A RECOMBINANT RETROVIRUS ENCODING ALKALINE-PHOSPHATASE CONFIRMS CLONAL BOUNDARY ASSIGNMENT IN LINEAGE ANALYSIS OF MURINE RETINA
A RECOMBINANT RETROVIRUS ENCODING ALKALINE-PHOSPHATASE CONFIRMS CLONAL BOUNDARY ASSIGNMENT IN LINEAGE ANALYSIS OF MURINE RETINA
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DOI：
10.1073/pnas.89.2.693
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发表时间：
1992-01-15
影响因子：
11.1
通讯作者：
CEPKO, CL
中科院分区：
文献类型：
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作者：
FIELDSBERRY, SC;HALLIDAY, AL;CEPKO, CL
重组逆转录病毒编码组织化学可检测的酶-半乳糖苷酶已被用于研究脊椎动物神经系统的谱系。当后代细胞以可复制的集群模式排列时，单个祖先后代的鉴定是直截了当的，但当后代广泛迁移和/或以不规则模式排列时，解释就会出现困难。为了更好地解决克隆边界问题，可以将产生不同反应产物的其他组织化学标记病毒与携带lacz的病毒联合使用。为此，我们创建了逆转录病毒载体DAP，它编码一种易于检测的酶，人胎盘碱性磷酸酶。在高病毒滴度、表达稳定性和体内感染细胞鉴定方面，DAP被发现至少与编码lacz的逆转录病毒（如BAG）一样有用。此外，它被发现对出生后的啮齿动物视网膜发育是中性的，并且相对于lacZ具有优越的染色特性。用DAP和BAG共同感染啮齿动物视网膜，可以检查标记视网膜细胞径向阵列的克隆性质，这些细胞以前被描述为单个感染祖细胞的产物。在1100个同时检测DAP-和bag -感染细胞的径向阵列中，只有1.2%是感染了一种以上病毒的结果。
Recombinant retroviruses encoding the histochemically detectable enzyme beta-galactosidase have been used to investigate lineage in the vertebrate nervous system. Identification of the descendants of individual progenitors is straightforward when progeny cells are arranged in a reproducible, clustered pattern, but difficulties in interpretation arise when progeny migrate extensively and/or in an irregular pattern. To better resolve clonal boundaries, additional histochemical marker viruses that engender distinctive reaction products can be used in combination with lacZ-bearing viruses. To this end, we have created a retrovirus vector, DAP, encoding an easily assayable enzyme, human placental alkaline phosphatase. DAP was found to be at least as useful as a lacZ-encoding retrovirus (e.g., BAG) with respect to high viral titer, stability of expression, and in identification of infected cells in vivo. Moreover, it was found to be neutral with respect to postnatal rodent retinal development and offered superior staining characteristics relative to lacZ. Coinfection of rodent retina with DAP and BAG allowed an examination of the clonal nature of radial arrays of labeled retinal cells that previously had been described as products of a single infected progenitor. Of 1100 radial arrays examined for the presence of both DAP- and BAG-infected cells, only 1.2% were the result of infection with more than one virus.