Expression of mouse small interfering RNAs in lettuce using artificial microRNA technology

Abstract

Artificial miRNA technology enables the generation of siRNAs to regulate the expression of targeted genes. However, the application of siRNAs to alter gene expression is challenging due to their instability and requires a means to efficiently deliver siRNAs into the host. Here, we report that the siRNAs targeted to animal mRNAs can be heterologously expressed and stably produced in lettuce. We have modified rice miRNA precursors to produce siRNAs in lettuce with the potential to target mRNAs of mouse complement 3 (C3) and coagulation factor 7 (CF7). Expression of primary and mature siRNAs in the transgenic lettuce lines was confirmed via Sanger sequencing. Our study demonstrates an applicable tool to alter gene expression in the targeted host and has potential utility in siRNA-based oral therapeutics. METHOD SUMMARY We customized a rice miRNA backbone, Osa-MIR528, to construct plant expression vectors containing amiRNAs targeting mouse messenger RNAs. This system, which previously was used for amiRNA expression in monocots, now is used to generate amiRNAs in agriculturally important dicots such as lettuce, enabling production of siRNAs targeting various medically important proteins that could potentially be delivered through the diet.