264 - Using in vivo RNA Interference to Investigate Ruminant Placental Function

Abstract: Pregnant sheep have been used extensively for investigating pregnancy physiology, providing valuable information about the progression of ruminant pregnancy. The ability to place indwelling catheters, within both maternal and fetal vessels, allows for steady-state investigation of blood flow, nutrient uptakes and utilization, and hormone secretion, under non-stressed and non-anesthetized conditions. As such, our understanding of the in vivo physiology of pregnancy in sheep is unrivalled by any other species. However, until recently, a significant deficit existed in determining the specific function or significance of individual genes expressed by the placenta in livestock. To that end, we developed and have been using in vivo RNA interference (RNAi) within the sheep placenta to examine the function and relative importance of genes involved in conceptus development (PRR15 and LIN28), placental nutrient transport (SLC2A1 and SLC2A3), and placenta derived hormones (CSH). The lentiviral vector LL3.7 is used to generate virus that is stably integrated into the infected cell’s genome, thereby expressing a short-hairpin RNA (shRNA), that when processed within the cell, combines with the RNA Induced Silencing Complex (RISC) resulting in specific mRNA degradation or translational blockage. To accomplish in vivo RNAi, day 9 hatched and fully expanded blastocysts are infected with the lentivirus for 4-5 hours, and then surgically transferred to synchronized recipient uteri. Only the trophectoderm cells are infected by the replication deficient virus, leaving the inner cell mass unaltered, and we typically obtain 70-80% pregnancy rates following transfer of a single blastocyst. Data will be presented from two projects. One is focused on generating a deficiency in placental glucose transporters at mid-gestation, and the other on the impact of CSH RNAi during late gestation, demonstrating the utility of this experimental approach for examining gene function within the placenta of livestock. Supported by NIH-NICHD grants HD093701 and HD094952.