Physiology and Biophysics Department, University of Colorado, School of Medicine, Aurora, CO, USA.
J Neurosci Methods. 2011 Apr 30;197(2):238-48. doi: 10.1016/j.jneumeth.2011.02.026. Epub 2011 Mar 8.
Since transgenes were first cloned into recombinant adenoviruses almost 30 years ago, a variety of viral vectors have become important tools in genetic research. Viruses adeptly transport genetic material into eukaryotic cells, and replacing all or part of the viral genome with genes of interest or silencing sequences creates a method of gene expression modulation in which the timing and location of manipulations can be specific. The hypothalamo-neurohypophyseal system (HNS), consisting of the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, regulates fluid balance homeostasis and is highly plastic, yet tightly regulated by extracellular fluid (ECF) osmolality and volume. Its reversible plasticity and physiological relevance make it a good system for studying interactions between gene expression and physiology. Here, four viral vectors were compared for their ability to transduce magnocellular neurosecretory neurons (MNCs) of the SON in adult rats. The vectors included an adenovirus, a lentivirus (HIV) and two serotypes of adeno-associated viruses (AAV5 and AAV2). Though adenovirus and AAV2 vectors have previously been used to transduce SON neurons, HIV and AAV5 have not. All four vectors transduced MNCs, but the AAV vectors were the most effective, transducing large numbers of MNCs, with minimal or no glial transduction. The AAV vectors were injected using a convection enhanced delivery protocol to maximize dispersal through the tissue, resulting in the transduction of neurons throughout the anterior to posterior length of the SON (∼1.5mm). AAV5, but not AAV2, showed some selectivity for SON neurons relative to those in the surrounding hypothalamus.
自 30 年前首次将转基因克隆到重组腺病毒以来,各种病毒载体已成为遗传研究的重要工具。病毒能够熟练地将遗传物质运送到真核细胞中,并且通过用感兴趣的基因或沉默序列替换病毒基因组的全部或部分,创建了一种基因表达调节方法,其中可以特异性地操纵时间和位置。下丘脑-神经垂体系统(HNS)由下丘脑的室旁核(PVN)和视上核(SON)组成,调节体液平衡稳态,具有高度可塑性,但受到细胞外液(ECF)渗透压和体积的严格调节。其可逆可塑性和生理相关性使其成为研究基因表达与生理之间相互作用的良好系统。在这里,比较了四种病毒载体在成年大鼠的 SON 中的大细胞神经分泌神经元(MNC)中转导的能力。这些载体包括腺病毒、慢病毒(HIV)和两种血清型腺相关病毒(AAV5 和 AAV2)。尽管腺病毒和 AAV2 载体以前已被用于转导 SON 神经元,但 HIV 和 AAV5 尚未被使用。所有四种载体均转导 MNC,但 AAV 载体的效率最高,可转导大量 MNC,并且仅有最小或没有神经胶质转导。使用对流增强递送方案(以最大限度地增加组织中的弥散)注射 AAV 载体,从而使 SON 的前至后全长(约 1.5mm)中的神经元均被转导。AAV5 而非 AAV2 相对于周围下丘脑神经元显示出对 SON 神经元的一些选择性。