Gonzalez-Sqalli Emmanuel, Caron Matthieu, Loppin Benjamin
Laboratoire de Biologie et Modélisation de la Cellule, École Normale Supérieure de Lyon, CNRS UMR5239, Université Claude Bernard Lyon 1, 9 rue du Vercors, 69007 Lyon, France.
G3 (Bethesda). 2024 Oct 15;14(12). doi: 10.1093/g3journal/jkae235.
The cricket Gryllus bimaculatus is an emerging model insect of the order Orthoptera that is used in a wide variety of biological research themes. This hemimetabolous species appears highly complementary to Drosophila and other well-established holometabolous models. To improve transgenesis applications in G. bimaculatus, we have designed a transformation marker gene inspired from the widespread Drosophila mini-white+. Using CRISPR/Cas9, we first generated a loss-of-function mutant allele of the Gb-white gene (Gb-w), which exhibits a white eye coloration at all developmental stages. We then demonstrate that transgenic insertions of a piggyBac vector containing a 3xP3-Gb-w+ cassette rescue eye pigmentation. As an application, we used this vector to generate G. bimaculatus lines expressing a centromeric histone H3 variant (CenH3.1) fused to EGFP and validated EGFP-CenH3.1 detection at cricket centromeres. Finally, we demonstrate that Minos-based germline transformation and site-specific plasmid insertion with the ΦC31 integrase system function in G. bimaculatus.
双斑蟋（Gryllus bimaculatus）是直翅目一种新兴的模式昆虫，被用于广泛的生物学研究主题。这种渐变态昆虫似乎与果蝇及其他成熟的全变态模式生物具有高度互补性。为了改进双斑蟋的转基因应用，我们设计了一个受广泛使用的果蝇mini-white+启发的转化标记基因。利用CRISPR/Cas9，我们首先生成了Gb-white基因（Gb-w）的功能缺失突变等位基因，该基因在所有发育阶段均表现出白眼颜色。然后我们证明，含有3xP3-Gb-w+盒的piggyBac载体的转基因插入可挽救眼色素沉着。作为一种应用，我们使用该载体生成了表达与EGFP融合的着丝粒组蛋白H3变体（CenH3.1）的双斑蟋品系，并验证了在双斑蟋着丝粒处对EGFP-CenH3.1的检测。最后，我们证明基于Minos的生殖系转化和使用ΦC31整合酶系统的位点特异性质粒插入在双斑蟋中起作用。
