Establishment Of A Novel System Based On Biotin And Avidin To Screen Positive Cells Expressing Target Genes

A valid method to efficiently screen gene-modified cells is critical for establishment of stable cell lines for functional studies and, possible gene therapies. Traditionally, Fluorescence-activated cell sorting (FACS) with fluorescence marker or other reporter genes including galactosidase and resistant genes of antibiotic were used for the selection of gene-modified cells. However, there are some disadvantages for these currently available selective methods. FACS is one of very efficient methods but an expensive instrument is required. The application of galactosidase gene is used for selection of single positive clone, and the detection of the gene is disruptive for cells checked. Antibiotic resistant cell lines are often target genes negative and they are not suitable for clinical application in vivo due to antibiotic resistant gene contained. An alternative selection of gene-modified cells with speed, efficiency, easy to operate would be desirable for generation of gene-modified cells.Here we have established a novel system for efficient screening gene modified cells, which might find wide applications in biological research and clinical therapy. Furthermore, the system is allowed for the selection of two target genes simutaniously. The system, based on the specific and high affinity between avidin/streptavidin and biotin (Ka=1014~1015M -1), contains two report elements, one is BirA enzyme that catalyzes covalently linkage of biotin toε-NH2 of BirAtag (GLNDIFEAQKIEWHE) and another one is a fusion protein A2TM which can express BirA tag on the surface of a cell by transmembrane protein HLA-A2. As long as both elements with two target genes are delivered into a cell, a biotin linked tag will be expressed on the surface of gene-modified cells, which can be easily hooked onto a streptavidin linked solid phase and selected efficiently.At first a four-plasmid lentivirus system is adopted as model to demonstrate the feasiblility and efficiency of selection system. To make the reporter elements expressed properly they are targeted into endoplasmic reticulum directly by association of ER signal with the N terminal of BirA and A2TM, meanwhile, an ER retention signal was fused to the C terminal of BirA for increasing the efficiency of biotinylation in the ER. The modified A2TM and BirA were inserted into each lentivector respectively. The remain cassette in each vector is used for expressing target gene. Once the genome of lentivectors enter cells through transient transfection or infection, along with two target genes, BirA was expressed in ER and biotinylated BirAtags fused with A2TM when they passed through ER. The biotinylated BirAtags were appeared on the surface of target cells by A2TM. The gene-modified cells were easily captured with paramagnetic beads coated with streptavidin. Use a handy magnet to pull the target cells to the tube wall, leaving the unwanted cells in the supernant. After isolation, the beads are detached from the cells. Meanwhile, to screen easily the cells modified more feasible, we also construct some correlative vectors which only express A2TM or BirA.The modified gene selection system have been demonstrated in three applications. Firstly, the cells expressing single modified gene were selected using the system. GFP report gene was cloned into one vector with surface expression of BirAtag and co-transfected with a vector BirA enzyme. Positive GFP expressing cells could be pulled down with streptavidin magnet beads. Similarlyβ2 microglobulin (β2m) protein linked with a T cell epitope peptide could be expressed in eitherβ2m negative HCT, a human colorectal carcinoma cell or B16AAD, a mouse melanoma cell line B16 transduced with HLA-A2. The cells screened will be used as target cells of CTLs specific to pMHC-I-flu/gp100 in indirectly killing assays. Secondly, the selection of cells with two modified genes was tested for the expression of both ICP47, a herpes simplex virus immediate early protein andβ2m-flu/gp100. Thus, the system could be used to obtain a large number of APCs such as CD40B cells and DCs which express bothβ2m-flu/gp100 and ICP47 genes for efficient presentation of specific epitope to T cells. Finally, the system has been demonstrated to deliver a TCR-like single domain antibody (VHH) specific to the melanoma epitope HLA-A2 MART1 or gp100 into NK92mi, a potential cell line for killing tumor cells in vitro and in vivo. In this application, to assign NK cells a specific killing function as that of CTLs, VHH were attached to the N terminal of A2TM for the expression on the suface of target NK cells. A GFP gene was inserted into a selection vector containing BirA enzyme. When selected, modified NK92mi would express both green protein and TCR like antibody on the surface of the cells and could be used for the study of specific killing of tumour cells.In summary, we have established a quick, easy and flexible system for selection of target-gene modified cells and explored its application for selection of either one target-gene modified cells, or two target-genes modified cells simutaneously. The further investigations for optimization of the system and wide applications in different gene delivery system are required for its routine use in the field of research and clinical therapy.