Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP).
Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP).
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DOI：
10.3389/fnmol.2012.00018
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发表时间：
2012
影响因子：
4.8
通讯作者：
Sanes JR
中科院分区：
文献类型：
--
作者：
Yamagata M;Sanes JR
在“跨突触伴侣的绿色荧光蛋白（GFP）重组”（GRASP）方法中，GFP的非荧光片段在两种不同的神经元中表达；这些片段在两者之间的突触处自我组装形成荧光团。GRASP已被证明可用于在两种无脊椎动物——秀丽隐杆线虫和黑腹果蝇中通过光学显微镜识别突触，但尚未应用于脊椎动物。在此，我们描述了在哺乳动物细胞中起作用的GRASP构建体，并实施了一种转基因策略，即Cre依赖性基因开关导致这两种片段在小鼠相互排斥的神经元亚群中表达。利用一种在视杆光感受器中选择性表达Cre的转基因品系，我们展示了视网膜外丛状层突触的标记。这种标记是特异性的，因为视杆细胞形成的突触至少在6个月内保持标记，而附近由插入的视锥光感受器在许多相同的中间神经元上形成的突触则未被标记。我们还制备了能标记重组GFP但不标记任何一种片段的抗血清，以放大GRASP信号，从而提高该方法的灵敏度。
In the “GFP reconstitution across synaptic partners” (GRASP) method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two invertebrate species, Caenorhabditis elegans and Drosophila melanogaster, but has not yet been applied to vertebrates. Here, we describe GRASP constructs that function in mammalian cells and implement a transgenic strategy in which a Cre-dependent gene switch leads to expression of the two fragments in mutually exclusive neuronal subsets in mice. Using a transgenic line that expresses Cre selectively in rod photoreceptors, we demonstrate labeling of synapses in the outer plexiform layer of the retina. Labeling is specific, in that synapses made by rods remain labeled for at least 6 months whereas nearby synapses made by intercalated cone photoreceptors on many of the same interneurons remain unlabeled. We also generated antisera that label reconstituted GFP but neither fragment in order to amplify the GRASP signal and thereby increase the sensitivity of the method.
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影响因子：
46.9
作者：
Cabantous, S;Terwilliger, TC;Waldo, GS
通讯作者：
Waldo, GS
影响因子：
46.9
作者：
Pédelacq, JD;Cabantous, S;Waldo, GS
通讯作者：
Waldo, GS
影响因子：
3.4
作者：
Blank, M;Koulen, P;Kröger, S
通讯作者：
Kröger, S
影响因子：
25
作者：
Madisen L;Zwingman TA;Sunkin SM;Oh SW;Zariwala HA;Gu H;Ng LL;Palmiter RD;Hawrylycz MJ;Jones AR;Lein ES;Zeng H
通讯作者：
Zeng H
影响因子：
16.2
作者：
Gordon, Michael D.;Scott, Kristin
通讯作者：
Scott, Kristin