有条件的遗传跨突触标记在胚胎小鼠脑
Summary
Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.
Abstract
Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.
Introduction
解剖路径跟踪是最常用的工具，以破译大脑和行为1之间的关系之一。进步的神经回路追踪技术已经赋予与神经科学家从小鼠基因2确定神经元群追踪神经回路的功能。尽管这些技术上的进步仍然具有挑战性解开尤其是在胚胎成熟的神经回路的形成。这是因为大多数开发迄今跟踪方法是基于立体定位注射跨突触示踪剂的或遗传修饰的嗜神经病毒（ 图1）2,3。虽然这些技术实现空间和时间分辨率的连通，几个固有的局限性，如技术上具有挑战性示踪剂注射入发育中的大脑中，注射部位的再现性，潜在炎症在注射部位和最祁门功夫，功夫造成嗜神经病毒tantly毒性限制了其使用4。
另一种方法是要表达的跨突触示踪剂如遗传改造的小鼠转基因。我们最近修改了此技术，开发了二进制遗传跨突触跟踪系统，以地图的任何基因鉴定神经元群5的神经回路。我们的试验策略是基于两个新敲入小鼠品系，其表达或者双向示踪剂大麦凝集素（BL）的6或逆行示踪剂破伤风毒素片段C的ROSA 26轨迹融合到绿色荧光蛋白（GTT）7后的Cre介导的重组。在这里，我们用这些小鼠品系选择性表达BL和GTT的产生亲吻促动素的神经元，即牵连调节生殖轴8,9的成熟神经肽。我们表明，这种技术适用于可视吻的发育和成熟在雌性小鼠大脑5的胚胎发育过程中peptin神经电路。
育种策略
在R26-BL-IRES-τlacZ（BIZ）和R26-GFP-TTC（GTT）示踪线敲入5株携带重组ROSA26等位基因。所述R26-BIZ和R26-GTT等位基因是转录沉默由于强烈的转录终止信号，它是由两个loxP序列侧翼5的存在。的BIZ和GTT转基因的表达是通过Cre重组酶介导的切除的转录终止信号的激活。在R26-BIZ和R26-GTT等位基因可以独立通过简单地用酶Cre车手穿越中使用。用于分析动物杂合的各自的Cre和R 26等位基因都可以使用。同窝承载1 的Cre或1 R 26等位基因，分别应用作对照。可替代地，也有可能产生吨riple敲入动物携带的Cre，R26-BIZ和R26-GTT等位基因，然而这将需要一个额外的十字架。
Protocol
Representative Results
Discussion
相比于立体定位注射示踪剂或neurotopic病毒的表达跨突触示踪剂作为转基因跟踪基因定义神经元群体的神经回路具有几个优点。首先，将示踪剂是作为内源蛋白质，因此不引起任何免疫反应和选择性的神经通路可在不同的动物具有较高的再现性进行分析。第二，由于这是一种非侵入性的方法也可用于从神经元用于立体定位注射不容易进入跟踪电路，例如在子宫内 。限制包括在跨突触传递一?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Michael Candlish for critical comments on the manuscript. This project was supported by the Deutsche Forschungsgemeinschaft grants BO1743/6 and SFB/TRR 152 P11 and Z02 to Ulrich Boehm.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Bisbenzimide (Hoechst 33258 dye) | Sigma | 14530-100MG | |
| Ethanol | Sigma | 32205-1L | |
| Cryo mold (Peel-a-way) | Polyscience Inc. | 18646A-1 | 22mm x 22mm x 20mm |
| DMSO | Sigma | D8418-100ML | |
| Dimethyl Formamide (DMF) | VWR Chemicals | 23470,293 | |
| EGTA | ROTH | 3054.3 | |
| Fluoromount G | Southern Biotech | 0100-01 | |
| Glutaraldehyde | Sigma | G5882-50ML | |
| Hydrogen peroxide | Sigma | 34988-7 | |
| Isopentane (Methyl 2-butane) | Sigma | M32631-2.5L | |
| Kaiser's Glycine gelatin | Merck | 1092420100 | |
| Methanol | Sigma | 494437-1L | |
| MgCl2 | Sigma | M2670-100G | |
| NaCl | ROTH | HN00.2 | |
| NBT | Sigma | 298-83-9 | |
| Nonidet P40 substitute | Fluka | 743.85 | |
| OCT | Leica | 14020108926 | |
| PAP pen | Dako | S2002 | |
| Parafarmaldehyde | Sigma | P6148-1KG | |
| Sodium deoxycholate | Sigma | D6750-25G | |
| Sucrose | Sigma | S7903-1KG | |
| Superfrost slides | Thermo Scientific | FT4981GLPLUS | |
| TSA kit | PerkinElmer | NEL700 | |
| TSA plus kit | PerkinElmer | NEL749A001KT | |
| Tris | ROTH | AE15.2 | |
| Triton-X 100 | ROTH | 3051.2 | |
| Tween 20 | ROTH | 9127.1 | |
| X-gal | ROTH | 2315.1 | |
| Cryostat | Leica | na | |
| Light microscope equipped with DIC imaging | Zeiss | Axioskop2 equipped with Axio Vision software | |
| Fluroscence microscope | Zeiss | Axioskop2 equipped with Axio Vision software | |
| Photoshop | Adobe | PS6 | |
| Goat anti-WGA (recognizes BL) | Vector Laboatories | AS-2024 | |
| Biotinylayted horse anti-goat IgG | Vector Laboatories | BA-9500 | |
| Biotinylated goat anti-rabbit IgG | Vector Laboatories | BA-1000 | |
| Rabbit anti-GFP (recognizes GTT) | Invitrogen | A11122 | |
| Rabbit anti-GnRH | Affinity Bio Reagent | PA1-121 | |
| Dylight488-donkey anti-rabbit IgG | Thermo Scientific | SA5-10038 | |
| SA-Alexa Fluor 546 | Life Technologies | S-11225 | |
| Primers | |||
| BL Fwd (for BIZ genotyping) | Eurofins MWG Operon | ATGAAGATGATGAGCACCAG GGC |
|
| BL Rev (for BIZ genotyping) | Eurofins MWG Operon | AGCCCTCGCCGCAGAACTC | |
| Cre Fwd (for Cre genotyping) | Eurofins MWG Operon | GTCGATGCAACGAGTGATGAG GTTCG |
|
| Cre Rev (for Cre genotyping) | Eurofins MWG Operon | CCAGGCTAAGTGCCTTCTCTAC ACCTGC |
|
| TTC Fwd (for GTT genotyping) | Eurofins MWG Operon | AGCAAGGGCGAGGAGCTGTT | |
| TTC Rev (for GTT genotyping) | Eurofins MWG Operon | GTCTTGTAGTTGCCGTCGTCCT TGAA |
|
| XY Fwd (for gender genotyping) | Eurofins MWG Operon | TGAAGCTTTTGGCTTTGA | |
| XY Rev (for gender genotyping) | Eurofins MWG Operon | CCGCTGCCAAATTCTTTG | |
| ROSA26 Fwd | Eurofins MWG Operon | CGAAGTCGCTCTGAGTTGTTATC | |
| ROSA26 Rev | Eurofins MWG Operon | GCAGATGGAGCGGGAGAAAT | |
| SA Rev | Eurofins MWG Operon | CGAAGTCGCTCTGAGTTGTTATC | |
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