| To obtain the cells express heterologous genes of interest is a fundamental technique in the preclinical medicine and biological research. But owing to the low transfected efficiency, selection of cell lines expressing desired genes from a large number of candidate clones is often an important but tedious work.In standard methods, plasmid commonly contains an antibiotic resistant gene to facilitate identification and selection of gene-modified cells. After transfection, transfected cells were cultured in the selective medium for a period of time, then randomly picking and screening clones of the survival, and amplifying the colonies expressing the gene of interest. Despite its classical use, it is hampered by time-consuming and tedious work for the selection of transfected cells.Recently, the magnetic cell sorting (MACS) technique has been widely used in cell sorting owing to its efficacious, commercially available and clinical approved system. The basis for selection of cells by a magnet was the antibody-coated magnetic beads bind to the antigen displayed on the surface of cells, and the magnetic labeled cells would be separated in a magnetic field. To facilitate the selection of cell lines expressing heterologous genes of interest, we applied the MACS technique in the transfected cells sorting. And a bicistronic vector has be constructed which contained a CD34 marker gene for MACS-based selection of transfected cells.CD34 is a surface glycophosphoprotein mainly expressed on hematopoietic stem/progenitor cells (HSC/HPC). An engineered variant of CD34, delected CD34 (dCD34), which has the extracellular domain for adhesion but completely lacks the cytoplasmic tails was amplified with PCR. The PCR products were ligated into the pGEM-T-easy vector.To permit the co-expresion of the inserted 5'-end heterologous gene and the 3'-end marker gene, the internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) was employed. IRES sequences allow the initiation of translation in a cap-independent manner: ribosomes bind internally at the initiating AUG without scanning the 5' nontranslated region of the transcript, and two genes connected by IRES sequences can be efficiently expressed from a single promoter. The CD34 gene and IRES fragment isolated from pIRES2-EGFP were inserted into pGEM-T-easy vector, and then pT-IRES-CD34 was digested with Sail, Xbal to generate IRES-CD34 fragment and cloned between the Xhol and Xbal site of pcDNA3.1(+) to generate p3.1-IRES-CD34. It contains an EMCV IRES and an artificial variant of CD34 following the multiple cloning site (MCS). After cells were transfected with the vector, the heterologous genes of interest and CD34 gene coexpressed, and then the cells express heterologous genes would be selected by MACS.To test the utility of p3.1-IRES-CD34, the enhanced green fluorescent protein (EGFP) gene was chosen as the heterologous genes of interest. EGFP gene was cloned with PCR from the plasmid pIRES2-EGFP and inserted into the MCS of the vector. NIH-3T3 Cells were transfected with the bicistronic expression vector p3.1-IRES-CD34-EGFP using the Lipofectamine transfection reagent according to the manufacturer's protocol. After transfection, magnetic cell sorting was performed to screen the transient and stable transfected cells.For transient transfected cells, after 48hr incubation in the growth medium, MACS selection was performed. At least 95% of the positive fraction cells showed GFP fluorescence, contrastively, in the negative fraction, only <3% cells were green under fluorescent microscopy. As evident from flow cytometry, after one round of MACS, the percentage of CD34-positive cells was highly increased (from 2% to 88%). And it indicated that the expression of EGFP and CD34 marker was coupled, the results were similar for the stable transfected cells. Additional, for the relatively low transfection efficiency cells, to yield comparatively pure positive populations by only one round of MACS, an improved method was adopted. As the vector...
|
PDF Full Text Request