Neuromedin U-deficient rats do not lose body weight or food intake.

Studies in genetically modified mice establish that essential roles of endogenous neuromedin U (NMU) are anorexigenic function and metabolic regulation, indicating that NMU is expected to be a potential target for anti-obesity agents. However, in central administration experiments in rats, inconsistent results have been obtained, and the essential role of NMU energy metabolism in rats remain unclear. This study aims to elucidate the role of endogenous NMU in rats. We generated NMU knockout (KO) rats that unexpectedly showed no difference in body weight, adiposity, circulating metabolic markers, body temperature, locomotor activity, and food consumption in both normal and high fat chow feeding. Furthermore, unlike reported in mice, expressions of Nmu and NMU receptor type 2 (Nmur2) mRNA were hardly detectable in the rat hypothalamic nuclei regulating feeding and energy metabolism, including the arcuate nucleus and paraventricular nucleus, while Nmu was expressed in pars tuberalis and Nmur2 was expressed in the ependymal cell layer of the third ventricle. These results indicate that the species-specific expression pattern of Nmu and Nmur2 may allow NMU to have distinct functions across species, and that endogenous NMU does not function as an anorexigenic hormone in rats.

An improved iliac lymph node method for production of monoclonal antibodies.

Monoclonal antibodies have been applied in a wide range of biological and medical studies since the advent of cell fusion technology. Although cell fusion techniques have been improved by using myelomas and reagents, researchers still find it difficult to produce monoclonal antibodies because of the long protocols, high costs, and low efficiency of obtaining hybridomas. To solve these problems, we first developed an iliac lymph node method in 1995 using rats. In this method, an antigen emulsion is injected intramuscularly into the tail base, and then B lymphocytes are isolated from the enlarged iliac lymph nodes. This method is approximately 10 times more productive than the conventional spleen method. Here, we present further improvements to the iliac lymph node method to render it easily applicable in both mice and rats. We found that the frequency of hybridomas secreting specific antibodies was over five times higher using the electro cell fusion method than using the polyethylene glycol (PEG) fusion method. This frequency using the iliac lymph node method with electro cell fusion is at least 50 times higher than that using the traditional spleen method, thereby leading to the reduction in the number of mice or rats to be sacrificed. In addition, only a single injection for immunization is necessary for the iliac lymph node method, opposed to three for the spleen method. Therefore, this method is rapid, inexpensive, and ethical for producing monoclonal antibodies.

Creation of X-linked Alport syndrome rat model with Col4a5 deficiency.

Alport syndrome is an inherited chronic human kidney disease, characterized by glomerular basement membrane abnormalities. This disease is caused by mutations in COL4A3, COL4A4, or COL4A5 gene. The knockout mice for Col4α3, Col4α4, and Col4α5 are developed and well characterized for the study of Alport syndrome. However, disease progression and effects of pharmacological therapy depend on the genetic variability. This model was reliable only to mouse. In this study, we created a novel Alport syndrome rat model utilizing the rGONAD technology, which generated rat with a deletion of the Col4α5 gene. Col4α5 deficient rats showed hematuria, proteinuria, high levels of BUN, Cre, and then died at 18 to 28 weeks of age (Hemizygous mutant males). Histological and ultrastructural analyses displayed the abnormalities including parietal cell hyperplasia, mesangial sclerosis, and interstitial fibrosis. Then, we demonstrated that α3/α4/α5 (IV) and α5/α5/α6 (IV) chains of type IV collagen disrupted in Col4α5 deficient rats. Thus, Col4α5 mutant rat is a reliable candidate for the Alport syndrome model for underlying the mechanism of kidney diseases and further identifying potential therapeutic targets for human renal diseases.

GONAD: A new method for germline genome editing in mice and rats.

Recent advances in the CRISPR/Cas9 system have demonstrated it to be an efficient gene-editing technology for various organisms. Laboratory mice and rats are widely used as common models of human diseases; however, the current standard method to create genome-engineered animals is laborious and involves three major steps: isolation of zygotes from females, ex vivo micromanipulation of zygotes, and implantation into pseudopregnant females. To circumvent this, we recently developed a novel method named Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). This method does not require the ex vivo handling of embryos; instead, it can execute gene editing with just one step, via the delivery of a genome-editing mixture into embryos in the oviduct, by electroporation. Here, we present a further improvement of GONAD that is easily applicable to both mice and rats. It is a rapid, low-cost, and ethical approach fulfilling the 3R principles of animal experimentation: Reduction, Replacement, and Refinement. This method has been reconstructed and renamed as "improved GONAD (i-GONAD)" for mice, and "rat improved GONAD (rGONAD)" for rats.