Lockowandt Marcus, Günther Dorothee M, Quintino Luis, Breger Ludvine S, Isaksson Christina, Lundberg Cecilia
CNS Gene therapy Unit, Wallenberg Neuroscience Center, Dept Experimental Sciences, Lund University BMC A11, 22184 Lund, Sweden.
CNS Gene therapy Unit, Wallenberg Neuroscience Center, Dept Experimental Sciences, Lund University BMC A11, 22184 Lund, Sweden.
J Neurosci Methods. 2020 Apr 15;336:108542. doi: 10.1016/j.jneumeth.2019.108542. Epub 2020 Feb 1.
To target specific neuronal populations by gene transfer is challenging. A complicating fact is that populations of neurons may have opposing roles despite being found adjacent to each other. One example is the medium spiny neurons of the striatum. These cells have different projection patterns, a trait used in this study to specifically target one population.
Here we present a way of labeling and further studying neurons based on their projections. This was achieved by pseudotyping lentiviral vectors with a chimeric glycoprotein allowing for retrograde transport in combination with optimizing the promoter element used.
We transduced on average 4000 neurons of the direct pathway in the striatum, with the viral vector allowing for microscopy and miRNA immunoprecipitation. In addition, we were able to optimize vector production, reducing the time and material used.
The optimized protocol is more reproducible compared to previously published protocols. Alternative methods to study specific populations of neurons are transgenic animals or, if available, specific promoter elements. However, very specific promoter elements are rarely available and often large, limiting the usefulness in viral vectors. Our optimized retrograde vectors allow for selection based on neuronal projections and are therefore independent of such elements.
We have developed a method that allows for specific analysis of neuronal subpopulations in the brain either by microscopy or by biochemical methods e.g. immunoprecipitation. This method is simple to use and can be combined with transgenic animals for studying disease models.
通过基因转移靶向特定神经元群体具有挑战性。一个复杂的事实是，尽管神经元群体彼此相邻，但它们可能具有相反的作用。一个例子是纹状体的中等棘状神经元。这些细胞具有不同的投射模式，本研究利用这一特性专门靶向其中一个群体。
在此，我们提出了一种基于神经元投射对其进行标记并进一步研究的方法。这是通过用嵌合糖蛋白对慢病毒载体进行假型化来实现的，该嵌合糖蛋白允许逆行运输，并结合优化所使用的启动子元件。
我们平均转导了纹状体中直接通路的4000个神经元，该病毒载体可用于显微镜检查和miRNA免疫沉淀。此外，我们能够优化载体生产，减少所用的时间和材料。
与先前发表的方案相比，优化后的方案具有更高的可重复性。研究特定神经元群体的替代方法是转基因动物，或者如果有可用的特定启动子元件。然而，非常特异的启动子元件很少可用，而且通常很大，限制了其在病毒载体中的实用性。我们优化的逆行载体允许基于神经元投射进行选择，并因此独立于此类元件。
我们开发了一种方法，可通过显微镜检查或生化方法（例如免疫沉淀）对大脑中的神经元亚群进行特异性分析。该方法易于使用，并可与转基因动物结合用于研究疾病模型。
