Phylogeographic study using nuclear genome sequences of Asip to infer the origins of ventral fur color variation in the house mouse Mus musculus.

While the house mouse (Mus musculus), widely distributed in Eurasia, is known to have substantial coat color variation between and within local populations, in both primary and secondary distribution areas, including the Japanese archipelago, the evolutionary history of the color variation is poorly understood. To address the ventral fur color variation, we quantified the lightness of museum skin specimens, and found that the southern subspecies, M. m. castaneus (CAS), has high and low lightness in dry and rainy geographic regions, respectively. The northern subspecies, M. m. musculus (MUS), has low and high levels of lightness in the high and middle latitudes of northern Eurasia, respectively. We examined sequence variation of the agouti signaling protein gene (Asip), which is known to be responsible for the ventral fur color. We performed phylogenetic analyses with 196 haplotype sequences of Asip (~180 kb) generated by phasing the whole-genome data of 98 wild mice reported previously. Network and phylogenetic tree construction revealed clustering of haplotypes representing the two subspecies, MUS and CAS. A number of subclusters with geographic affinities appeared within the subspecies clusters, in which the essential results were consistent with those reconstructed with whole mitochondrial genome data, indicating that the phased haplotype genome sequences of the nuclear genome can be a useful tool for tracing the dispersal of geographical lineages. The results of phylogeographic analysis showed that CAS mice with darker ventral fur possessed similar Asip haplotypes across the geographic distribution, suggesting that these haplotypes are major causes of the historical introduction of Asip haplotypes for darker ventral fur in mice from northern India to the peripheral areas, including the Japanese archipelago. Similarly, MUS in East Asia, which has a white abdomen, formed an Asip haplogroup with that from northern Iran, also with a white abdomen.

A transgenic-cloned pig model expressing non-fluorescent modified Plum.

Genetically modified pigs that express fluorescent proteins such as green and red fluorescent proteins have become indispensable biomedical research tools in recent years. Cell or tissue transplantation studies using fluorescent markers should be conducted, wherein the xeno-antigenicity of the fluorescent proteins does not affect engraftment or graft survival. Thus, we aimed to create a transgenic (Tg)-cloned pig that was immunologically tolerant to fluorescent protein antigens. In the present study, we generated a Tg-cloned pig harboring a derivative of Plum modified by a single amino acid substitution in the chromophore. The cells and tissues of this Tg-cloned pig expressing the modified Plum (mPlum) did not fluoresce. However, western blot and immunohistochemistry analyses clearly showed that the mPlum had the same antigenicity as Plum. Thus, we have obtained primary proof of principle for creating a cloned pig that is immunologically tolerant to fluorescent protein antigens.

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.