Generation of knockout rabbits with X-linked severe combined immunodeficiency (X-SCID) using CRISPR/Cas9.

Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.

Generation of colonic IgA-secreting cells in the caecal patch.

Gut-associated lymphoid tissues are responsible for the generation of IgA-secreting cells. However, the function of the caecal patch, a lymphoid tissue in the appendix, remains unknown. Here we analyse the role of the caecal patch using germ-free mice colonized with intestinal bacteria after appendectomy. Appendectomized mice show delayed accumulation of IgA(+) cells in the large intestine, but not the small intestine, after colonization. Decreased colonic IgA(+) cells correlate with altered faecal microbiota composition. Experiments using photoconvertible Kaede-expressing mice or adoptive transfer show that the caecal patch IgA(+) cells migrate to the large and small intestines, whereas Peyer's patch cells are preferentially recruited to the small intestine. IgA(+) cells in the caecal patch express higher levels of CCR10. Dendritic cells in the caecal patch, but not Peyer's patches, induce CCR10 on cocultured B cells. Thus, the caecal patch is a major site for generation of IgA-secreting cells that migrate to the large intestine.

Pitfalls in mouse norovirus (MNV) detection in fecal samples using RT-PCR, and construction of new MNV-specific primers.

The murine norovirus (MNV), which belongs to the Caliciviridae family, is prevalent in laboratory mice. Since this virus affects macrophages and dendritic cells, infected mice are not suitable for immunological investigations, making it important to detect MNV infections accurately. When we tested RNA extracts derived from mouse feces for MNV detection using nested RT-PCR with a set of MNV-specific primers reported by Goto et al. (Exp. Anim. 58: 135-140, 2009), we found that these primers amplified not only an MNV-specific signal but also amplified a relatively weak signal with a size almost identical to that of the specific signal. Analysis of the nucleotide sequence of this amplified signal revealed that it was at least 98% identical to the exophosphatase gene of a commensal bacterium, Bacteroides vulgatus. Subsequent analysis showed that the signal amplified with a pair of nested primers was from DNA derived from B. vulgatus, which is sometimes present in SPF laboratory mouse feces, and the nested primers used were both partly homologous with the B. vulgatus nucleotide sequence. We thus designed a new set of nested RT-PCR primers that was not cross-reactive with the B. vulgatus genome. PCR products amplified by the newly designed primers were at least 89.3% identical to the MNV RNA polymerase gene in all cases. Our findings demonstrated that the primer set we designed was suitable for detecting an MNV-specific signal without cross-reacting with B. vulgatus DNA in mouse feces.

A diagnostic method for pneumocystis carinii a causative agent of pneumonia in immunodeficient rats.

Immunodeficient animals are in demand in current biomedical research, and they contribute to medical progress. In Pneumocystis infections, a specific histological diagnostic tool may be immunochemistry (IC). However, it was recently reported that the antibody (3F6) was not suitable for detecting Pneumocystis in rats. We purchased another antibody [PNC007] from a commercial source for IC. We could detect positive signals at identical locations with IC and Toluidine blue O in lungs of infected rats. These results corresponded to the results obtained with PCR. We should study the relationship between unexpected positive signals seen in IC and trophic forms in lungs of infected rats. We could clinically diagnose pneumonia caused by Pneumocystis carinii with the diagnostic method we developed.

Evaluation of a forced-air-ventilated micro-isolation system for protection of mice against Pasteurella pneumotropica.

Studies to date have established that the physical environment inside cages can be controlled adequately by setting the intra-cage ventilation at 60 air changes per hour in a forced-air-ventilated micro-isolation system (FVMIS). In this study, the capability of FVMIS to prevent inter-cage transmission of microorganisms was evaluated using Pasteurella pneumotropica as a reference microorganism. One FVMIS rack and a conventional rack were used, and cages with mice positive for P. pneumotropica and those with P. pneumotropica-free mice were housed on both racks. The mice were examined for P. pneumotropica contamination every 4 weeks after initiating the experiment for 12 weeks using a polymerase chain reaction method. Some P. pneumotropica-free mice housed in open air cages in the conventional rack became positive for P. pneumotropica (four of 28 animals after 4 weeks; eight of 28 animals after 12 weeks), but all P. pneumotropica-free mice housed in the FVMIS cages remained negative for the bacterium throughout the experiment. The results demonstrate that FVMIS can prevent inter-cage transmission of P. pneumotropica when proper cage handling practice is under taken.