Trial of cloning and functional analysis of a novel vascular endothelial growth factor.

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1.Cloning of Interleukin 6(I1-6)receptor/gp130 and TielThe DNA fragment coding the intracellular region of gp130 and that coding the extracellular region of Tiel were amplified by PCR.2.Construction of a chimera gene of gp130 and Tiel and its introduction into pro-B cellsThe DNA fragments of gp130 and Tiel were subcloned into an expression vector of pcDNA3 to construct a chimera gene. Then we transfected this plasmid to IL-3 dependent pro-B cells(Ba/F3)by electroporation method. However, we could not detect the chimera protein by immunoblotting with anit-gp130 antibody. Since RNA expression of a chimera gene was detected, its stability at protein level appeared to be unstable.3.Molecular cloning of the genes that are induced by gp130We cloned several IL-6 induced genes by subtraction method, one of which was Ral guanine nucleotide dissociation stimulator(RalGDS). The induction was fully dependent on Stat3 activation. The analysis of RalGDS function in signal transduction through gp130 now is under way.factor(TNF)α, one vascular endothelial growth factor.4.Cloning of inhibitory molecules for signal transduction of tumor necrosisWhile TNFα induces the apoptosis in NIH3T3 mouse fibroblasts, v-Src-transformed NIH3T3 cells are resistant. We constructed a retroviral cDNA expression library from v-Src-transformed cells and infected it to NIH3T3 cells. Then we selected several clones in the presence of TNFα and recovered cDNA from each clone. One of them was an antisense-fragment coding a novel possible serine threonine phosphatase. Full length of the cDNA was cloned by PCR method, sequenced, and named as PET.When antisense of PET was introduced into NIH3T3 cells, they became partially resistant to TNFα. Now, the analysis of its expression in mouse tissues and function are under way.