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

1.TFL基因的表达。小鼠TFL基因在脾脏中高表达。 T 细胞和 B 细胞之间的表达水平相似。人TFL基因在T细胞和B细胞淋巴瘤细胞中以不同水平表达。结果表明，TFL 基因可能与恶性淋巴瘤选定亚型的发病机制有关。我们还制备了 TFL 蛋白的多克隆抗体。该抗体被证明可以与 TFL 蛋白相互作用。 2.TFL与细胞生长。我们将 TFL/GFP 表达载体引入 BaF3 细胞中。过表达TFL基因的BaF3细胞的增殖受到抑制，并且GFP蛋白的表达也降低。结果表明TFL基因可能具有抑癌基因的功能。 3. TFL蛋白的定位。我们还将TFL/GFP表达载体导入NIH3T3细胞中，并通过双色荧光染色分析TFL蛋白的定位。 TFL 蛋白被证明位于细胞核中，尤其是在核仁中。 4. TFL-突变体的功能。 TFL 蛋白的 C 末端有锌指 (Zn) 基序。我们通过删除Zn基序构建了TFL Zn-/GFP载体，并进行了类似的实验。由该突变载体引入的 BaF3 细胞的增殖水平与对照细胞相似。此外，TFL蛋白不仅仅定位于NIH3T3细胞的细胞核中。这些结果表明，Zn 基序可能对 TFL 蛋白的功能至关重要。 5. 基因敲除小鼠的产生。我们将小鼠TFL基因外显子2（包括翻译起始密码子）替换为PGK-neo盒，构建了TFL基因打靶载体。我们将该载体导入胚胎干（ES）细胞，并通过同源重组选择ES细胞。这些细胞被显微注射到囊胚中，并用于产生具有种系突变的小鼠。我们将来会分析 TFL 敲除小鼠。

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.