pCT-H2B-GFP（CMV）细胞器核定位荧光定位载体质粒 BioVector NTCC质粒载体菌种细胞基因保藏中心

pCT-H2B-GFP（CMV）细胞器核定位荧光定位载体质粒 BioVector NTCC质粒载体菌种细胞基因保藏中心
I. Introduction and Background
Molecular trafficking is a dynamic process in eukaryotic cells. The Cyto-Tracers™ provide a means of live monitoring of molecules in real-time. SBI has created a line of lentivector-based Cyto-Tracers that utilize GFP-fusion proteins to mark cellular compartments, organelles, vesicles and structures to enable more long-term and more in-depth experimentation. Using Cyto-Tracers, cellular structures can be visualized in real-time by fluorescence microscopy without the use of chemical staining. You can also monitor the dynamic movement of a protein-of-interest in relationship to a given subcellular compartment or structure by labeling the protein-of-interest with separate fluorescent proteins. The Cyto-Tracers are built upon an HIV-based lentiviral expression system to enable broad tropism for delivery, stable expression via integration, robust synthesis, and expression by using appropriate promoter elements. To ensure the expression of these constructs in a variety of cell types, constructs are available with two extensively-tested promoters. The Murine Stem Cell Virus promoter (MSCV) is useful for stem cells and hematopoietic cell types. The Cytomegalovirus promoter (CMV)-driven constructs can be used for other, easier to infect cell types. Cyto-Tracers can be used in transfections or packaged into virus to create stable GFP tracer cell lines in primary cells, tumor cell lines and stem cells. This allows researchers to perform preliminary plasmid-based transfection experiments before proceeding to package lentivirus particles, which allow long-term and in depth studies

Vector Components
The components of the pCT-CMV/MSCV-GFP-Fusion-EF1-Puro vectors are displayed in the table below.

GFP-Fusion Protein Characteristics Each construct encodes a fusion protein consisting of an organelle-specific signaling peptide and the copGFP. The organelle-specific signaling peptide directs the copGFP to the appropriate subcellular location. SBI also offers a GFP untagged vector for researchers who would like to create their own tag for subcellular locations not currently available. copGFP is a novel natural green monomeric GFP-like protein derived from copepod (Pontellina sp.). The copGFP has optimized human codons for a high level of expression of the fluorescent protein. CopGFP is a non-toxic, non-aggregating protein with fast protein maturation, high stability at a wide range of pH (pH4-12), and does not require any additional cofactors or substrates. The copGFP protein has very bright fluorescence that exceeds at least 1.3 times the brightness of EGFP, the widely used Aequorea victoria GFP mutant. The copGFP protein emits green fluorescence with the following characteristics:
excitation wavelength max= 482 nm
emission wavelength max = 502 nm
Due to its exceptional properties, copGFP is an excellent fluorescent marker that can be used instead of EGFP for monitoring molecular dynamics.
II. Available CytoTracers


III. Protocol
A. Transfection of pCT-Vector into Target Cells The pCT vectors can be transfected into target cells for use in pilot experiments or for use in cells that are easy to transfect. We recommend using SBI’s PureFection™ nanotechnology-based transfection reagent according to the protocol: http://www.systembio.com/downloads/PureFection_protocol_ver2. pdf
B. Transduction of pCT-Vector into Target Cells For cells that are more difficult to transfect, such as stem cells, primary cells, or hematopoietic cells, or to create stable GFP tracer cell lines, we recommend packaging the pCT-vector into virus and then transducing the target cells with the virus. For a protocol and list of necessary supplies and reagents, please refer to the Lentivector Expression Systems manual: http://www.systembio.com/lentiviral-technology/deliverysystems/ppack/?id=product_11#product_8_tab_1_4

IV. Examples
A. GFP fluorescence from Cyto-Tracers with different subcellular location-specific tags

B. pCT-vectors can be either transduced or transfected into cells

【Supplier来源】BioVector NTCC Inc.
TEL：+86-010-53513060
【Website网址】 http://www.biovector.net