Conditional expression of the TVA receptor allows clonal analysis of descendents from Cre-expressing progenitor cells
Conditional expression of the TVA receptor allows clonal analysis of descendents from Cre-expressing progenitor cells
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DOI：
10.1016/j.ydbio.2011.03.004
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发表时间：
2011-05-15
影响因子：
2.7
通讯作者：
Cepko, Constance L.
中科院分区：
文献类型：
--
作者：
Beier, Kevin T.;Samson, Maria Elena S.;Cepko, Constance L.
了解祖细胞在发育过程中产生的后代的数量和类型，为研究细胞命运决定发生的时间和地点提供了基础。直系关系可以通过鉴定表达重组酶（如Cre或Flp）的细胞后代来揭示。这种方法提供了有关基因表达历史的数据，但不能提供后代之间的克隆分辨。另一种方法是使用逆转录病毒标记，这种方法可以识别克隆，但不允许追踪基因表达历史。在这里，我们报告了这些方法的组合，以规避每种方法的局限性。通过利用Cre表达的特异性，并通过仅选择具有Cre病史的细胞子集进行逆转录病毒感染，可以标记具有基因表达史的克隆。该方法利用禽肿瘤病毒受体a （TVA）的一个条件等位基因，允许在Cre活性后用带有ASLV-A包膜糖蛋白（EnvA）假型的哺乳动物逆转录病毒载体感染小鼠细胞。我们量化了该系统在体内和体外的效率和特异性。我们还生成了一系列逆转录病毒载体，编码多种组织化学和荧光报告基因，能够跟踪克隆混合物，从而更好地分辨克隆边界。这种方法和新的载体可用于进一步了解产生特定子细胞的祖细胞的基因表达模式，并为操纵已识别的发育细胞亚群提供平台。(C) 2011爱思唯尔公司版权所有。
An understanding of the number and types of progeny produced by progenitor cells during development provides a foundation for studies of when and where cell fate determination takes place. Lineal relationships can be revealed by the identification of descendents of cells that express a recombinase, such as Cre or Flp. This method provides data concerning gene expression history, but does not provide clonal resolution among the descendents. An alternative method employs retroviral labeling, which permits the identification of clones, but does not allow for the tracking of gene expression history. Here we report a combination of these methods to circumvent each method's limitations. By employing the specificity of Cre expression, and by selecting only a subset of cells with a Cre history for retroviral infection, clones with a gene expression history can be labeled. The method utilizes a conditional allele of the avian tumor virus receptor A (TVA), which allows infection of mouse cells following Cre activity, with mammalian retroviral vectors pseudotyped with the ASLV-A envelope glycoprotein (EnvA). We quantified the efficiency and specificity of this system in vivo and in vitro. We also generated a series of retroviral vectors encoding a variety of histochemical and fluorescent reporter genes that enable the tracking of mixtures of clones, thus enabling better resolution of clonal boundaries. This method and new vectors can be used to further our understanding of the gene expression patterns of progenitor cells that make particular daughter cells, as well as provide a platform for manipulating identified subsets of developing cells. (C) 2011 Elsevier Inc. All rights reserved.