Comprehensive Resource for the Drosophila 4th chromosome

125年来，黑腹果蝇在生物学问题的遗传分析方面一直处于领先地位。第4 染色体是果蝇遗传分析的最后一个领域。第四个小而缺乏重组 一直被忽视。它包含105个基因。74%的蛋白质编码基因具有人类 同源物，其中68%与疾病相关。例如，Eyeless属于PAX/RAX系列 其中人RAX 2的体细胞丢失导致年龄相关性黄斑变性。人类ANK 2的突变， 锚蛋白同源物是先天性长QT综合征的主要原因。完全理解 健康需要检查这些基因。为了推进这一努力，PI最近产生了独特的 染色体用于研究携带突变的标记单细胞克隆（MARCM）4号。这里他 建议与IU和UMN的同事合作，以促进遗传和分子分析， 所有基因都在第四天具体目标是为果蝇第四代产生全面的资源 染色体该资源将包含大约730个股票，分为五个集合。1.使用CRISPR的FRT 用于功能丧失研究和MARCM的79个蛋白质编码基因中的每一个的突变（每个基因两个突变）。 基因= 158只股票）2. 26种非编码RNA中的每一种具有CRISPR突变的FRT，用于功能丧失， MARCM（26只股票）。3.用现有的MIMIC将蛋白质编码基因和非编码RNA转化为 T2A.GAL4：GFP和在剩余部分中插入具有T2A.GAL4：GFP的CRIMIC用于荧光标记， 作为报告基因（蛋白质或RNA）和功能获得研究（120个储备）。4.功能股收益 由以下组成：a）每个蛋白质编码基因和非编码RNA的UASt和UASp/UASz，和B）UASt和 保守蛋白质编码基因和非编码RNA的两个最接近的人cDNA的UASp/UASz（400 股票）。5.用于克隆分析的平衡染色体和辅助染色体，如FRT-GAL 80， MARCM，FRT-attP用于设计克隆，FRT-ovoD用于有/无UAS.FLP的生殖系克隆（20个原种）。 该资源将实现：功能测定的丧失和获得、组织特异性和时间基因调节 体细胞和生殖细胞系组织，标记RNA和蛋白质的跟踪以及所有遗传分析方式。到 协助确定任务的优先次序，我们有一个咨询委员会，并将接受社区的意见。表征 新种群的研究是在研究人员的分子遗传学专业知识范围内进行的。除了促进 发展和疾病的基础研究，我们的资源将有直接的转化应用， 了解人类健康。例如，T2A.GAL4：GFP插入破坏了果蝇基因并导致了果蝇的死亡。 GAL 4在天然模式中的表达可以与UAS人cDNA储备物组合，用于分析 “人源化”疾病或治疗模型。这一资源将随时提供给研究人员， 促进我们对生物学和人类健康基础的保守分子机制的理解。

For 125 years, Drosophila melanogaster has led the way in the genetic analysis of biological questions. The 4th chromosome is the final frontier for genetic analysis in Drosophila. Small and devoid of recombination the 4th has long been ignored. Nevertheless it contains 105 genes. 74% of the protein coding genes have human homologs and 68% of these have a disease association. For example Eyeless belongs to the PAX/RAX family where somatic loss of human RAX2 leads to age-related macular degeneration. Mutations in human ANK2, homolog of Ankyrin are the primary cause of congenital Long QT syndrome. A complete understanding of health requires the examination of these genes. To advance this effort, the PI recently generated unique chromosomes for the study of marked single cell clones (MARCM) carrying mutations on the 4th. Here he proposes to collaborate with colleagues at IU and UMN to facilitate the genetic and molecular analyses of every gene on the 4th. The Specific Aim is to generate a comprehensive resource for the Drosophila 4th chromosome. The resource will contain roughly 730 stocks divided into five collections. 1. FRT with a CRISPR mutation for each of the 79 protein coding genes for loss of function studies and MARCM (two mutations per gene = 158 stocks) 2. FRT with a CRISPR mutation for each of 26 noncoding RNAs for loss of function and MARCM (26 stocks). 3. Conversion of protein coding genes and noncoding RNAs with an existing MIMIC to T2A.GAL4:GFP and insertion of a CRIMIC that has T2A.GAL4:GFP in the remainder for fluorescent tagging, as reporter genes (protein or RNA) and gain of function studies (120 stocks). 4. Gain of function stocks composed of: a) UASt and UASp/UASz for each protein coding gene and non-coding RNA and b) UASt and UASp/UASz for the two closest human cDNAs for conserved protein coding genes and noncoding RNAs (400 stocks). 5. Balancer chromosomes and auxiliary chromosomes for clonal analyses such as FRT-GAL80 for MARCM, FRT-attP for designer clones and FRT-ovoD for germ line clones with/without UAS.FLP (20 stocks). The resource will enable: loss and gain of function assays, tissue-specific and temporal gene regulation in somatic and germ line tissues, the tracking of tagged RNA and proteins and all manner of genetic analyses. To assist in prioritizing tasks, we have an Advisory Committee and will accept community input. Characterization of new stocks takes place within the molecular genetics expertise of the investigators. In addition to facilitating basic research on development and disease, our resource will have direct translational application to understanding human health. For example, T2A.GAL4:GFP insertions that disrupt the fly gene and result in the expression of GAL4 in native patterns can be combined with UAS human cDNA stocks for the analysis of “humanized” disease or treatment models. This resource will be made readily available to researchers to advance our understanding of biology and conserved molecular mechanisms underlying human health.