that mouse gametes, embryos, and ovarian tissues may be contaminated with MPV and therefore caution is necessary when infected germplasm is used for assisted reproductive technologies such as embryo transfer, establishing embryonic stem cell lines, in vitro fertilization, ovary transplantation, and intracytoplasmic sperm injection.


Subject(s)
Animals, Laboratory/virology , Embryo, Mammalian/virology , Germ Cells/virology , Mice , Ovary/virology , Parvoviridae Infections/veterinary , Parvovirus/genetics , Rodent Diseases/virology , Animals , Female , Humans , Male , Parvoviridae Infections/virology , Polymerase Chain Reaction/veterinary , Reproductive Techniques, Assisted/standards
10.
Biotechniques ; 41(6): 715-9, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17191616

ABSTRACT

Transgene delivery systems, particularly those involving retroviruses, often result in the integration of multiple copies of the transgene throughout the host genome. Since site-specific silencing of trangenes can occur; it becomes important to identify the number and chromosomal location of the multiple copies of the transgenes in order to correlate inheritance of the transgene at a particular chromosomal site with a specific and robust phenotype. Using a technique that combines restriction endonuclease digest and several rounds of PCR amplification followed by nucleotide sequencing, it is possible to identify multiple chromosomal integration sites in transgenic founder animals. By designing genotyping assays to detect each individual integration site in the offspring of these founders, the inheritance of transgenes integrated at specific chromosomal locations can be followed efficiently as the transgenes randomly segregate in subsequent generations. Phenotypic characteristics can then be correlated with inheritance of a transgene integrated at a particular chromosomal location to allow rational selection of breeding animals in order to establish the transgenic line.


Subject(s)
Animals, Genetically Modified , Chromosome Mapping , Lentivirus/genetics , Transgenes , Virus Integration , Animals , Base Sequence , Genetic Vectors , Molecular Sequence Data , Rats , Rats, Inbred Lew
11.
Biologicals ; 32(4): 177-82, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15572099

ABSTRACT

Mouse antibody production (MAP) tests have become the standard assay for the detection of murine viral contamination in biologic materials, such as cell lines and transplantable tumors. However, newly developed PCR assays offer the advantage of lower cost, faster turn around times, and eliminate the use of live animals. In this study, the MAP test and a panel of PCR assays were compared for the detection of 11 different viral contaminants of cell lines and transplantable tumors. The PCR assays had either better or comparable results to the MAP test for all agents tested. The results of this study confirm that PCR assays are an effective method for detection of viral contamination and can be used as an alternative to the MAP test.


Subject(s)
Antibody Formation/immunology , Equipment Contamination , Polymerase Chain Reaction/methods , Animals , Cells, Cultured , DNA Viruses/genetics , DNA Viruses/isolation & purification , DNA, Viral/analysis , DNA, Viral/genetics , Humans , Mice , RNA Viruses/genetics , RNA Viruses/isolation & purification , RNA, Viral/analysis , RNA, Viral/genetics
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