Design and cloning strategies for constructing shRNA expression vectors.

ABSTRACT:

Background

Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %).

Results

We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20-40 % for other strategies. By mod

SUBMITTER: McIntyre GJ 

PROVIDER: S-EPMC1343552 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature