Development of approaches for inducible trophoblast-specific gene modulation: the role of trophoblast Lat1 in the regulation of placental function and fetal growth

工程 摘要 这个 胎盘 导致胎儿异常生长的确切机制仍未完全确定，但 氨基酸转运可能与胎儿宫内发育迟缓和胎儿过度生长有关。大中性氨基酸 酸性转运体小亚基1(LAT1)介导必需氨基酸和甲状腺经胎盘转运 激素由转运体系统L组成。重要的是，胎盘系统L的氨基酸转运体活性是 在妇女中，胎儿宫内发育迟缓降低，胎儿过度生长增加。然而，目前还不清楚是否 胎盘LAT1表达/活性的变化与胎盘功能、胎儿 生长和后代心脏代谢结果。重要的是，全局LAT1缺失会导致胚胎死亡 在小鼠妊娠中期，很难确定LAT1对胎盘功能的作用。其他基因， 虽然不是胚胎致死的，但可能会影响早期胎盘的发育和胎盘的功能 已建立的胎盘，需要工具在怀孕的特定时间点‘关闭’或‘开启’基因。因此， 迫切需要开发方法来实现可诱导的、滋养层细胞特异性的基因调控。这个 这项建议的目的是开发和验证可诱导滋养层细胞特异性基因的新方法。 并验证恢复正常滋养层细胞LAT1表达拯救小鼠的中心假设 全球LAT1缺失和滋养细胞特异性LAT1基因敲除对胚胎的致死性 胎盘的建立减少了胎盘中必需氨基酸的运输，并抑制了胎盘 MTOR，线粒体呼吸和蛋白质合成，限制胎儿生长和计划后代 新陈代谢和心血管功能。我们提出了三个具体目标：目标1：建立和验证小鼠 通过可诱导的滋养层细胞特异性LAT1基因调控。我们的方法将是用 多西环素诱导的滋养细胞特异性LAT1基因在LAT1/-胚胎中的敲除或挽救 转座酶促进的转基因、慢病毒介导的囊胚和四倍体转导 分别进行互补实验。目的2：确定滋养层细胞特异性LAT1的调节作用 对胎盘功能、胎儿生长和后代长期结局的影响。我们的方法将是(1)诱导 LAT1基因敲除后胎盘的建立和(2)挽救滋养层细胞LAT1基因的表达 全球LAT1基因敲除(LAT1-/-)胚胎。我们将确定氨基酸和甲状腺的经胎盘转运。 激素，胎盘mTOR信号活性，线粒体呼吸和蛋白质合成，胎儿生长和 子代长期代谢和心血管功能目标3：确定LAT1的调节作用 原代人滋养层细胞合胞化及其功能的研究。我们的方法将是隔离主要的 人足月胎盘滋养层细胞及其合胞化、氨基酸和甲状腺的测定 PHT对照细胞的激素摄取、mTOR信号、线粒体呼吸和蛋白质合成 SiRNA介导的LAT1基因敲除的细胞和LAT1过表达的PHT细胞。

PROJECT SUMMARY The placental precise mechanisms causing abnormal fetal growth remain to be fully established, however changes in amino acid transport may contribute to both IUGR and fetal overgrowth. The Large Neutral Amino Acid Transporter Small Subunit 1 (LAT1) mediates transplacental transfer of essential amino acids and thyroid hormones by the transporter System L. Importantly, placental System L amino acid transporter activity is decreased in human IUGR and increased in fetal overgrowth in women. However, it remains unknown if changes in the expression/activity of placental LAT1 are mechanistically linked to placental function, fetal growth and offspring cardiometabolic outcomes. Importantly, global Lat1 deletion leads to embryonic lethality in mid-gestation in mice, making it difficult to determine the role of LAT1 for placental function. Other genes, albeit not embryonically lethal, may influence both early placental development and the function of the established placenta, requiring tools to `turn-off' or `turn-on' genes at specific time points of gestation. Thus, there is an urgent need to develop approaches to achieve inducible, trophoblast-specific gene modulation. The objective of this proposal is to develop and validate novel approaches for inducible trophoblast-specific gene modulation in mice and to test the central hypothesis that restoring normal trophoblast Lat1 expression rescues the embryonic lethality of global Lat1 deletion and that trophoblast-specific Lat1 knockdown after the establishment of the placenta decreases placental transport of essential amino acids and inhibits placental mTOR, mitochondrial respiration and protein synthesis, restricts fetal growth and programs offspring metabolism and cardiovascular function. We propose three Specific Aims: Aim 1: Develop and validate mice with inducible trophoblast-specific Lat1 gene modulation. Our approach will be to generate mice with doxycycline-inducible, trophoblast-specific Lat1 gene knockdown or rescue in Lat1-/- embryos using piggyBac transposase-enhanced transgenesis, lentivirus-mediated transduction of blastocysts and tetraploid complementation assay, respectively. Aim 2: Determine the effect of trophoblast-specific Lat1 modulation on placental function, fetal growth and offspring long-term outcomes. Our approach will be to (1) induce Lat1 knockdown after the establishment of the placenta and (2) rescue trophoblast Lat1 gene expression in global Lat1 knockout (Lat1-/-) embryos. We will determine transplacental transport of amino acids and thyroid hormones, placental mTOR signaling activity, mitochondrial respiration and protein synthesis, fetal growth and offspring long-term metabolic and cardiovascular function Aim 3: Establish the effect of LAT1 modulation on primary human trophoblast syncytialization and function. Our approach will be to isolate primary human trophoblast (PHT) cells from term placentas and determine syncytialization, amino acid and thyroid hormone uptake, mTOR signaling, mitochondrial respiration and protein synthesis in control PHT cells, in PHT cell with siRNA mediated LAT1 knockdown and in PHT cells with LAT1 overexpression.