Construction And Expression Of Three Forms Of Tumour Necrosis Factor Alpha Conditional Expression Plasmids

Tumour necrosis factor alpha (TNF-a) is a pleiotropic cytokine that is firstly expressed on the cell surface as a26kD transmembrane protein, namely transmembrane TNF-a (tmTNF-a). The extracellular portion of tmTNF-a can be cleaved by TNF converting enzyme (TACE) and released as a soluble form, called secretory TNF-a (sTNF-a. We have previously reported that tmTNF-a and sTNF-a play a different role in inflammation. sTNF-a exerts pro-inflammatory activity via TNFR1, while tmTNF-a displays anti-inflammatory effect through TNFR2.To further investigate the role of both forms of TNF-a in the process of malignant transformation of nonresolving inflammation, we have constructed wild type, transmembrane and soluble TNF-a conditional expression plasmids which were expressed by eukaryocyte and used to make transgenic mice. This study provides useful animal model for the future related research.The main results of this study are as follows:1. Construction and cloning of three forms of TNF-a conditional expression plasmids:The cDNA encoding wtTNF-a, tmTNF-a or sTNF-a was inserted into conditional expression plasmid pBSA-IRES-GFP. The three recombinant plasmids pBSA-wttnf-IRES-GFP, pBSA-tmtnf-IRES-GFP and pBSA-stnf-IRES-GFP were confirmed through DNA sequencing analysis.2. Construction and cloning of CRE eucaryotic expression plasmid:To express the three recombinant conditional TNF-a containing plasmids, we performed PCR to gain CRE DNA fragment from CMV-CRE transgenic mouse and inserted it into eucaryotic expression plasmid pcDNA3.1(pcDNA-CRE) and confirmed its right sequence by DNA sequencing analysis.3. Conditional expression of the three forms of TNF-a by eucaryocyte: Co-transfection of pcDNA-CRE with the three recombinant conditional plasmids in COS-7cells, the results of flow cytometry showed that wild type TNF-or tmTNF-transfected cells expressed higher membrane TNF-a except sTNF-transfected cells and all the three co-transfectants expressed GFP. In contrast, neither membrane TNF-a nor GFP was detected in the cells solely transfected pBSA-wttnf-IRES-GFP, pBSA-tmtnf-IRES-GFP or pBSA-stnf-IRES-GFP. Furthermore, we found that sTNF-a was secreted in the supernatants of cells co-transfected with pcDNA-CRE and pBSA-wttnf-IRES-GFP or pBSA-stnf-IRES-GFP detected by ELISA assay, but not in the supernatant of tmTNF-transfectants. The data indicate that wtTNF-a gene expresses both tmTNF-a and s TNF-a, tmTNF-a mutant gene only produces tmTNF-a and sTNF-a mutant only secrets sTNF-a.4. Cytotoxicity of sTNF-α:Cytotoxicity of supernatants of cultured cells co-transfected by CRE and the three conditional TNF plasmids against L929cells was detected by MTT bioassay. The culture supernatants of wtTNF-and sTNF-transfectants have been shown significant cytotoxicity that could be largely attenuated by specific TNF-a blocking antibody. However, little cytotoxic effect was observed in tmTNF-transfectants or the cells solely transfected with pBSA-wttnf-IRES-GFP, pBSA-tmtnf-IRES-GFP or pBSA-stnf-IRES-GFP.5. Cytotoxicity of tmTNF-a:Cytotoxicity of tmTNF-a expressed by the transfectants against L929cells was determined by MTT bioassay. Membrane TNF-a displayed cytotoxicity on the surface of cells co-transfected by CRE and wtTNF or tmTNF except sTNF, and the cytotoxic activity was largely blocked by neutralization of membrane TNF-a. Conversely, little cytotoxic effect was detected in the cells solely transfected with pBSA-wttnf-IRES-GFP, pBSA-tmtnf-IRES-GFP or pBSA-stnf-IRES-GFP.6. Establishment of transgenic mice:We entrusted the Guangzhou Cyagen Company to make transgenic mice by using our constructed recombinant conditional plasmids. We got three wtTNF- transgenic founder mice identified by genome PCR. The transgenic mice were then crossbred with CMV-CRE transgenic mice that express CRE universally. Among14littermates,4mice carried wtTNF gene, but no CRE gene and8mice carried CRE gene, but no wtTNF gene. There was no double gene positive mouse. The preparation of the other two strains of transgenic mice (pBSA-tmtnf-IRES-GFP, pBSA-stnf-IRES-GFP) are going on.The results of this study provide transgenic animal model for further investigation of the role of the two forms of TNF-a in malignant transformation of nonresolving inflammation and other related diseases.