載體說明

RNAi-Ready pSIREN-RetroQ-DsRed-Express 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-DsRed-Express is provided as a linearized vector digested with BamH I and EcoR I. 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-RetroQDsRed- Express 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.

This vector also expresses a variant of Discosoma sp. red fluorescent protein (DsRed-Express; 1), which has been engineered for improved solubility (excitation maximum = 557 nm; emission maximum = 579 nm). The DsRed-Express 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 red fluorescent protein constitutively. The DsRed-Express fluorescent marker allows you to directly monitor the delivery efficiency of your gene silencing construct using either fluorescence microscopy or flow cytometry 8–12 hours after transfection.

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-DsRed-Express also contains a bacterial origin of replication and E. coli Ampr gene for propagation and selection in bacteria.

載體應用

RNAi-Ready pSIREN-RetroQ-DsRed-Express is used for targeted gene silencing when a ds DNA oligonucleotide encoding an appropriate shRNA is inserted. To construct recombinant pSIREN-RetroQ-DsRed-Express, 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'-BamH I and 3'-EcoR I overhangs. Then ligate the annealed oligonucleotide into RNAi-Ready pSIREN-RetroQ-DsRed-Express.

Your pSIREN-RetroQ-DsRed-Express construct can be transfected as a plasmid expression vector to screen for functional shRNA oligonucleotides. For gene silencing experiments using viral delivery, transfect the pSIREN-RetroQDsRed- Express 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); 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 DsRed-Express 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.