Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

Phylogeography of Nanorana parkeri (Anura: Ranidae) and multiple refugia on the Tibetan Plateau revealed by mitochondrial and nuclear DNA.

Quaternary climatic changes have been recognized to influence the distribution patterns and evolutionary histories of extant organisms, but their effects on alpine species are not well understood. To investigate the Pleistocene climatic oscillations on the genetic structure of amphibians, we sequenced one mitochondrial and three nuclear DNA fragments in Nanorana parkeri, a frog endemic to the Tibetan Plateau, across its distribution range in the southern plateau. Mitochondrial cytochrome b (Cytb) and three nuclear genes (c-Myc2, Rhod, and Tyr) revealed two distinct lineages (i.e. the lineages East and West), which were strongly geographically structured. The split of the two divergent lineages was dated back earlier than the Middle Pleistocene, probably being associated with climatic and ecological factors. Species distribution modeling, together with the phylogeographic structuring, supported the hypothesis of multiple refugia for N. parkeri on the Tibetan Plateau during the Pleistocene glaciations, and suggested the Yarlung Zangbo valley and the Kyichu catchment to be the potential refugia. Our findings indicate that Pleistocene climatic changes have had a great impact on the evolution and demographic history of N. parkeri. Our study has important implications for conservation of this and other frog species in the Tibetan Plateau.

Real-time dynamic optical imaging of ACC-M tumor cells killed by HSV-tk/ACV system.

HSV-tk/ACV induced and killed human adenoid cystic carcinoma cell (ACC-M) in vivo and in vitro, which were observed through optical imaging and green fluorescence protein (GFP) tagging technique. ACC-M was transfected with TK-GFP, and the single clone cell ACC-M-TK-GFP was selected by G418. With fluorescent stereomicroscope, whole-body fluorescent imaging system and fluorescent microscope, we could observe ACV treated ACC-M-TK-GFP cells in cell level and nude mice. The therapies of tumor were visualized clearly with optical imaging. This study proves that optical imaging is a very good approach for studying the effect of HSV-tk/ACV on the ACC-M tumor cells and decreasing the amount of vessel about tumors cell. Optical imaging will become a visual groundwork for monitoring tumor growth and evaluating in vivo curative effect of antitumor drugs.

In vivo monitoring the process of tumor growth, metastasis and bacterial infection expressing GFP via real-time optical imaging.

Noninvasive molecular fluorescence imaging in vivo which combines Optical imaging with genetic marker technology can real time monitor the development of tumor, through the use of human adenoid cystic carcinoma cell (ACC-M) and lung carcinoma cells SPC-A1 were thansfected by green fluorescent protein (GFP). This study established three types of model: Experimental metastases by tail vein injection of ACC-M-EGFP, spontaneous metastases by abdomen subcutaneous injection of SPC-A1-EGFP and subcutaneous tumor growth by subcutaneous injection of SPC-A1-EGFP. Tumor-bearing mice were viewed with whole-body fluorescent imaging system. The results showed that growth and metastasis of tumor were visualized clearly with this system. While this simple, nonintrusive technique can show in great detail the temporal behavior of the infectious process of red fluorescent protein (DsRed2)-expressing bacteria from outside intact infected animals. Therefore, this study provides a platform for monitoring tumor growth and metastasis and evaluating efficacy of antitumor drugs in vivo.