AI Article Synopsis
- The hU6 promoter is commonly used to express shRNAs in mammalian cells and needs a purine, typically guanine, at the transcription start site.
- A library of shRNAs was created with various nucleotide compositions to test the promoter's requirements for effective transcription.
- The findings indicate that having a purine at position +2 rather than at +1 is more crucial for generating effective shRNAs that can successfully silence target genes, such as PKACalpha.
Article Abstract
The human U6 (hU6) promoter is widely used to express short hairpin RNAs (shRNAs) in mammalian cells. To verify the validity of the generalized concept-the hU6 promoter essentially requires a purine (usually guanine) at +1 for transcription, we enzymatically constructed an arbitrary shRNA library with the following features: (1) to have any one of adenine, cytosine, guanine, and thymine at the site; (2) to comprise shRNAs of 25-30 nucleotides in stem length which are transcribed through the promoter. cDNA of the catalytic subunit of cAMP-dependent protein kinase (PKACalpha) was used as material for library construction. We then used luciferase reporter cell lines to screen shRNAs which effectively reduced PKACalpha activity. Consequently, a purine was mostly present at +2, not at +1, of the clones isolated, suggesting that a purine at +2 rather than +1 adjacent to the hU6 promoter provides effective shRNAs for target gene silencing.
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| http://dx.doi.org/10.1016/j.bbrc.2006.08.187 | DOI Listing |
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