Abstract 4195: Tetracycline inducible expression of synovial sarcoma associated SYT-SSX2 fusion gene in a new mouse model

Abstract

Abstract Synovial sarcomas are marked by the pathognomic t(X;18) translocation leading to expression of a unique SYT-SSX fusion protein. We previously reported synovial sarcomas in mice by conditionally expressing the human SYT-SSX2 fusion protein within specific cell/tissue types. However, expression of SYT-SSX2 is constitutive in this system after Cre mediated removal of the stop signal. To develop a more versatile model where SYT-SSX2 expression can be turned on and off by exogenous stimuli within any target tissue, we combined Cre-LoxP based conditional approach and tetracycline-inducible system to engineer a mouse expressing SYT-SSX2 from a tetracycline responsive promoter (TetO) within the lineage of Cre expressing cells (SSMT mouse line). This was accomplished by targeting TetO - LoxP - PGK-Neo-PolyA_LoxP_SYT-SSX2 - IRES - EGFP construct in the 3′UTR of the gene for large subunit of RNA polymerase II (Polr2a). Since the construct was placed after stop codon, Polr2a coding region was not perturbed precluding any unwanted phenotype. The floxed PGK-Neo-PolyA cassette in the SSMT mouse ensures Cre dependent removal of transcriptional stop signal in target tissue. Expression of SYT-SSX2, however, requires additional presence of the reverse tetracycline transactivator (rtTA) protein and exogenously supplied tetracycline. rtTA binds to the TetO promoter in the presence of tetracycline to activate transcription of downstream SYT-SSX2. Subsequent withdrawal of tetracycline abrogates SYT-SSX2 expression. SSMT mice were bred to RTA mouse line obtained from Jackson laboratories that express rtTA conditionally in the presence of Cre. Therefore, expression of SYT-SSX2 within SSMT/RTA mice is a two step process where the first step is irreversible; 1) removal of floxed stop signal from both the SSMT and RTA construct and 2) Presence of tetracycline or its analogue doxycycline. To confirm this experimentally, embryonic fibroblasts with the following genotypes were collected; SSMT/RTA, SSMT, RTA, and ROSA-YFP. ROSA-YFP is a reporter mouse line expressing yellow fluorescent protein (YFP) after exposure to Cre recombinase. All fibroblasts were divided into the following treatment groups; 1) exposed to modified Cre recombinase protein (TAT-Cre), 2) exposed to TAT-Cre + doxycycline, 3) exposed to doxycycline, and 4) no exposure. Cre exposure was a onetime 2 hour exposure at 4µm while doxycycline exposure was continuous at 1 µg/ml. Expression of SYT-SSX2 was monitored based on fluorescence of enhanced green fluorescent protein (EGFP) whose expression is linked to SYT-SSX2 via IRES. Our results clearly demonstrate Cre dependence as well as doxycycline inducibility of SYT-SSX2 expression. By inducing and abolishing SYT-SSX2 in various cell types and time points, we can effectively use this system to gain further insight into the biology of synovial sarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4195.