视网膜神经细胞发育和成熟的单细胞的剖析
Summary
描述一个孤立单一的视网膜细胞，随后他们的cDNA扩增的方法。单细胞转录揭示了在组织细胞的异质性目前的程度，并揭示了罕见的细胞群的新的标记基因。所附的协议，可以调整，以适应多种不同的细胞类型。
Abstract
高度专业化的，但非常小的细胞群体在许多组织中发挥的重要作用。细胞类型特异性标志物和基因表达程序极为罕见的细胞亚群的识别一直是一个挑战，使用标准的整个组织的方法。单个细胞的基因表达图谱允许获得了前所未有的细胞类型，包括只有一个很小的比例，共组织1-7。此外，这种技术可用于研究基因的表达，瞬时表达细胞小的数字，在动态发展过渡8的方案。
这种细胞的多样性的问题反复出现在中枢神经系统（CNS）神经连接可以相当不同的细胞之间发生9。不同的细胞类型的确切的数字是不准确不得而知，但据估计可能有多达1000个不同类型的皮质我tself 10。复杂的神经回路的功能（S）可能依靠一些罕见的神经类型和他们表达的基因。通过确定新的标志物和分子分类不同的神经元，单细胞的方法是在中枢神经系统的细胞类型分析特别有用。它也可能有助于阐明神经发育机制在神经祖发展的早期阶段，通过识别差异表达的基因和基因途径。
作为一种简单，方便地访问组织神经元具有相当的多样性，脊椎动物的视网膜是一个极好的模型系统研究细胞发育，神经细胞的分化和神经多样化的进程。然而，这种细胞的多样性，如中枢神经系统的其他部分，可以决定采取一种特定的细胞命运，推动视网膜祖细胞的遗传途径提出一个问题，特别是考虑到杆感光弥补马jority视网膜细胞总人口的11。在这里，我们报告单（ 图1）视网膜细胞中表达的转录的识别方法。单细胞分析技术允许在不同的细胞群体的视网膜2,4,5,12评估为异质目前的金额的。此外，这种方法揭示了一个新的候选基因，可能发挥的作用（S）在决策过程中的细胞命运发生在视网膜祖细胞亚群8。随着协议的一些简单的调整，这种技术可以用于许多不同的组织和细胞类型。
Protocol
1。细胞分离流程图，概述了协议，显示图1。特别是在本协议所使用的试剂的目录编号，请参考表1。剖析在PBS洗澡的视网膜。在解剖中，最好是删除的，因为玻璃体视网膜可以阻碍分离和镜头。它并不总是非常重要的，以消除所有的视网膜色素上皮（RPE），并在某些情况下，它可能无法彻底清除它。然而，感光细胞的单细胞分析实验中，RPE应该被删除。未能取出?…
Discussion
被认为是考虑到他们的基因表达6,8更均匀的人口数量不断扩大的研究揭示强大的细胞，细胞变异。至少有一个实例，该基因表达的“噪音”已被证明发挥了重要的生物功能13。使用传统的全组织方法掩盖单个细胞之间的基因表达差异。这些实验产生一个“平均”的细胞，这可能不是代表14的表达谱。在这里，我们提出了一个从单一的视网膜细胞的基因表达模式的分离和鉴定…
Offenlegungen
The authors have nothing to disclose.
Materials
Reaction mixtures:
Cell Lysis buffer
| 0.45 μ | 10X reaction buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 2 mM MgCl2) |
| 0.23 μl | 10% NP-40 |
| 0.23 μl | 0.1M DTT |
| 0.05 μl | RNase Inhibitor (40 U/μl) |
| 0.05 μl | SUPERase-In (20U/μl) |
| 0.13 μl | Modified Oligo d(T) primer (TATAGAATTCGCGGCCGCTCGCGATTTTTTTTTTTTTTTTTTTTTTTT) |
| 0.09 μl | dNTPs (2.5 mM each) |
| 3.27 μl | dH20 (use molecular biology grade for all reaction mixtures) |
Tailing Reaction Mixture
| 0.18 μl | 100 mM dATP |
| 0.6 μl | 10X reaction buffer(100 mM Tris-HCl pH 8.3, 500 mM KCl, 2 mM MgCl2) |
| 4.62 μl | dH2O |
| 0.3 μl | TdT (400 U/μl) |
| 0.3 μl | RNase H (2 U/μl) |
PCR Reaction Mixture
| 10 μl | 10x Ex-Taq HS Buffer with Mg2+ |
| 10 μl | 2.5 mM dNTPs |
| 0.2 μl | Modified Oligo d(T) primer (10 μg/μl) |
| 1 μl | Ex-Taq HS Polymerase (5U/μl) |
| 68.8 μl | dH2O |
10X One-Phor All Buffer
| 0.5M | Potassium acetate |
| 0.1M | Tris acetate (pH 7.6) |
| 0.1M | Magnesium acetate |
Solutions:
10X Phosphate buffered saline (1 liter)
80g NaCl
2g KCl
14.4 g Na2PO4
2.4 g KH2PO4
adjust to pH 7.4 with NaOH and bring the volume to 1 liter with water
| Name of the reagent | Company | Catalog number | Comments |
| Vertical needle puller | David Kopf Instruments | Model 750 | |
| Microcapillary tubes | Sigma | P0674 | |
| Aspirator tube assembly | Sigma | A5177 | |
| Corning filter tips (100-1000 μl | Fisher Scientific | 07-200-265 | |
| Hank’s balanced salt solution (1X) | Lonza BioWhittaker | 10-508F | |
| HEPES buffer (1M) | MP Biomedicals | 091688449 | |
| Bovine serum albumin | Sigma | A9418 | |
| DNase I | Roche | 04716728001 | |
| papain | Worthington | LS03126 | |
| Superscript III | Invitrogen | 18080-044 | |
| RNase Inhibitor | Applied Biosystems | AM2682 | These two RNase inhibitors seem to work the best. Contaminants present in other inhibitors can contribute to a background smear. |
| SUPERase-In | Applied Biosystems | AM2694 | These two RNase inhibitors seem to work the best. Contaminants present in other inhibitors can contribute to a background smear. |
| Modified Oligo d(T) primer (gel purified) | Oligos ETC | We have used primers purchased from many different companies and have seen the most reliable and reproducible results from Oligos ETC. | |
| T4 gene 32 protein | New England Biolabs | M0300L | |
| Exonuclease I | New England Biolabs | M0293S | |
| TdT | Roche | 3 333 574 | As with other reagents, using a TdT enzyme from other companies gave more variable results. |
| RNase H | Invitrogen | 18021-014 | |
| Ex-Taq HS Polymerase | Takara | RR006A | |
| Biotin N6-ddATP | Enzo Biosciences | 42809 |
Table 1. Specific reagents and equipment.
Referenzen
- Tietjen, I. Single-cell transcriptional analysis of neuronal progenitors. Neuron. 38, 161-175 (2003).
- Trimarchi, J. M. Molecular heterogeneity of developing retinal ganglion and amacrine cells revealed through single cell gene expression profiling. J. Comp. Neurol. 502, 1047-1065 (2007).
- Trimarchi, J. M., Cho, S. H., Cepko, C. L. Identification of genes expressed preferentially in the developing peripheral margin of the optic cup. Dev. Dyn. 238, 2327-2327 (2009).
- Cherry, T. J., Trimarchi, J. M., Stadler, M. B., Cepko, C. L. Development and diversification of retinal amacrine interneurons at single cell resolution. Proc. Natl. Acad. Sci. U.S.A. 106, 9495-9500 (2009).
- Roesch, K. The transcriptome of retinal Muller glial cells. J. Comp. Neurol. 509, 225-238 (2008).
- Ramos, C. A. Evidence for diversity in transcriptional profiles of single hematopoietic stem cells. PLoS Genet. 2, e159 (2006).
- Chiang, M. K., Melton, D. A. Single-cell transcript analysis of pancreas development. Dev. Cell. 4, 383-393 (2003).
- Trimarchi, J. M., Stadler, M. B., Cepko, C. L. Individual retinal progenitor cells display extensive heterogeneity of gene expression. PLoS ONE. 3, e1588 (2008).
- Nelson, S. B., Sugino, K., Hempel, C. M. The problem of neuronal cell types: a physiological genomics approach. Trends Neurosci. 29, 339-345 (2006).
- Stevens, C. F. Neuronal diversity: too many cell types for comfort. Curr. Biol. 8, R708-R710 (1998).
- Cepko, C. L., Austin, C. P., Yang, X., Alexiades, M., Ezzeddine, D. Cell fate determination in the vertebrate retina. Proc. Natl. Acad. Sci. U.S.A. 93, 589-595 (1996).
- Kim, D. S. Identification of molecular markers of bipolar cells in the murine retina. J. Comp. Neurol. 507, 1795-1810 (2008).
- Chang, H. H., Hemberg, M., Barahona, M., Ingber, D. E., Huang, S. Transcriptome-wide noise controls lineage choice in mammalian progenitor cells. Nature. 453, 544-547 (2008).
- Levsky, J. M., Singer, R. H. Gene expression and the myth of the average cell. Trends Cell Biol. 13, 4-6 (2003).
- Livesey, F. J., Cepko, C. L. Vertebrate neural cell-fate determination: lessons from the retina. Nat. Rev. Neurosci. 2, 109-118 (2001).
- Masland, R. H., Raviola, E. Confronting complexity: strategies for understanding the microcircuitry of the retina. Annu. Rev. Neurosci. 23, 249-284 (2000).
- Blackshaw, S. Genomic analysis of mouse retinal development. PLoS Biol. 2, e247 (2004).
- Livesey, F. J., Young, T. L., Cepko, C. L. An analysis of the gene expression program of mammalian neural progenitor cells. Proc. Natl. Acad. Sci. U.S.A. 101, 1374-1379 (2004).
- Chowers, I. Identification of novel genes preferentially expressed in the retina using a custom human retina cDNA microarray. Invest. Ophthalmol. Vis. Sci. 44, 3732-3741 (2003).
- Clarke, G. C., Leavitt, R. A., Andrews, B. R., Hayden, D. F., Lumsden, M. R., J, C., McInnes, R. R. A one-hit model of cell death in inherited neuronal degenerations. Nature. 406, 195-199 (2000).
- McEvoy, J. F. -. O., Zhang, J., Nemeth, J., Brennan, K., Bradley, R., Krafcik, C., Rodriguez-Galindo, F., Wilson, C., Xiong, M., Lozano, S. Coexpression of normally incompatible developmental pathways in retinoblastoma genesis. Cancer Cell. 20, 260-275 (2011).
- Gelder, R. N. V. a. n. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. U.S.A. 87, 1663-1667 (1990).
- Ginsberg, S. D., Che, S. Expression profile analysis within the human hippocampus: comparison of CA1 and CA3 pyramidal neurons. J. Comp. Neurol. 487, 107-118 (2005).
- Iscove, N. N. Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat. Biotechnol. 20, 940-943 (2002).
- Subkhankulova, T., Livesey, F. J. Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level. Genome Biol. 7, R18 (2006).
Tags
Single-cell ProfilingDeveloping Retinal NeuronsMature Retinal NeuronsCell-type Specific MarkersGene Expression ProgramsIndividual CellsTransient Gene Expression ProgramsCellular DiversityCentral Nervous SystemDistinct Cell TypesComplex Neural CircuitsRare Neuronal TypesMolecular ClassificationAnalysis Of Cell TypesNervous SystemMechanisms Of Neural Development
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Goetz, J. J., Trimarchi, J. M. Single-cell Profiling of Developing and Mature Retinal Neurons. J. Vis. Exp. (62), e3824, doi:10.3791/3824 (2012).