Influence of Rack Design and Disease Prevalence on Detection of Rodent Pathogens in Exhaust Debris Samples from Individually Ventilated Caging Systems.

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.

Germline transmission of a novel rat embryonic stem cell line derived from transgenic rats.

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.

A restriction enzyme-PCR-based technique to determine transgene insertion sites.

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.

Mutational insertion of a ROSA26-EGFP transgene leads to defects in spermiogenesis and male infertility in mice.

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.

Breeding colony refinement through phenotypic and genotypic characterization of the SPRD-Pkdr1/Rrrc rat model of polycystic kidney disease.

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.

Detection of mouse parvovirus in Mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay.

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.

Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus.

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.

Comparison of the mouse antibody production (MAP) assay and polymerase chain reaction (PCR) assays for the detection of viral contaminants.

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.