Construction And Activity Detection Of Dual Fluorescent Protein Reporter Gene Vector Drived By CXCR4 Promoter

Chemokines and chemokine receptors are closely related to lymphocyte transshipment, angiogenesis.A large amount of clinical studies showed the expression level of CXCR4 in breast cancer cells is significantly higher than that of normal mammary epithelial cells.Meanwhile, its corresponding ligand CXCL12 in the most common organs of breast cancer metastasis like lung, lymph nodes, brain, liver, and bone marrow is overexpression.It is this activity affects the behavior of the tumor like CXCR4 can make the cells migrate to organs that express high levels of its respective ligand CXCL12.However, the study of CXCR4 focused on the tissue mixed positive and negative cells, the reasearch can’t indicate the expression of CXCR4 gene in single positive cells. The purpose of this study is to establish a method for separation of CXCR4 single positive cells, to facilitate the study of the gene expression and transfer behavior in the body. A dual fluorescence vector which can simultaneously express red fluorescent protein and green fluorescent protein is designed and constructed. Then CXCR4 gene promoters were obtained successfully by PCR from 4T1 cell genomic DNA and replace the CMV to drive green fluorescent protein. The dual fluorescence carrier was transfected into 4T1 breast cancer cell. So the expression of CXCR4 gene cells expressing green fluorescent and red fluorescent, so we can mark CXCR4 positive cells. Then the positive cells were sortied by using flow cytometry and cultured in vitro. Lay the foundation for the research of metastasis.The results show that, the dual fluorescence vector is successfully constructed, and it is effective. The dual fluorescent protein reporter gene vector drived by CXCR4 promoter is also successfully constructed, and it is effective.Then the positive cells were sortied by using flow cytometry and cultured in vitro.We find that the ductility of positive cells is weak and the pseudopodia is short, while the ductility of negative cell is strong and the pseudopodia is long. So the CXCR4 positive cells are easy for invasion and metastasis. In vitro culture fully confirmed that CXCR4 positive cells and CXCR4 negative cells have growth pattern, these results provide a reliable basis for the study in vivo.The significance of the study is using the promoter for the cell sorting instead of specific antibody screening. This methord can keep cancer cells primitiveness and integrity and it is advantageous to the growth of in vivo culture. The dual fluorescent reporter gene carrier can also be used for other animal gene promoter replacement, sorting different gene expression of positive cells, it is a useful report gene carrier.According to the results the high transcriptional activity of CXCR4 gene promoter in breast cancer cell indicates its potential utility as a novel candidate for transcriptional targeting of breast cancer.