Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
Department of Genetics, Harvard Medical School, Boston, United States.
Elife. 2018 Mar 22;7:e35574. doi: 10.7554/eLife.35574.
We generated a library of ~1000 stocks in which we inserted a construct in the intron of genes allowing expression of under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single cDNA construct. Third, loss-of-function phenotypes associated with many insertions can be reverted by excision with . Fourth, driven reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with or any DNA. These stocks comprise a powerful resource for assessing gene function.
我们构建了一个约 1000 个品系的文库，在这些品系中，我们在基因的内含子中插入了一个构建体，使在 GAL4 的内源性启动子控制下表达，同时在 polyadenylation 信号 3'处阻止转录。这允许许多应用。首先，在必需基因中的约 90%的插入会导致严重的功能丧失表型，这是一种有效的基因突变方法。有趣的是，通过 CRISPR 工程化的 12/14 条染色体不携带第二致死突变。其次，在测试的致死插入中，有 26/36（70%）可以通过单个 cDNA 构建体挽救。第三，许多带有功能丧失表型的插入可以通过切除 来逆转。第四，驱动的报告具有高灵敏度的基因表达的组织和细胞类型特异性。我们报告了数百个以前未报道过的基因的表达。最后，插入的盒可以用 或任何 DNA 替换。这些品系构成了评估基因功能的强大资源。
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