Can In-Fusion be used to clone shRNA (small hairpin RNA)?

An shRNA double-stranded DNA oligonucleotide (?50 bp) can be cloned via In-Fusion technology into a linearized shRNA expression vector.

-For In-Fusion cloning of short synthetic oligos (between 50 and 150 bp), the suggested oligo-to-vector molar ratio is 5–15:1. Depending on the oligo length, the optimal ratio must be determined empirically.
-High-quality, non-phosphorylated oligonucleotides purified by desalting are compatible with In-Fusion Cloning. However, 3' exonuclease activity in the In-Fusion enzyme mix requires terminal 3' OH groups.

Note: Not all antisense oligonucleotides designed and tested for direct cell transfection, such as siRNAs, will be equally efficient when expressed as an shRNA from a vector. It is usually recommended to redesign the siRNA oligo for expression as an shRNA with various orientations of the target sequence as a sense or antisense strand.

-For efficient knockdown, at least four different shRNA constructs are typically designed and tested first in transient transfection (using easy-to-transfect cells, if applicable), prior to establishing a stable cell line or running in vivo experiments.
-In order to distinguish recombinant shRNA vectors, a diagnostic restriction site (MluI) can be inserted into the shRNA oligo downstream from the RNA Polymerase III Termination Signal.

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