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RNAi Vector
Vector Description All of the plasmids within the HuSH shRNA collection are cloned into OriGene's non-proprietary pRS vector, allowing both transient and stable transfection, as well as stable delivery of the shRNA expression cassette into host cells via a replication-deficient retrovirus. These vectors can be purchased independently. • HuSH Green Fluorescence Protein (pRS-shGFP 29) Hush knockdown using these control plasmids Vector Diagram The HuSH pRS plasmid vector contains both 5 and 3 LTRs of Moloney murine leukemia virus (MMLV) that flank the puromycin marker and the U6-shRNA expression cassette. Upon transient transfection of the plasmids into a packaging cell line, replication deficient viruses can be obtained and used to infected target cells. A puromycin-N-acetyl transferase gene is located downstream of SV40 early promoter, resulting in the resistance to the selection of antibiotics puromycin. For our RNAi products, the shRNA expression cassette consists of a 29 bp target gene specific sequence, a 7 bp loop, and another 29 reverse complementary sequence, all under human U6 promoter. A termination sequence (TTTTTT) is located immediately downstream of the second 29 bp reverse complementary sequence to terminate the transcription by RNA Pol III. The 29 bp gene specific sequence was sequence-verified to ensure its match to the target gene. The basic backbone of the this vector series is presented diagrammatically below in Figure 1 and the full vector sequence is available.
Figure 1 Vector Diagram The HuSH pGFP-RS plasmid vector was created with an integrated GFP element to readily verify transfection of the HuSH plasmid into cells. It also incorporates both a kanamycin and puromycin resistance elements for greater selection capabilities. The sequence is available for download.
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