Functional analysis of a novel lymphoma-associated gene, TFL, located on the long arm of chromosome 6

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1. Expression of the TFL gene. The mouse TFL gene was highly expressed in the spleen. The expression was at a similar level between T-cells and B-cells. The human TFL gene was expressed in T-cell and B-cell lymphoma cells at various levels. The results indicated that the TFL gene may be implicated in the pathogenesis of selected subtypes of malignant lymphoma. We also made a polyclonal antibody to TFL protein. This antibody was shown to interact with TFL protein. 2. TFL and cell growth. We introduced TFL/GFP expression vector into BaF3 cells. Proliferation of BaF3 cells with overexpressed TFL gene was suppressed, and expression of GFP protein was also decreased. The results suggested that the TFL gene might function as a tumor suppressor gene. 3. Localization of TFL protein. We also introduced TFL/GFP expression vector into NIH3T3 cells and analyzed localization of TFL protein by dual-color fluorescence staining. The TFL protein was shown to be localized in the nucleus, especially in nucleoli. 4. Function of TFL-mutant. The TFL protein has zinc-finger (Zn) motif at its C-terminal. We constructed TFL Zn-/GFP vector by deleting Zn motif, and carried out similar experiments. BaF3 cells introduced by this mutant vector proliferated at a similar level to control cells. Furthermore, the TFL protein was not localized only in the nucleus of NIH3T3 cells. These results suggested that Zn motif may be essential to the function of the TFL protein 5. Generation of knockout mice. We replaced mouse TFL gene exon 2 including a translation initiation codon by PGK-neo cassette, and constructed the TFL gene targeting vector. We introduced this vector into embryonic stem (ES) cells and selected ES cells with homologous recombination. These cells were microinjected into blastocyst and used to generate mice with a germ-line mutation. We will analyze TFL knockout mice in future.