Department of Health and Kinesiology, Texas A&M University, College Station, Texas, United States of America.
PLoS One. 2013 Apr 22;8(4):e61472. doi: 10.1371/journal.pone.0061472. Print 2013.
Physical activity is associated with disease prevention and overall wellbeing. Additionally there has been evidence that physical activity level is a result of genetic influence. However, there has not been a reliable method to silence candidate genes in vivo to determine causal mechanisms of physical activity regulation. Vivo-morpholinos are a potential method to transiently silence specific genes. Thus, the aim of this study was to validate the use of Vivo-morpholinos in a mouse model for voluntary physical activity with several sub-objectives. We observed that Vivo-morpholinos achieved between 60-97% knockdown of Drd1-, Vmat2-, and Glut4-protein in skeletal muscle, the delivery moiety of Vivo-morpholinos (scramble) did not influence physical activity and that a cocktail of multiple Vivo-morpholinos can be given in a single treatment to achieve protein knockdown of two different targeted proteins in skeletal muscle simultaneously. Knocking down Drd1, Vmat2, or Glut4 protein in skeletal muscle did not affect physical activity. Vivo-morpholinos injected intravenously alone did not significantly knockdown Vmat2-protein expression in the brain (p = 0.28). However, the use of a bradykinin analog to increase blood-brain-barrier permeability in conjunction with the Vivo-morpholinos significantly (p = 0.0001) decreased Vmat2-protein in the brain with a corresponding later over-expression of Vmat2 coincident with a significant (p = 0.0016) increase in physical activity. We conclude that Vivo-morpholinos can be a valuable tool in determining causal gene-phenotype relationships in whole animal models.
身体活动与疾病预防和整体健康有关。此外，有证据表明身体活动水平是遗传影响的结果。然而，目前还没有一种可靠的方法可以在体内沉默候选基因，以确定身体活动调节的因果机制。活体形态发生素是一种潜在的瞬时沉默特定基因的方法。因此，本研究的目的是验证活体形态发生素在一种自愿身体活动的小鼠模型中的应用，并有几个次要目标。我们观察到，活体形态发生素在骨骼肌中实现了 Drd1-、Vmat2-和 Glut4-蛋白的 60-97%敲低，活体形态发生素的递送部分（ scramble ）不会影响身体活动，并且可以在单次治疗中给予多种活体形态发生素鸡尾酒，同时实现骨骼肌中两种不同靶向蛋白的蛋白质敲低。敲低骨骼肌中的 Drd1 、Vmat2 或 Glut4 蛋白不会影响身体活动。单独静脉内注射活体形态发生素不会显著降低大脑中的 Vmat2 蛋白表达（ p = 0.28 ）。然而，使用缓激肽类似物增加血脑屏障通透性与活体形态发生素联合使用，可显著降低大脑中的 Vmat2 蛋白（ p = 0.0001 ），随后 Vmat2 蛋白过度表达，同时身体活动显著增加（ p = 0.0016 ）。我们得出结论，活体形态发生素可以成为确定全动物模型中因果基因表型关系的有价值的工具。
