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.

中文翻译：

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lncRNA-HIT 条件性功能缺失等位基因的开发和功能表征

对人类和小鼠转录组的分析已将长链非编码 RNA (lncRNA) 确定为这两个物种的主要功能成分。小鼠肢体的转录分析导致我们发现了lncRNA-HIT，我们之前的体外分析表明该 lncRNA 在肢体、颅面和泌尿生殖组织发育中具有潜在作用（Carlson 等人，2015 年）。为了验证这一假设，我们开发了一种条件性lncRNA-HIT功能缺失等位基因，它使用 Cre 重组酶激活lncRNA-HIT特异的 shRNA 。lncRNA-HIT shRNA 等位基因的激活导致lncRNA-HIT的强烈敲低以及 mCherry 报告基因的共同激活，证实了 shRNA 等位基因以组织和细胞类型特异性方式降低内源性 lncRNA 水平的功效。表达激活的 shRNA 和 mCherry 共同报告基因的胚胎的发育分析揭示了与 shRNA 激活位点相对应的多种畸形，影响颅面、肢体和泌尿生殖组织发育。这些结果证实了lncRNA-HIT shRNA 等位基因以组织和细胞类型特异性方式敲低内源转录物的功效，并表明lncRNA-HIT在这些组织的发育中是必需的。