Development and functional characterization of a lncRNA-HIT conditional loss of function allele.

Analysis of the human and murine transcriptomes has identified long noncoding RNAs (lncRNAs) as major functional components in both species. Transcriptional profiling of the murine limb led to our discovery of lncRNA-HIT, which our previous in vitro analyses suggested a potential role for this lncRNA in the development of limb, craniofacial, and genitourinary tissues (Carlson et al., 2015). To test this hypothesis, we developed a conditional lncRNA-HIT loss of function allele which uses Cre recombinase to activate an shRNA specific for lncRNA-HIT. Activation of the lncRNA-HIT shRNA allele resulted in a robust knock-down of lncRNA-HIT as well as co-activation of a mCherry reporter, confirming the efficacy of the shRNA allele to reduce endogenous lncRNA levels in a tissue- and cell-type specific manner. Developmental analyses of embryos expressing the activated shRNA and mCherry co-reporter revealed multiple malformations corresponding to the sites of shRNA activation, affecting craniofacial, limb, and genitourinary tissue development. These results confirm the efficacy of lncRNA-HIT shRNA allele to knock-down endogenous transcripts in tissue- and cell type specific manner and indicate a requirement for lncRNA-HIT in the development of these tissues.