shRNA Info

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shRNA Vector Map

The current shRNA library was made in the laboratory of Greg Hannon in the pSHAG-MAGIC2c vector (short hairpin activated gene silencing - Mating Assisted Genetically Integrated Cloning System), which is a modified version of the original pSHAG-MAGIC vector constructed in the laboratory of Stephen Elledge. Both vectors are self-inactivating stem cell virus (MSCV) retroviral vectors containing a number of convenient design features to allow in vivo DNA transfer from the donor (the shRNA containing) vector to a recipient vector. The modified vector permits the insertion of the PCR amplified oligo containing the hairpin and common 5' and 3' ends.

• LTR - long tandem repeats of the murine stem cell virus
• Ψ - Nhe1/Xba1 deletion
• I-Sce1 - rare restriction endonuclease sites flanking the cloning cassette, used in the MAGIC shRNA movement strategy
• U6 - U6 promoter
• leader - 27 nucleotide U6 leader sequence which has a positive effect on transcription
• miR5'-miR3' - the shRNA construct
• Cmr - chloramphenicol resistance gene
• HR2
• PGK-puro - murine phosphoglycerate kinase (PKG) promoter used to express the puromycin resistance gene for antibiotic selection in eukaryotic cells.
• sinLTR - self-inactivating long terminal repeat of the murine stem cell virus
• RK6γ - conditional plasmid origin which requires expression of the PIR1 gene to function
• F1 otiT - facilitates F'-dependent transfer of the shRNA cassette during bacterial mating
• Kan - kanamycin resistance gene