Drosophila Genomics Resource Center, Indiana University, Bloomington, IN 47405, USA.
Department of Biology, Indiana University, Bloomington, IN 47405, USA.
G3 (Bethesda). 2021 Aug 7;11(8). doi: 10.1093/g3journal/jkab161.
The generation of Drosophila stable cell lines has become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100 Drosophila cell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the available Drosophila melanogaster cell lines. Here, we describe a toolkit and procedure that combines CRISPR/Cas9 and theaaa PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic the in vivo attP sites located at 25C6 and 99F8 of the Drosophila genome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existing D. melanogaster cell lines.
果蝇稳定细胞系的产生对于补充体内实验和作为遗传筛选工具变得非常宝贵。最近利用 attP/PhiC31 整合酶系统的进展使得可以创建果蝇细胞，其中重组介导的盒交换 (RMCE) 可用于生成稳定整合的转基因细胞系，该细胞系在所需基因座处含有转基因的单个拷贝。当前的技术除了繁琐和引入外源元件外，还仅限于少数胚胎来源的细胞系。尽管如此，由于有 100 多种以上的果蝇细胞系可供使用，包括越来越多的 CRISPR/Cas9 修饰的细胞系，因此需要一种更通用的方法来从任何可用的果蝇黑素细胞系中生成稳定整合的转基因系。在这里，我们描述了一种结合 CRISPR/Cas9 和 aaa PhiC31 整合酶系统的工具包和程序。我们已经生成并分离了单个细胞克隆，该克隆将 Actin5C::dsRed 盒侧翼带有 attP 位点，插入到 Kc167 和 S2R+细胞系的基因组中，该基因组模拟了位于 Drosophila 基因组 25C6 和 99F8 处的体内 attP 位点。此外，我们利用侧翼带有 attB 位点的两个独立 GFP 表达构建体测试了 attP 对接位点的功能，该构建体允许 RMCE，从而插入任何感兴趣的 DNA。最后，为了证明我们方法和现有构建体的通用性，我们已经成功地将侧翼带有 attP 位点的 Actin5C::dsRed 盒整合到两个不同的 CNS 细胞系 ML-DmBG2-c2 和 ML-DmBG3-c2 中。总体而言，这里报道的试剂和方法允许在现有的 D. melanogaster 细胞系中以最小的细胞基因组变化有效地生成稳定的转基因盒。
