果蝇个体小，发育周期短，饲养方便，利用其进行生物医学研究具有以下优势：1、果蝇基因与人类高度同源，75%以上的人类致病基因都与果蝇同源。2、经过近百年的积累，果蝇拥有丰富的遗传学工具。3、果蝇是多细胞动物，有体细胞、干细胞等不同类型细胞，因此，不仅适合基因在发育中的功能研究，也可以方便地研究基因在不同类型细胞之间介导的相互作用。4、果蝇卵巢是成熟的体内干细胞模型，具有成本低廉、性能稳定、不受伦理学限制、干细胞位置确定、易于标记等优势，便于开展干细胞的体内研究。 在多年的研究过程中，我们发现对高表达、冗余基因及多个基因的条件性调控，仍缺乏有效的手段。因此，我参与研发了新一代转基因干扰技术，可对体细胞和生殖干细胞内多拷贝、高表达基因进行高效敲低，并且首次实现多个基因同时调控。为了实现高效、可遗传突变，并验证转基因干扰结果，参与开发了基于CRISPR/Cas9的生殖细胞特异的基因编辑技术，并对这一技术进行了优化；此外，开发了脱靶效应低、能精确对基因组进行编辑的Cas9D10A切口酶系统。为了在生殖细胞中特异激活目的基因，我在本实验室开发的体细胞基因激活技术的基础上，开发了果蝇生殖细胞特异基因激活系统，并首次应用到生殖干细胞研究上。 PIWI最初是在果蝇中发现的，是参与piRNA信号通路、转座子调控及异染色质形成的关键蛋白，在果蝇生殖干细胞维持和自我更新调控中发挥重要作用。大量临床研究及生物信息学分析发现，人源piwi在包括卵巢癌、结肠癌在内的癌症中表达升高，但其分子机制尚不清楚。因此，本研究利用自主开发的果蝇遗传学工具实现高效的piwi表达调控，研究其在果蝇卵巢生殖干细胞调控中的作用机制。利用新研发的转基因干扰技术，可显著敲低piwi在生殖干细胞中的表达；进一步通过嵌合体分析，首次发现piwi参与干细胞竞争，piwi表达量降低导致干细胞竞争能力下降。随后，通过新技术构建的piwi突变体，得到了同样结论。特别重要的是，在生殖干细胞中以传统过表达piwi编码序列的方式，不能提高piwi表达量，而通过我开发的生殖细胞特异基因定点激活技术，能有效激活piwi的表达；并且发现，升高piwi表达，可以增强生殖干细胞竞争力。最后，我们研究了piwi参与干细胞竞争的机制，发现JAK-STAT信号通路介导了piwi参与的干细胞竞争。本论文的研究，不仅进一步丰富了果蝇领域基因表达调控的遗传研究手段，也为生殖干细胞的研究提供了新思路。
In addition to small size, short developmental cycle and easy culture, Drosophila melanogaster has many advantages over other model organisms for biomedical research. Specifically, 1. Drosophila genes are highly homologous to human genes, and more than 75% of human pathogenic genes have homologs in Drosophila. 2. With more than 100 years of research experience, Drosophila has many genetic tools superior to other model organisms. 3. Drosophila are multicellular animals with different types of cells, such as somatic cells and stem cells. Therefore, we can not only study the intrinsic roles of genes in development, but also explore the interactions between different cells. 4. Drosophila ovary is a mature in vivo model for stem cell studies, with advantages such as low cost, stable performance, and no ethical limitations. More importantly, Drosophila ovarian stem cells can be easily identified by location and immunofluorescence staining, bringing convenience for in vivo studies of ovarian stem cells. During long-term studies, we found it’s challenging to efficiently modulate the highly expressed genes, redundant genes and multiple genes formed complex using current tools. Therefore, we developed the next-generation transgenic RNAi technique, which can efficiently modulate multiple copied and highly expressed genes, and modulate multiple genes without genetic recombination. To confirm the RNAi result, we also developed the heritable mutation method, and then optimized this approach. On the other hand, we developed Cas9D10A nickase system, which almost avoids off-target effects when generating indel mutants. To specifically increase gene transcription in germline, I optimized the targeting activator system developed by our lab, and applied it in female germline stem cells. PIWI was first found in Drosophila and is a key protein involved in piRNA biogenesis, transposon control and heterochromatin formation. It also plays an important role in Drosophila germline stem cell maintenance and self-renewal. Large numbers of clinical studies and bioinformatic analysis found that human PIWI gene expression is elevated in many cancers including ovarian cancer and colon adenocarcinoma. But the molecular mechanism how PIWI works in cancers is still unclear. In this study, we used the technologies above to modulate expression of piwi, to study the function and mechanism of PIWI in the regulation of germline stem cells. With the novel RNAi technique, we significantly knocked down piwi, and together with the mosaic analysis, we found it is involved in adult female germline stem cell competition. PIWI deficiency reduced the competition ability of germline stem cells （GSCs）. This phenomenon was further confirmed by piwi mutant. More importantly, the expression of piwi cannot be improved by the overexpression of piwi coding sequence in GSCs, but can be effectively activated by the activation technique I developed. By activating piwi expression, it was further demonstrated that increased PIWI expression can enhance the competitiveness of GSCs. Finally, we explored the mechanism and found piwi mediates JAK-STAT pathway to control female germline stem cell competition.Taken together, in this thesis we not only developed the new genetic tools for gene regulation in Drosophilo, but also provided new ideas of the mechanism of germline stem cell.
