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| Cat#: | SI505A-1-SBI |
| Quantity: | 10 ug |
| Price: | 522 € |
| Supplier: | SBI |
· Stably express Short-Hairpin RNA (shRNA) in any cell or model organism
· Target a specific gene for knockdown or make a library of siRNA sequences to screen
· Select transfected / transduced target cells by puromycin
· Confirm Transfection / Transduction with Fluorescence using copGFP and sort and select transfected /transduced cells by flow cytometry
· HIV (human immunodeficiency virus) based, replication-incompetent design
· shRNA expression promoter by CMV promoter, which is higly active in common cell lines such as HeLa, HEK293, HT1080 etc.
pGreenPuro: shRNA expression vector for GFP sorting and Puromycin selection:
The pGreenPuro™ Vector (right map) contains a puromycin resistance gene to enable drug selection of target cells stably expressing cloned shRNA, and a copGFP gene. The copGFP is a novel fluorescent protein, derived from copepod plankton (Panalina sp.), which is similar to EGFP but has a brighter color. This gene serves as a fluorescent reporter for the transfected or transduced cells. The open reading frames of puromycin resistance and copGFP genes are connected by a T2A sequence and are transcribed from the CMV promoter as a bicistronic transcript. The two proteins are then separated through translational cleavage at the T2A site.
The pGreenPuro™ Vector is designed to express a single-stranded shRNA sequence with a fold-back stem-loop structure (also known as a “hairpin”) from a RNA polymerase III H1 promoter (Abbas-Terki 2002, Qin 2003, Wiznerowicz 2003). The hairpin-type siRNA (shRNA) template oligonucleotides need to be cloned into unique BamHI/EcoRI sites located just downstream of an H1 promoter. The pGreenPuro™ vector needs to be linearized by restriction digest with BamHI and EcoRI, and purified to remove the stuffer fragment. When linearized, the vector contains two unique 5’ overhangs to facilitate directional cloning of shRNA template oligos with minimal self-ligation background.
When the shRNA construct is expressed from constitutive H1 promoter and terminated with the TTTTT sequence, the shRNA transcript folds into the hairpin structure, which is recognized by the DICER enzyme, cleaved to form a functional ds siRNA and transferred to a RISC complex for selective digestion of complementary target mRNAs (Brummelkamp 2002, Paddison 2002).
Pre-packaged pGreenPuro Vector (CMV)
pGreenPuro Vector with EF1 Promoter (stem & primary cells)
Lentivirus Packaging Kit
Lentivirus Purification and Quantitation Kits
copGFP Antibody
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