High frequency of germline recombination in Nestin-Cre transgenic mice crossed with Glucagon-like peptide 1 receptor floxed mice

by Yusuke Kajitani, Takashi Miyazawa, Tomoaki Inoue, Nao Kajitani, Masamichi Fujita, Yukina Takeichi, Yasutaka Miyachi, Ryuichi Sakamoto, Yoshihiro Ogawa

The Cre-loxP strategy for tissue-specific gene inactivation has become a widely employed tool in several research studies. Conversely, inadequate breeding and genotyping without considering the potential for non-specific Cre-recombinase expression may lead to misinterpretations of results. Nestin-Cre transgenic mice, widely used for the selective deletion of genes in neurons, have been observed to have an incidence of Cre-line germline recombination. In this study, we attempted to generate neuron-specific Glucagon-like peptide 1 receptor (Glp1r) knock-out mice by crossing mice harboring the Nestin-Cre transgene with mice harboring the Glp1r gene modified with loxP insertion, in order to elucidate the role of Glp1r signaling in the nervous system. Surprisingly, during this breeding process, we discovered that the null allele emerged in the offspring irrespective of the presence or absence of the Nestin-Cre transgene, with a high probability of occurrence (93.6%). To elucidate the cause of this null allele, we conducted breeding experiments between mice carrying the heterozygous Glp1r null allele but lacking the Nestin-Cre transgene. We confirmed that the null allele was inherited by the offspring independently of the Nestin-Cre transgene. Furthermore, we assessed the gene expression, protein expression, and phenotype of mice carrying the homozygous Glp1r null allele generated from the aforementioned breeding, thereby confirming that the null allele indeed caused a global knock-out of Glp1r. These findings suggest that the null allele in the NestinCre-Glp1r floxed breeding arose due to germline recombination. Moreover, we demonstrated the possibility that germline recombination may occur not only during the spermatogenesis at testis but also during epididymal sperm maturation. The striking frequency of germline recombination in the Nestin-Cre driver underscores the necessity for caution when implementing precise breeding strategies and employing suitable genotyping methods.

Association between lignan polyphenol bioavailability and enterotypes of isoflavone metabolism: A cross-sectional analysis

by Tomoko Fujitani, Mariko Harada Sassa, Zhaoqing Lyu, Yukiko Fujii, Kouji H. Harada

Lignan polyphenols derived from plants are metabolized by bacteria in the gut to mammalian lignans, such as enterolactone (ENL) and enterodiol (END). Mammalian lignan intake has been reported to be associated with obesity and low blood glucose levels. However, the factors that are responsible for individual differences in the metabolic capacity for ENL and END are not well understood. In the present study, the effects of enterotypes of isoflavone metabolism, equol producers (EQP) and O-desmethylangolensin producers (O-DMAP), on lignan metabolism were examined. EQP was defined by urinary daidzein (DAI) and equol concentrations as log(equol/DAI) ≥ –1.42. O-DMAP was defined by urinary DAI and O-DMA concentrations as O-DMA/DAI > 0.018. Isoflavone and lignan concentrations in urine samples from 440 Japanese women were measured by gas chromatography-mass spectrometry. Metabolic enterotypes were determined from the urinary equol and O-DMA concentrations. Urinary END and ENL concentrations were compared in four groups, combinations of EQP (+/–) and O-DMAP (+/–). The urinary lignan concentration was significantly higher in the O-DMAP/EQP group (ENL: P