The efficient establishment of genetically-modified clones.

<p>(<b>A</b>) 293T clones expressing GFP were rapidly established by infection and sorting with magnetic beads. The 293T cells were first lentivirally transduced with 3.7lnGFP and 3.7CMVBirA. After infection, 293T cells expressing GFP were dissociated with 0.5 mM EDTA and mixed with streptavidin-labeled beads. Only GFP-positive cells adhered to the streptavidin-labeled beads. Single 293T cells isolated with the streptavidin-labeled beads were cultured to form a stable cell line within ten days. (<b>B</b>) HCT-15 clones expressing GFP were established by infection and sorted with magnetic beads. HCT-15 cells were infected with 3.7lnGFP and 3.7CMVBirA. After infection, the GFP-positive HCT-15 cells were isolated with streptavidin-labeled beads. (<b>C</b>) Establishment of a 293T cell line stably expressing BirA. 293T cells were first infected with 3.7CMVBirA lentivirus, then transiently transfected with 3.7lnGFP vector and mixed with streptavidin-labeled beads. The modified cells were isolated using streptavidin-labeled magnetic beads. (D) Stable expression of transgene in HCT-15 cells. After 20 days of selection, the stable expression of GFP was determined by FACS analysis. Shown are the data represented as a dot-plot analysis.</p

Isolation of genetically-modified cell lines with streptavidin-labeled magnetic beads.

<p>(<b>A</b>) Schematic structures of the lentivectors used for delivery of the genes of interest into cells. 3.7EF-1α-ER-<b>Δ</b>LNGFR-CMV-transgene was a lentivector derived from the backbone of pll3.7. The first transgene is expressed in a cassette with an CMV promoter. A truncated form of the membrane-anchored human low-affinity nerve growth factor receptor (LNGFR) tagged with BirA tag (GLNDIFEAQKIEWHE) peptide was driven by a EF-1α promoter in a separate cassette. 3.7EF-1α-transgene-CMV-ER-BirA was a lentivector containing the second transgene expression cassette and the BirA enzyme as a reporter gene. (<b>B</b>) The 293T cells were co-transfected with 3.7lnGFP and 3.7CMVBirA lentivectors and isolated with streptavidin-labeled magnetic beads. Merged images (left panels), GFP fluorescence images (right panels).</p

FACS analysis of genetically-modified cell lines.

<p>(<b>A</b>) FACS staining of transfected 293T cells with antibodies against the marker proteins LNGFR and Myc. 293T Cells were transfected with 3.7lnGFP, 3.7CMVBirA, or both 3.7lnGFP and 3.7CMVBirA and selected with streptavidin-labeled beads. The cells were then analyzed by flow cytometry to detect the level of surface expression of Myc or LNGFR. Histogtams indicate Myc and LNGFR expression (top four panels, middle four panals), shaded histograms represented untransfected 293T cells. The percentage of positive cells is indicated in each panel. (<b>B</b>) Flow cytometric analysis of coexpression of GFP and biotinylated ΔLNGFR. The cells were stained with streptavidin-PE-Cy5. GFP and streptavidin double positive cells are indicated by gate and percentage. Shown are the data represented as a dot-plot analysis of stained tansfected 293T cells.</p

HCT-15 cells modified with a single transgene (β2m-gp100) were specifically targeted by TCR-modified NK cells.

<p>(<b>A</b>) Modified HCT-15 cells were isolated with streptavidin-labeled beads, and single cell-derived clones were grown for 8 days. (<b>B</b>) FACS analysis of HCT-15 cells transduced with 3.7ln β2mgp100 and 3.7cmvBirA lentivirus. The cells were stained with mAbs W6/32, which is specific for HLA-A, B and C, BBM1, which is specific for β2m and streptavidin-PE-Cy5 conjugate. Mock-infected HCT-15 cells were infected with lentivirus without a target gene (shaded histograms). HCT-15 cells were infected with lentivirus 3.7ln β2mgp100 and 3.7CMVBirA (left panels) and selected by streptavidin-labeled beads (right panels). (<b>C</b>) Specific killing of HCT-15 cells expressing β2mgp100 by NK92MI/gp100 cells. NK92MI-pef-GPA7A is a NK92MI cell line expressing GPA7A, a TCR-like antibody specific for HLA-A2 loaded with gp100 peptide. The parental NK92MI cell line was used as a control. The results are presented as percentage ±SD from five individual experiments. (<b>D</b>) Stable expression of β2mgp100 gene in lentivirus transduced HCT-15 cells. Transduced cells were passed 20 passages and stained with W6/32 antibody (black line). Mock-transduced HCT-15 served as a negative control (shaded histogram).</p

Impedance Detection and Modeling of Chemotherapeutic Agents by a Cancer Cell-Based Biosensor

<div><p>Impedance detection on adherently growing cells by micro-electrodes provides useful analytical methods for cancer research. Colon adenocarcinoma is one of the most common types of gastrointestinal cancer. To improve the survival rate of colon adenocarcinoma patients, chemotherapy becomes more and more important among the standard clinical treatments. In this study, a cell based-biosensor was developed to determine the cytotoxicity of therapy medicines to colorectal cancer using the LS180 cell line. By analyzing the varying parameters in the membrane and cell nucleus in an impedance circuit model, the anticancer mechanisms of the drugs were deduced by impedance sensing. By real-time monitoring of the biosensor, time- and concentration- dependent impedance changes were detected under treatment medicines, such as oxaliplatin, 5-fluorouracil, and the more effective combination regimen. The cell-based biosensor combined with circuit modeling provides a promising approach for drug screening and developing new therapeutic regimens with anticancer medicines.</p> </div