All of the plasmids within the HuSH shRNA collection are cloned into OriGene's non-proprietary pRS or pGFP-V-RS 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. The pGFP-V-RS vector offes additional feature of turbo-GFP expression that facilitates easy monitoring of transfection.
The HuSH pRS plasmid vector (Figure 1) 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 sequence is for download.
The HuSH pGFP-V-RS plasmid vector (Figure 2) was created with an integrated turboGFP element to readily verify transfection efficiency. It also incorporates both a kanamycin and puromycin resistance elements for greater selection capabilities. Please note the bacterial selection marker for pGFP-V-RS based vectors are kanamycin instead of ampicillin as in pRS-based vectors.
The sequence is available for download.
Additional control vectors are available and can be purchased seperately.
- pRS vector harboring effective shRNA against Green Fluorescence Protein (pRS-shGFP 29)
- pRS vector harboring effective shRNA against Luciferase Protein (pRS-shLuc 29)
- pRS vector harboring a non-effective GFP Protein (pRS-shGFP 29 Non-Effective)
- vector harboring a non-effective GFP Protein (pRS-shGFP 29 Non-Effective)