Laboratory of Molecular Biology, National Institute of Mental Health, NIH, Bethesda, United States.
Neural Cell-Fate Determinants Section, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, United States.
Elife. 2020 Apr 14;9:e53041. doi: 10.7554/eLife.53041.
Existing genetic methods of neuronal targeting do not routinely achieve the resolution required for mapping brain circuits. New approaches are thus necessary. Here, we introduce a method for refined neuronal targeting that can be applied iteratively. Restriction achieved at the first step can be further refined in a second step, if necessary. The method relies on first isolating neurons within a targeted group (i.e. Gal4 pattern) according to their developmental lineages, and then intersectionally limiting the number of lineages by selecting only those in which two distinct neuroblast enhancers are active. The neuroblast enhancers drive expression of split Cre recombinase fragments. These are fused to non-interacting pairs of split inteins, which ensure reconstitution of active Cre when all fragments are expressed in the same neuroblast. Active Cre renders all neuroblast-derived cells in a lineage permissive for Gal4 activity. We demonstrate how this system can facilitate neural circuit-mapping in .
现有的神经元靶向遗传方法通常无法达到绘制大脑回路所需的分辨率。因此，需要新的方法。在这里，我们介绍了一种可迭代应用的精细神经元靶向方法。如果需要，第一步实现的限制可以在第二步进一步细化。该方法依赖于首先根据发育谱系将靶基因群（即 Gal4 模式）内的神经元分离出来，然后通过选择仅激活两个不同神经母细胞增强子的那些来交叉限制谱系的数量。神经母细胞增强子驱动分割 Cre 重组酶片段的表达。这些片段与非相互作用的分割整合酶对融合，当所有片段在同一神经母细胞中表达时，确保活性 Cre 的重组。活性 Cre 使谱系中的所有神经母细胞衍生细胞都允许 Gal4 活性。我们展示了该系统如何有助于. 中的神经回路映射。
