Abstract BACKGROUND Isolation of cell lines from patients’ tumors is difficult, due in large part, to contaminating fibroblasts that often overgrow the malignant cells. The same is true of xenografted tumors. To overcome this problem, we developed biochemically selectable mouse cells by using genetically-engineered mice. METHODS We constructed a series of hypoxanthine phosphoribosyl transferase (hprt) null immunodeficient mice. These include nude hprt-null, SCID hprt-null and NOD-SCID hprt-null mice. Tumors from patients were implanted into these mice for growth. During growth of xenografted human cancers, hprt-null murine stromal cells replace their human counterparts to facilitate the growth of human cancer cells. Explanted tumors were then grown in tissue culture flasks with selection media (hypoxanthine, aminopterin, thymine; HAT media) to eliminate the mouse biochemically-defective fibroblasts and produce pure human cancer cell lines. RESULTS We successfully established several pancreatic cancer cell lines and an ovarian cancer cell line from patients using the hprt null immunodeficient mice. CONCLUSIONS The new cell line isolating system using genetically-engineered mice provides a greater chance to isolate pure cancer cell lines from patients’ tumors. This may allow personalized chemotherapy selection. Citation Format: Hirohiko Kamiyama, Sherri Rauenzahn, Anirban Maitra, James R. Eshleman. Genetically-engineered mice as a cell line isolating tool. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3481. doi:10.1158/1538-7445.AM2014-3481