Summary

细胞系标记的共定位和番茄信号

Published: December 28, 2016
doi:

Summary

我们制定了两套在ROSA26 tdtomato(在所有细胞中广泛表达)/ Cre重组(特别是软骨细胞表达)小鼠跟踪组合:一个2.3Col1a1-GFP(具体以成骨细胞)和一个与免疫(具体以骨细胞)。该数据证明软骨细胞的直接转化成骨细胞。

Abstract

细胞谱系追踪系统已在发育生物学研究中主要使用。利用Cre重组酶的允许记者在特定细胞系和所有后代的活化。在这里,我们使用的细胞谱系追踪技术来证明软骨细胞长骨以及使用2种酶Cre,COL10A1 Cre重组聚蛋白多糖-Cre重组ERT2( 此Agg-Cre重组ERT2)的下颌骨髁发育过程中直接转变成成骨细胞和骨细胞,越过与ROSA26 tdTomato。无论Col10和蛋白聚糖被公认的标记物软骨细胞。

在此基础上,我们开发了一种新方法,细胞谱系通过分析细胞特异性标志物的表达在用荧光免疫-以限定细胞的命运一起跟踪。 RUNX2(对于早期骨细胞的标记)和牙本质基质蛋白1(DMP1;对于晚期骨细胞的标记)为用于鉴定软骨衍生骨细胞和它们的分化状态。这种组合不仅拓宽细胞谱系追踪中的应用,而且还简化了化合物的小鼠的产生。更重要的是,亲本细胞的后代的数量,位置和分化状态同时显示,提供比细胞谱系单独跟踪的详细信息。总之,细胞谱系追踪技术和免疫荧光共应用是用于体内研究细胞生物学的有力工具。

Introduction

在开发过程中,软骨内骨形成占骨架体积的80%以上。人们普遍认为,这首先肥大软骨细胞的凋亡。接着,从底层骨髓细胞侵入并启动血管生成,随后通过骨髓和骨膜衍生细胞-1,2-新骨沉积。肥大软骨细胞的细胞命运(HCS),然而,一直是人们争论的一个问题,几十年来3。最初,碳氢化合物被认为是软骨细胞分化途径的端部,和凋亡普遍认为是碳氢化合物的最终命运。现在,一些研究人员认为,至少有一些碳氢化合物能够生存,并有助于软骨内成骨。虽然他们提议生长板软骨不得不转分化成基于超微结构,免疫组织化学染色的成骨细胞的能力,以及在体外研究46中 ,这些方法瓦特埃雷在证明对成骨细胞谱系的贡献软骨明确。

该细胞谱系追踪技术提供了一种更严格的方式来研究细胞命运。简要地说,一个重组酶,这是只在特定类型的细胞中表达,刺激报道基因的表达。以这种方式,这种类型的细胞及其后代被永久标记7。该Cre的loxP系统在谱系追踪常用。酶Cre(重组酶酶)将切除两个loxP位点之间的停止序列和激活报告在特定的细胞系( 图1A)。在一些情况下,研究者可以选择有利的时间点,通过使用一种药物,例如他莫昔芬激活酶Cre,引起酶Cre熔合到雌激素受体(酶Cre ERT2)8的改进形式。荧光记者已成为谱系追踪实验,因为他们大幅降低复杂性的标准和提高细胞命运跟踪8,9的准确性和效率。 tdTomato是变荧光报告之间的最佳选择,因为它具有最亮的荧光蛋白和最强的萤光,使它很容易地可视化7( 图1A)。

通过使用ROSA26 tdTomato谱系追踪系统,我们组和其他研究者已经证明,碳氧化合物可以在开发过程中10-14改变其表型成骨细胞。要做到这一点,我们开发了两套与ROSA26 tdtomato(普遍存在的表达在所有的细胞)/ Cre的(特定于软骨细胞)的小鼠的跟踪组合:2.3Col1a1-GFP(特定于成骨细胞)和免疫荧光(特定于骨细胞)。该数据表明,这两种方法都是可行的方法,来研究细胞的命运体内

Protocol

所有协议进行审查和机构动物护理和使用委员会(IACUC)在得克萨斯A&牙科M大学学院批准。 1.动物育种在这项研究中使用三种动物模型。为了研究胚胎软骨细胞的形成髁的命运,第一次使用COL10A1 Cre重组 15只和ROSA26 tdTomato越过他们(B6; 129S6- 亿吨(ROSA)26Sor TM9(CAG-tdTomato)HZE / J)小鼠获得Col10a1-综合招聘考试及ROS…

Representative Results

软骨细胞直接转变成在颌骨髁状突和长骨骨细胞(成骨细胞和骨细胞)。 聚集蛋白聚糖 ,对软骨的关键基因,主要表现为早期和成熟的软骨细胞18。其结果是,在2周的年龄在此Agg-Cre重组ERT2三苯氧胺的注射液; ROSA26 tdTomato激活小鼠红番茄记者在所有的软骨细胞和它们?…

Discussion

由于技术限制,它始终是难以调查细胞在体内的行为。然而,细胞谱系追踪技术已被证明是用于研究细胞生物学7-9的有力工具。在这项研究中,我们进一步通过与免疫相结合的方法进行改进。以这种方式,细胞命运可以由多个相关的标志物,从而拓宽谱系追踪中的应用来定义。此外,免疫荧光和番茄信号的此共定位同时显示的创始人细胞,它们的位置,以及它们的分化状态的后代?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This study was supported by NIH grant DE025014 to JQF.

Materials

Tamoxifen  Sigma T5648 activate the Cre event 
Paraformaldehyde Sigma  P6148 fix the sample
Ethylenediaminetetraacetic acid Alfa Aesar A10713 decalcify the hard tissue
Sucrose  Sigma S0389 dehydrate the tissue
Hyaluronidase from bovine testes  Sigma H4272 retrieve antigen for immunochemical staining
Bovine serum albumin Sigma  A3059 blocking solution 
primary antibody for Runx2  Cell Signal D1L7F primary antibody for immunochemical staining
primary antibody for DMP1 provided by Dr. Chunlin Qin primary antibody for immunochemical staining
anti-rabbit IgG Sigma 18140 control for immunochemical staining
secondary antibody  Invitrogen A11008 second antibody for immunochemical staining
OCT Tissue-Tek 4583 embed the sample for frozen section
Tween 20 Fisher Scientific BP337 PBST
non-fluorescing antifade mountant Life technologies P36934 mounting slides
DAPI Life technologies P36931 nuclear staining
Hydrophobic Barrier Pen Vector Laboratories circle the section on the slide for for immunochemical staining 
Xylazine AnaSed anesthetization 
Ketaset  Zoetis anesthetization 
cryosection machine Leica CM1860 UV
confocal microscope Leica DM6000 CFS 

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Cite This Article
Jing, Y., Hinton, R. J., Chan, K. S., Feng, J. Q. Co-localization of Cell Lineage Markers and the Tomato Signal. J. Vis. Exp. (118), e54982, doi:10.3791/54982 (2016).

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