Short hairpin RNAs (shRNAs) — synthetic molecules that are modelled on small, non-coding microRNA molecules with a 'hairpin' secondary structure — can silence gene expression by RNA interference (RNAi), much as small interfering RNAs (siRNAs) do. As now reported in Nature Structural Biology, Thomas Rosenquist's group, in collaboration with Greg Hannon's group, explored whether germline transmission of shRNA constructs was feasible in mammals, as this would enable stable, long-term silencing of gene expression.

The initial attempt of Rosenquist and colleagues to achieve germline transmission using standard transgenics methods, in which linearized constructs were injected into pronuclei to create transgenic founder animals, was unsuccessful. Choosing Neil1 (a DNA N-glycosylase that initiates base-excision repair) as the target gene, they turned to a different approach — based on the use of embryonic stem (ES) cells. The authors created a single shRNA expression construct against the Neil1 target gene, which was introduced into mouse ES cells by electroporation. Stable ES cell lines showed ∼80% reduction of Neil1 protein, which correlated with a similar reduction in mRNA levels, and the cells were approximately twofold more sensitive to ionizing radiation — consistent with Neil1's role in DNA repair.

To obtain transgenic animals, the authors injected cells from two independent ES cell lines into blastocysts. The chimeric mice that contained a high percentage of ES-derived cells were outcrossed, and germline transmission of the shRNA expression construct was detected in several F1 offspring. Neil1 protein and mRNA levels were reduced in several tissues of F1 mice, as was observed in the ES cells. In addition, siRNA was detected, by northern blotting, in animals that carried the shRNA expression vector, but not in those that lacked the vector.

The authors conclude, therefore, that shRNAs can be used to create germline transgenic mice in which a target gene is silenced by RNAi. These findings now open the door for tissue-specific, inducible and reversible suppression of gene expression in mice.