neomycin selection marker  (TaKaRa)

Journal: Journal of Carcinogenesis

Article Title: Human endogenous retroviral K element encodes fusogenic activity in melanoma cells

doi: 10.4103/1477-3163.109032

Figure Lengend Snippet: Inhibition of K type human endogenous retrovirus (HERV-K) expression blocks intercellular fusion-mediated colony formation. Human melanoma cell line A375 (a) and 624Mel (b) was transiently transfected and selected to stably express either control pS-puro-scrambled (1) or HERV-K shRNA pS-puro-H-Ki construct (2). (a). RT-polymerase chain reaction (RT-PCR) using total cellular RNA was performed using specific HERV-K POL and envelope (ENV) primers as described (15). Gel electrophoresis of RT-PCR products revealed that POL and ENV RNA levels were reduced in shRNA expressing cells of both A375 (a) and 624Mel (b) cells. GAPDH was used as internal control (b). Western blot analysis of HERV-K ENV protein. Total cellular protein was separated on SDS-PAGE, and blotted to Nilon membrane, and incubated with HERV-K ENV antibody. Actin was used as loading control. As expected, HERV-K POL and ENV transcripts (a) and ENV protein (b) levels were reduced in cells expressing shRNA. The knockdown levels of both RNA (a) and protein (b) were estimated as more than 50% (c). To examine cell fusion, 106 A375 or 624Mel cells stably expressing pEYFP-N3 were mixed with equal numbers of the same cells expressing either pS-puro-scrambled or pS-puro-H-Ki. Mixed cells were cultured to confluency in medium without puromycin or G418 selection. Cells were then trypsinized and passaged in culture medium that includes both puromycin and G418 to select pS-puro-scrambled-pEYFP-N3 and pS-puro-H-Ki-pEYFP-N3 fused cells. Colony formation assay was used to count “fused” clones under double selection

Article Snippet: To examine cell fusion, we also generated A375 and 624Mel cell lines stably expressing pEYFP-neo-N3 (Clontech, Mountain View, CA) that carried yellow fluorescent protein (YFP) with a neomycin (G418) selection marker.

Techniques: Inhibition, Expressing, Transfection, Stable Transfection, shRNA, Construct, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Western Blot, SDS Page, Incubation, Cell Culture, Selection, Colony Assay, Clone Assay

Journal: Journal of Carcinogenesis

Article Title: Human endogenous retroviral K element encodes fusogenic activity in melanoma cells

doi: 10.4103/1477-3163.109032

Figure Lengend Snippet: Live cell two color fusion assay and immune-neutralization of cell fusion using K type human endogenous retrovirus envelope monoclonal antibodies. Two color fusion assay (a and b). A375 cells stably expressing pS-puro-scrambled or pS-puro-H-Ki were labeled with blue Hoechst 33342, and A375 cells stably expressing pEYFP-N3 were labeled with green BODIFY using CellTracker probes for long-term tracing of living cells reagents according to manufacturer's instruction (Invitrogen, Carlsbad, CA). After dye labeling, 107 Hoechst 33342-labeled pS-puro-scrambled or pS-puro-H-Ki cells were each mixed with 10 7 green BODIFY-labeled pEYFP-N3 expressing cells. Mixed cells were cultured for 12 h without puromycin or G418 selection. Cells were then cultured in medium containing both puromycin and G418 for 48 h to select for puro-neo fused green (Green BODIFY) and blue (Hoechst 33342) double stained cells. Upper left, blue-green double colored cells were present when pEYFP-N3 expressing cells were mixed with pS-puro-scrambled (a), but blocked with pS-puro-H-Ki expressing cells (b). Lower left and right, blue and green stained cells, respectively. Photographed by fluorescence microscope. Upper right, photographed through phase contrast microscope

Article Snippet: To examine cell fusion, we also generated A375 and 624Mel cell lines stably expressing pEYFP-neo-N3 (Clontech, Mountain View, CA) that carried yellow fluorescent protein (YFP) with a neomycin (G418) selection marker.

Techniques: Single Vesicle Fusion Assay, Neutralization, Stable Transfection, Expressing, Labeling, Cell Culture, Selection, Staining, Fluorescence, Microscopy

Journal: Journal of Carcinogenesis

Article Title: Human endogenous retroviral K element encodes fusogenic activity in melanoma cells

doi: 10.4103/1477-3163.109032

Figure Lengend Snippet: Antibody neutralization assay. 10 4 each of A375 cells stably expressing pS-puro-scrambled and pEYFP-N3-neo were added per well in 96 well plate. Different dilutions of two K type human endogenous retrovirus envelope monoclonal antibodies HERM-1811-5 and HERM-1821-5 were added to culture medium. PBS was used as control. Cells were cultured for 48 h, followed by selection of pS-puro-scrambled-pEYFP-N3 fused cells using puromycin and G418 double selection for 48 h. The numbers of blue-green double color cells (fused cells) as shown in 3a upper left were calculated and compared between antibody treated and control

Article Snippet: To examine cell fusion, we also generated A375 and 624Mel cell lines stably expressing pEYFP-neo-N3 (Clontech, Mountain View, CA) that carried yellow fluorescent protein (YFP) with a neomycin (G418) selection marker.

Techniques: Neutralization, Stable Transfection, Expressing, Cell Culture, Selection