RNAi-Ready pSIREN-RetroQ-ZsGreen1 is a self-inactivating retroviral expression vector designed to express a small hairpin RNA (shRNA) using the human U6 promoter (PU6; RNA Pol III-dependent). RNAi-Ready pSIREN-RetroQ-ZsGreen1 is provided as a linearized vector digested with BamHI and EcoRI. It is used for targeted gene silencing when a ds DNA oligonucleotide encoding an appropriate shRNA is ligated into the vector. You can transfect your pSIREN-RetroQ-ZsGreen1 construct as a plasmid expression vector, or—upon transfection into a packaging cell line—this vector can transiently express, or integrate and stably express a viral genomic transcript containing the human U6 promoter and the shRNA.

In addition, this vector independently expresses a Zoanthus sp. green fluorescent protein (ZsGreen1; 1), which has been engineered for brighter fluorescence (excitation maximum = 496 nm; emission maximum = 506 nm). The ZsGreen1 gene is positioned just downstream of the immediate early promoter of cytomegalovirus (PCMV IE). As a result, cells transfected with this vector will express the green fluorescent protein constitutively.The ZsGreen1 fluorescent marker allows you to directly monitor the delivery efficiency of your gene silencing construct using either fluorescence microscopy or flow cytometry.

This retroviral vector is optimized to eliminate promoter interference through self-inactivation. The hybrid 5' LTR consists of the cytomegalovirus (CMV) type I enhancer and the mouse sarcoma virus (MSV) promoter. This construct drives high levels of transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (2–5) in these cells. The self-inactivating feature of the vector is provided by a deletion in the 3' LTR enhancer region (U3). During reverse transcription of the retroviral RNA, the inactivated 3' LTR is copied and replaces the 5' LTR, resulting in inactivation of the 5' LTR CMV enhancer sequences. This may reduce the phenomenon known as promoter interference (6) and allow for more efficient expression.

Also included in the viral genomic transcript are the necessary viral RNA processing elements including the LTRs, packaging signal (Psi+), and tRNA primer binding site. RNAi-Ready pSIREN-RetroQ-ZsGreen1 also contains a bacterial origin of replication and E. coli Ampr gene for propagation and selection in bacteria.

载体应用

RNAi-Ready pSIREN-RetroQ-ZsGreen1 is used for targeted gene silencing when a ds DNA oligonucleotide encoding an appropriate shRNA is inserted. To construct recombinant pSIREN-RetroQ-ZsGreen1, first design, generate, and anneal complementary shRNA oligonucleotides using the protocols in the Knockout RNAi Systems User Manual (PT3739-1). The annealed oligonucleotide should contain 5'-BamHI and 3'-EcoRI overhangs. Then ligate the annealed oligonucleotide into RNAi-Ready pSIREN-RetroQ-ZsGreen1.

Your pSIREN-RetroQ-ZsGreen1 construct can be transfected as a plasmid expression vector to screen for functional shRNA oligonucleotides. For gene silencing experiments using viral delivery, transfect the pSIRENRetroQ- ZsGreen1 construct into a packaging cell line (see the Retroviral Gene Transfer and Expression User Manual, PT3132-1, for a list of packaging cell lines available from Clontech Laboratories, Inc.); RNA from the vector is packaged into infectious retroviral particles. These retroviral particles can infect a wide range of target cells and transmit the shRNA but cannot replicate within these cells due to the absence of viral structural genes.

The ZsGreen1 fluorescent marker in this vector allows direct monitoring of the delivery of your gene silencing construct. Use fluorescence microscopy or flow cytometry to easily detect or enrich for cells containing your recombinant shRNA vector.