[Construction of murine CXCR3 gene transfected cell line, identifiction and study of its biological function]

To construct recombinant murine CXCR3 gene retroviral vector and obtain L929-mCXCR3 gene transfected cell line for stably expressing murine CXCR3. We further study on L929-mCXCR3 migration effect resulted from interaction by CXCR3 and its ligand IP-10.

One female BALB/c mouse (7 weeks) was injected with 0.5 mg Con A intravenously (i.v.) via a tail vein. Twelve hours later the mouse was sacrificed and the spleen was removed.The spleen was pressed through a 150 microm stainless steel mesh. The isolated splenocytes were cultured in RPMI1640 supplemented with 50 U/mL human IL-2 for 3 days.Total RNA was extracted with TRIzol. Murine CXCR3 gene of full length was amplified by RT-PCR, then, it was inserted into retrovirus vector pEGZ-term. The recombinant vector together with its helper virus vector were co-transfected into package cell 293T with Lipofectamine(TM); 2000.The supernatant of 293T was collected for infecting L929 cells (repeated three times), and cell clones stably expressing murine CXCR3 molecule were screened by zeocin(500 mg/L). We used FCM and RT-PCR to verify expression of CXCR3 from protein level and gene level, respectively. Studied migration ability of L929-mCXCR3 interacted with its ligand IP-10 by transwell system.

We have constructed recombinant murine CXCR3 gene retroviral vector and obtained L929-mCXCR3 gene transfected cell line which can stably expressing murine CXCR3 molecule. Positive expression rate of membrane is 97.0%, and it can directly migrate induced by IP-10, the chemotatic index is 4.356%.

Construction of L929-mCXCR3 cell line has laid a good foundation on research of biologic characteristics of CXCR3 signal path , establishment of tumor metastasis model and preparation of anti-murineCXCR3 monoclonal antibody.