OR06-06 Pituitary Tumors and Immortalized Cell Lines Generated by Cre-Inducible Expression of SV40 T-Antigen

Pituitary and hypothalamic cell lines have been developed by targeted oncogenesis. This involved using cell-specific transcriptional regulatory sequences to drive expression of large and small SV40 T-antigens in transgenic mice. Invariably, tumors develop in some of the mice, and the cells in these tumors can sometimes be adapted to grow in culture into stable, immortalized cell lines that maintain some of the features of differentiated cells. Cell lines that represent pre-gonadotropes (αT3-1), gonadotropes (LβT2), precursors to the POU1F1 lineage (GHFT1, Pit1-zero), differentiated cells of the POU1F1 lineage (Pit1-triple, TaT1, and Pit1-PRL), and GnRH neurons (GT1-1) have been made by this approach. Tumors often develop early and cause infertility or death. To increase the opportunity for generating cell lines and to make it feasible to follow the process of tumorigenesis, we developed a mouse strain that expresses SV40 T-antigens in response to cre-recombinase. Using CRISPR/Cas9 we inserted an 8 kb cassette with coding sequences for SV40 T-antigens and IRES-GFP into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26(LSL-SV40-GFP). 30% of the progeny born from hybrid zygotes injected with template DNA, CRISPR/Cas9, and sgRNA had correctly targeted the Rosa26 locus. These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. The majority of Rosa26(LSL-SV40-GFP/+); Prop1-cre and Rosa26(LSL-SV40-GFP/+); Tshb-cre mice developed dwarfism and large tumors by 4 wks. The pituitaries of Rosa26(LSL-SV40-GFP/+); Tshb-cre mice appear grossly normal at birth, but they are enlarged and showing evidence of increased vascularization by 2 wks. Flow-sorted GFP-positive cells from Rosa26(LSL-SV40-GFP/+); Prop1-cre and Rosa26(LSL-SV40-GFP/+); Tshb-cre mice express Prop1 and TSH, respectively. Tumors from Rosa26(LSL-SV40-GFP/+); Tshb-cre mice were adapted to growth in cell culture. We have established a thyrotrope-like cell line that expresses Cga and Pou1f1. These studies demonstrate the utility of the novel, Rosa26(LSL-SV40-GFP) mouse line for reliable targeted oncogenesis and development of unique cell lines. The authors have nothing to disclose.