pGreenPuro shRNA vector BioVector NTCC质粒载体菌种细胞基因保藏中心

pGreenPuro shRNA vector BioVector NTCC质粒载体菌种细胞基因保藏中心 pGreenPuro shRNA慢病毒荧光表达载体-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心
pGreenPuro™ shRNA Expression Lentivector The pGreenPuro™ expression vector is an improved third generation of HIV-based expression lentivector developed for gene therapy applications (Sodroski, J.G., 1997, 1999, Federico, 2003, Heiser, 2004, Machida, 2003). See section F for safety guidelines. The pGreenPuro™ vector (see detailed functional map in Appendix) provide the following features: • H1 expression cassette provides constitutive and efficient RNA polymerase III-dependent transcription of shRNA transcripts in a wide range of cell lines. • CMV promoter promotes high level of expression of both copGFP (fluorescent reporter) and puromycin-N-acetyl transferase (drug selectable marker) in the same vector for detection and selection of either transduced or transfected cells. • Hybrid RSV-5’LTR promoter provides a high level of expression of the full-length viral construct in 293 cells.
• Genetic elements (cPPT, GAG, LTRs) necessary for packaging, transducing, and stable integration of the viral expression construct into genomic DNA. • SV40 origin for stable propagation of the pGreenPuro™ plasmid in 293 producer cells. • The pUC origin for high copy replication and maintenance of the plasmid in E.coli cells. • The ampicilin-resistance gene for selection in E.coli cells. • WPRE element enhances stability and translation of the CMV-driven transcripts. • The SV40 polyadenylation signal enables efficient termination of transcription and processing of recombinant transcripts. The pGreenPuro™ Vector (Cat. # SI505A-1) contains a puromycin resistance gene to enable drug selection of target cells stably expressing the siRNA, 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. Two approaches have been developed for in vivo expression of siRNA from plasmid and viral vectors. In one approach, the sense and anti-sense strands are transcribed separately from two independent promoters and form the siRNA duplex (Lee 2002, Miyagishi 2002). With the second approach, a single-stranded shRNA sequence with a fold-back stem-loop structure (also known as a “hairpin”) is expressed from a single promoter (Abbas-Terki 2002, Qin 2003, Wiznerowicz 2003). This sequence is then converted into double-stranded siRNA after intracellular processing cleaves the loop (Brummelkamp 2002, Paddison 2002). In both approaches, the siRNA molecules are transcribed from constitutive RNA polymerase III promoters (i.e., U6 and/or H1) and terminated with TTTTT (T5) sequences (Tuschl 2002). The U6 and H1 promoters are different in size but contain the same conserved sequence elements (Myslinski 2001).
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 (Figure 1). 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 (Figure 1). 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) (Figure 2). Two PCR primers are designed for regions flanking the shRNA insert in order to provide a simple way for screening of plasmid clones for the presence of shRNA inserts by PCR (Figure 2).
电话：+86-010-53513060 网址：www.biovector.net [Supplier来源] http://www.biovector.net