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一种用于果蝇中转录后翻译的 mRNA 细胞特异性分析的多功能方法。

A versatile method for cell-specific profiling of translated mRNAs in Drosophila.

机构信息

Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

出版信息

PLoS One. 2012;7(7):e40276. doi: 10.1371/journal.pone.0040276. Epub 2012 Jul 6.

DOI:10.1371/journal.pone.0040276
PMID:22792260
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3391276/
Abstract

In Drosophila melanogaster few methods exist to perform rapid cell-type or tissue-specific expression profiling. A translating ribosome affinity purification (TRAP) method to profile actively translated mRNAs has been developed for use in a number of multicellular organisms although it has only been implemented to examine limited sets of cell- or tissue-types in these organisms. We have adapted the TRAP method for use in the versatile GAL4/UAS system of Drosophila allowing profiling of almost any tissue/cell-type with a single genetic cross. We created transgenic strains expressing a GFP-tagged ribosomal protein, RpL10A, under the control of the UAS promoter to perform cell-type specific translatome profiling. The GFP::RpL10A fusion protein incorporates efficiently into ribosomes and polysomes. Polysome affinity purification strongly enriches mRNAs from expected genes in the targeted tissues with sufficient sensitivity to analyze expression in small cell populations. This method can be used to determine the unique translatome profiles in different cell-types under varied physiological, pharmacological and pathological conditions.

摘要

在黑腹果蝇中，很少有方法可以快速进行细胞类型或组织特异性表达谱分析。一种翻译核糖体亲和纯化（TRAP）方法已被开发用于许多多细胞生物中的活性翻译 mRNA 分析，尽管它仅在这些生物中用于检查有限的细胞或组织类型。我们已经对 TRAP 方法进行了改编，使其可用于黑腹果蝇的多功能 GAL4/UAS 系统，允许通过单一遗传杂交来分析几乎任何组织/细胞类型。我们创建了表达 GFP 标记的核糖体蛋白 RpL10A 的转基因品系，该基因受 UAS 启动子的控制，以进行细胞类型特异性翻译组分析。GFP::RpL10A 融合蛋白有效地掺入核糖体和多核糖体中。多核糖体亲和纯化强烈富集来自目标组织中预期基因的 mRNA，具有足够的灵敏度来分析小细胞群体中的表达。该方法可用于在不同的生理、药理学和病理学条件下确定不同细胞类型中的独特翻译组谱。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/7552db07a682/pone.0040276.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/68a626afdff0/pone.0040276.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/ad9f9851927f/pone.0040276.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/f8b414477856/pone.0040276.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/5cbe0f8ffba6/pone.0040276.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/7552db07a682/pone.0040276.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/68a626afdff0/pone.0040276.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/ad9f9851927f/pone.0040276.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/f8b414477856/pone.0040276.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/5cbe0f8ffba6/pone.0040276.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/72d4/3391276/7552db07a682/pone.0040276.g005.jpg

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