Name: single-cell rna-seq of defined subsets of retinal ganglion cells
Uploaded: 2017-05-22T12:30:00.000+00:00
Duration: 11 min 26 s
Description: Watch this Scientific Journal Video about Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells at JoVE.com

私たちは、Jennifer BairとEinat SnirならびにIowa Institute for Human Geneticsがサンプルの調製と取り扱いを支援したことを認めたいと思います。

<ol>
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著者は何も開示することはない。

我々のプロトコールは、迅速で使い易いガイドを通して、標本に対する傷害をほとんど起こさずに、高品質の配列決定のために同定された形態学的クラスの単一細胞を調製する方法を実証する。現在の原稿では、本質的に感光性の網膜神経節細胞を形態学的に特徴付け、単離し、RNA-Seqについて調製する。細胞のストレスは、網膜の取り扱い中に起こり得る。このため、4時間以内に各組織を?...

ニューロンの多様性は、中枢神経系、特に網膜前駆細胞1,2,3の1つの集団から生じる1つの神経膠細胞型および6つのニューロン細胞タイプからなる高度に特殊化された組織である脊椎動物網膜において観察される。細胞の多くのサブタイプは、機能的、形態学的および遺伝的に分類することができる。このプロトコールの目的は、細胞型の遺伝的変異性をそれらの同定可能な機能的および/または形態学的特徴と結びつけることである。多くの遺伝子が細胞の分類のために同定されているが、多くの亜型は、全体集団のわずかな部分しか表さないので、特徴付けされていない。これらの特定のサブタイプ内の遺伝子の同定は、網膜内のニューロンの多様性のより大きな理解を可能にし、また、他の場所での神経細胞の多様化を明らかにする可能性がある。フーさらに、単細胞研究は、全細胞集団のうちの低い発現のために見過ごされた可能性のある新しい細胞タイプの発見を可能にする。4,5,6,7。 単一細胞トランスクリプトミックスの利点の1つは、特定の細胞亜型を定義する独特のマーカーまたはマーカーの組み合わせが発見され得ることである。これらは、異なる操作のためにその細胞型に遺伝的にアクセスするために使用することができます。例えば、我々は、このプロトコールを使用して、光色素メラノプシンを発現する網膜神経節細胞のサブセットの細胞型特異的遺伝子を特徴づける。メラノプシン発現網膜神経節細胞における蛍光マーカーの使用は、既知の遺伝子の発現のために一緒に集まっているので、これらの細胞の研究を可能にする。興味深いことに、この細胞集団の5つの既知のサブタイプが存在するマウスの網膜8にある。したがって、各タイプの細胞からRNAを単離するために、トランスジェニックモデル内に確立された形態学的分類を使用して、細胞分離の前に各サブタイプを同定した。この技術は、細胞内のストレス応答と切断された樹状突起によるコンタミネーションを引き起こす可能性のある組織の解離を必要とせずに、細胞の特徴付けと網膜からの直接的な分離を可能にする。 RNA-Seq法が発展しているので、ここ数年の間に数多くの新しい技術が明らかになりました。これらのツールは、手作業4,7,10,11,12,13の問題に近づいている間に、最大の細胞獲得とより高いコスト効率を可能にします。しかし、これらの技法は優れた踏み台になっていますが、このプロトコルが対応できる多くのハードルがあります。第1に、現在の手順の多くは、解離した組織から細胞を単離し、主成分分析または事後的な階層的クラスタリングのいずれかを用いて細胞分類を決定しようと試みる。これらのツールを使用してサブタイプを分類することは、信頼できる結果をもたらさない可能性があり、遺伝子マーカーと機能的細胞タイプとの相関についてこのデータを検証する新しい方法を見つけ出す可能性があります。他のプロトコールでの解離の必要条件は、組織損傷を引き起こし、神経突起を切断してmRNAの潜在的な消失を引き起こすことがある。さらに、解離した細胞調製物において、ストレス応答は、これらの細胞の転写物に影響し始める可能性がある14 。このプロトコルは、分離の前に機能的細胞型を決定することによってこれらの課題を克服し、h網膜組織を無傷に保つことによって細胞の恩恵を受ける。 1つの手法が2014年に導入され、生きた細胞のトランスクリプトームのインビボ分析で構成されていました15 。この技術は、組織への機械的破壊を最小限に抑えてトランスクリプトームの検査を可能にするが、非常に特異的なレポーターマウスを用いることなくトランスクリプトームを調べる前に組織内の特定の細胞型を分類する能力が欠けている。私たちのプロトコールでは、特定のレポーターは必要ありません。細胞を分離して分離する前に、細胞の充填と電気生理学を利用するためです。この以前のプロトコールのもう一つの制限は、光活性化可能なエレメントを励起するために特定の波長を必要とするということであるが、我々のプロトコールは、容易に入手可能であるかまたは各ラボで個別に選択できる蛍光レポーターおよび蛍光染料の使用を可能にする。それでもなお、他の研究所は、電気の2つの方法細胞多様性研究のための生体生理学およびトランスクリプトミクス。分離の前に細胞の機能を特徴づけるためのパッチクランプ記録の使用は、解離したニューロン16で実施され、場合によっては、これらの研究のためにマイクロアレイ分析17の使用に先行していた。それらが組織解離または利用可能なプローブへの試料のハイブリダイゼーションに依存するマイクロアレイ技術の使用を必要とするので、それらのアプローチによって同じ合併症が生じる。最新の進歩の1つは、パッチクランプ記録とRNA-Seq技術を組み合わせて全脳スライス18の細胞を理解するPatch-Seqの開発です。この手法はここに提示されたプロトコールと類似していますが、我々のアプローチでは、細胞の健康のために組織を無傷のままにすることができます。ここでは、最適化のためのプロトコルを提示するこれはRNA-Seqを使用して高い読み取り深度とマッピングカバレッジを得るための高品質の単一細胞ライブラリーを生成します。

<table><tbody><tr><td>Ames&#039; Medium</td><td>Sigma Aldrich</td><td>A1420-10X1L</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S8875</td><td></td></tr><tr><td>K-gluconate</td><td>Spectrum Chemical</td><td>PO178</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>Diethyl pyrocarbonate (DEPC)</td><td>Sigma Aldrich</td><td>D5758</td><td></td></tr><tr><td>Alexa Fluor 594 Hydrazide</td><td>Invitrogen</td><td>A10442</td><td></td></tr><tr><td>Collagenase</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS005273</td><td></td></tr><tr><td>Hyaluronidase</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS002592</td><td></td></tr><tr><td>Petri dish (35 mm diameter)</td><td>Thermo Fisher Scientific</td><td>153066</td><td></td></tr><tr><td>Ophthalmologic scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>#5 Forceps</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>Microplate Shaker</td><td>Fisher Scientific</td><td>13-687-708</td><td></td></tr><tr><td>Glass Micropipette</td><td>Sutter</td><td>BF120-69-10</td><td></td></tr><tr><td>Micropipette Puller</td><td>Sutter</td><td></td><td>P-1000 horizontal pipette puller</td></tr><tr><td>1 mL syringe</td><td>Fisher Scientific</td><td>14-823-2F</td><td></td></tr><tr><td>Flexible tubing</td><td>Fisher Scientific</td><td>14-171</td><td></td></tr><tr><td>TCL lysis buffer</td><td>Qiagen</td><td>1031576</td><td>Lysis Buffer 1</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma Aldrich</td><td>M3148</td><td></td></tr><tr><td>RNase-free Water</td><td>Qiagen</td><td>129112</td><td></td></tr><tr><td>0.2 mL PCR tubes</td><td>Eppendorf</td><td>30124359</td><td></td></tr><tr><td>Ethyl Alcohol, Pure</td><td>Sigma Aldrich</td><td>E7023</td><td>Ethanol</td></tr><tr><td>Analog Vortex Mixer</td><td>Thermo Fisher Scientific</td><td>02215365</td><td>Vortex</td></tr><tr><td>Mini Centrifuge</td><td>Thermo Fisher Scientific</td><td>05-090-100</td><td></td></tr><tr><td>Agencourt RNAClean XP Beads</td><td>Beckman Coulter</td><td>A63987</td><td>RNA magnetic beads</td></tr><tr><td>MagnaBlot II Magnetic Separator</td><td>Promega</td><td>V8351</td><td>Magnetic stand</td></tr><tr><td>1.5 mL MCT Graduated Tubes</td><td>Thermo Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Smart-Seq v4 Ultra Low Input RNA Kit</td><td>Clontech</td><td>634888</td><td>Reagents for Reverse Transcription and PCR Amplification</td></tr><tr><td>10x Lysis Buffer</td><td></td><td></td><td>Lysis Buffer 2</td></tr><tr><td>5x Ultra Low First-strand Buffer</td><td></td><td></td><td>Buffer 1</td></tr><tr><td>3&#039; SMART-Seq CDS Primer II A</td><td></td><td></td><td>Primer II</td></tr><tr><td>SMART-Seq v4 Oligonucleotide</td><td></td><td></td><td>Oligonucleotide</td></tr><tr><td>SMARTScribe Rverse Transcriptase</td><td></td><td></td><td>Reverse Transcriptase (RT)</td></tr><tr><td>2x SeqAmp PCR Buffer</td><td></td><td></td><td>PCR Buffer</td></tr><tr><td>PCR Primer II A</td><td></td><td></td><td>PCR Primer</td></tr><tr><td>SeqAmp DNA Polymerase</td><td></td><td></td><td>DNA Polymerase</td></tr><tr><td>Mastercycler pro S</td><td>Eppendorf</td><td>950030020</td><td>Thermocycler</td></tr><tr><td>Agencourt AMPure XP Beads</td><td>Beckman Coulter</td><td>A63881</td><td>DNA magnetic beads</td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2939AA</td><td></td></tr><tr><td>HS Bioanalyzer Chips &amp;amp; Reagents</td><td>Agilent Technologies</td><td>5067-4626</td><td></td></tr><tr><td>Qubit HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Q32851</td><td>For the calculation of sample concentrations</td></tr><tr><td>Qubit Assay Tubes</td><td>Thermo Fisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q32866</td><td></td></tr><tr><td>Nextera XT DNA Sample Preparation Kit</td><td>Illumina</td><td>FC-131-1024</td><td>Reagents for Tagmentation and Index Coupling</td></tr><tr><td>TD Buffer</td><td></td><td></td><td>Buffer 2</td></tr><tr><td>ATM</td><td></td><td></td><td>Tagmentation Mix</td></tr><tr><td>NT Buffer</td><td></td><td></td><td>Tagmentation Neutralizing Buffer</td></tr><tr><td>NPM</td><td></td><td></td><td>PCR Master Mix</td></tr><tr><td>Nextera XT Index Kit</td><td>Illumina</td><td>FC-131-1001</td><td>Indices for Tagmentation</td></tr><tr><td>N501</td><td></td><td></td><td>White 1</td></tr><tr><td>N502</td><td></td><td></td><td>White 2</td></tr><tr><td>N701</td><td></td><td></td><td>Orange 1</td></tr><tr><td>N702</td><td></td><td></td><td>Orange 2</td></tr><tr><td>HiSeq 2500</td><td>Illumina</td><td>SY-401-2501</td><td>For completing sequencing of samples</td></tr></tbody></table>

single-cell rna-seq of defined subsets of retinal ganglion cells

細胞タイプ特異的マーカーの発見は、細胞機能および細胞異種起源の洞察を提供することができる。近年、ニューロンの多様性の理解を促進するために、発現が細胞の様々な亜集団を定義する遺伝子を同定することが重要である。網膜は、中枢神経系の多様性の研究のための優れたモデルとして機能する。なぜなら、それは複数の主要な細胞タイプから構成されているからである。各主要クラスの細胞の研究により、これらの集団の同定を容易にする遺伝子マーカーが得られた。しかし、これらの主要な網膜細胞クラスのそれぞれには、複数のサブタイプの細胞が存在し、これらのサブタイプのほとんどは、形態マーカーまたは機能によって特徴付けられているが、遺伝マーカーは知られていない。個々の網膜サブタイプの遺伝マーカーの知識は、特定の視覚機能に関連する脳標的の研究およびマッピングを可能にし、また、その遺伝子ネットワークに洞察を与えることができる細胞の多様性を維持する。サブタイプの遺伝子マーカーを同定するために使用される現在の手段は、配列決定後の細胞型の分類などの欠点を有する。これはデータ分析の課題であり、クラスタに同じ機能のセルが確実に含まれるように厳密な検証方法が必要です。本発明者らは、単離および配列決定の前に細胞の形態および機能性を同定するための技術を提案し、これはサブタイプ特異的マーカーの同定を容易にする。この技術は、神経細胞以外の細胞型だけでなく、細胞の稀少な集団にも及ぶ可能性があります。このプロトコルは、数多くのライブラリが単一セルに対して2000万回以上の読み取り深度を提供しているので、優れた品質のデータをもたらします。この方法論は、単一細胞RNA-Seqによって提示される多くのハードルを克服し、直接的かつ非常に効率的な方法で細胞型をプロファイルすることを目指す研究者に適している可能性がある。

細胞タイプは、色素注入後に容易に分類される 図1は、蛍光トレーサ充填の前後のGFP + RGCの例を示す。この細胞は、トランスジェニック系統におけるGFPの発現に基づいて同定された（ 図1A ）。この細胞の細胞体上に細い先端の引っ張りガラス電極を用いて密封した。サブタイプを特徴付けるために?...

Watch this Scientific Journal Video about Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells at JoVE.com

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

ここでは、単離の前、およびその後の単一細胞転写物の特徴付けのために、ニューロン細胞タイプを分類するためのコンビナトリアルアプローチを提示する。このプロトコルは、RNAシーケンシング（RNA-Seq）のためのサンプルの調製を最適化し、細胞多様性の強化された理解のために特別に設計された方法論を記載する。

網膜神経節細胞の定義されたサブセットの単一細胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

single-cell-rna-seq-of-defined-subsets-of-retinal-ganglion-cells

Research

JoVE Journal

Neuroscience

13.7K ビュー.  Iowa State University. ここでは、単離の前、およびその後の単一細胞転写物の特徴付けのために、ニューロン細胞タイプを分類するためのコンビナトリアルアプローチを提示する。このプロトコルは、RNAシーケンシング（RNA-Seq）のためのサンプルの調製を最適化し、細胞多様性の強化された理解のために特別に設計された方法論を記載する。 

ビデオ: Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

神経科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

単一セルの開発のプロファイリングと成熟網膜神経細胞

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

ホールマウントラット網膜における免疫組織化学的およびカルシウムイメージング法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

フローサイトメトリーによる一次マウス網膜神経節細胞（RGCs）の単離

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

生物工学

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

次世代シーケンシングのためのマウス網膜内皮細胞の単離

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

発生生物学

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

scRNA-SeqおよびscATAC-Seqを用いた網膜遺伝子発現およびクロマチンアクセシビリティの多重化解析

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...

Isolating Murine Retinal Ganglion Cells Using Fluorescence-Activated Cell Sorting

Source: Chintalapudi, S. R., et al. <a href="https://app.jove.com/v/55785">Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry.</a> J. Vis. Exp. (2017)
This video demonstrates the procedure for isolating murine primary retinal ganglion cells (RGCs) by using flow cytometry. This method allows for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

蛍光活性化細胞選別を用いたマウス網膜神経節細胞の単離

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of ...

JoVE Encyclopedia of Experiments

神経細胞培養技術

一次神経細胞培養

Quantification of Retinal Ganglion Cell Somas Following Cauterization-Induced Ocular Hypertension 

<a href='https://app.jove.com/v/63442/full-circle-cauterization-limbal-vascular-plexus-for-surgically'>Source: Lani-Louzada, R., et al.&#160; Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022).</a>This video demonstrates the quantification of retinal ganglion cells from chemically fixed rat retinas subjected to full-circle cauterization of the limbal vascular plexus. The process includes permeabilization, antibody staining, and nuclear counterstaining, followed by visualization under an epifluorescence microscope to assess the impact of elevated intraocular pressure on retinal ganglion cell viability.

Source: Lani-Louzada, R., et al.&#160; Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. ...