Figure 2.

Schematic representing how microinjection of RNA into zygotes can experimentally confirm causal function for RNAs, specifically miRNAs, in transmitting epigenetically inherited phenotypes to offspring in mice. First, zygotes are produced by fertilization either naturally (and then flushed from the inseminated mother) or via IVF with sperm and eggs from naive/control animals and then cultured in vitro. In parallel, RNA from environmentally exposed sperm can be purified and further fractionated to isolate specifically miRNA-size (20–24 nt) sperm RNAs. Alternatively, candidate miRNAs of interest altered in the sperm of animals exposed to the environmental condition of interest can be synthetically produced. Purified sperm RNAs or synthetic RNAs are then injected into the zygotes produced. As the control, zygotes are injected with control sperm RNA, scrambled synthetic miRNAs, or other RNAs for comparisons with the experimentally injected zygotes. The resulting embryos can be analyzed for changes in gene expression and for embryonic phenotypes or transferred to surrogate mothers for full-term development. Finally, fully developed animals are analyzed for the phenotypes transmitted naturally by the paternal environmental exposure of interest.