A growing body of evidence indicates that deregulation of stem cell fate determinants is a hallmark of many types of malignancies. The neural stem cell fate determinant TLX plays a pivotal role in neurogenesis in the adult brain by maintaining neural stem cells. Here, we report a tumorigenic role of TLX in brain tumor initiation and progression. Increased TLX expression was observed in a number of glioma cells and glioma stem cells, and correlated with poor survival of patients with gliomas. Ectopic expression of TLX in the U87MG glioma cell line and Ink4a/Arf-deficient mouse astrocytes (Ink4a/Arf(-/-) astrocytes) induced cell proliferation with a concomitant increase in cyclin D expression, and accelerated foci formation in soft agar and tumor formation in in vivo transplantation assays. Furthermore, overexpression of TLX in Ink4a/Arf(-/-) astrocytes inhibited cell migration and invasion and promoted neurosphere formation and Nestin expression, which are hallmark characteristics of glioma stem cells, under stem cell culture conditions. Our results indicate that TLX is involved in glioma stem cell genesis and represents a potential therapeutic target for this type of malignancy.

Alternative cancer models that are close to humans are required to create more valuable preclinical results during oncology studies. Here, a new onco-pig model via developing a CRISPR-Cas9-based Conditional Polycistronic gene expression Cassette (CRI-CPC) system to control the tumor inducing simian virus 40 large T antigen (SV40LT) and oncogenic HRAS G12V . After conducting somatic cell nuclear transfer (SCNT), transgenic embryos were transplanted into surrogate mothers and five male piglets were born. Umbilical cord analysis confirmed that all piglets were transgenic. Two of them survived and they expressed a detectable green fluorescence. The test was made whether CRI-CPC models were naturally fertile and whether the CRI-CPC system was stably transferred to the offspring. By mating with a normal female pig, four offspring piglets were successfully produced. Among them, only three male piglets were transgenic. Finally, their applicability was tested as cancer models after transduction of Cas9 into fibroblasts from each CRI-CPC pig in vitro, resulting in cell acquisition of cancerous characteristics via the induction of oncogene expression. These results showed that our new CRISPR-Cas9-based onco-pig model was successfully developed.