Laboratory of Evolutionary and Functional Genomics, School of Life Sciences, Chongqing University, Chongqing, 401331, China.
Chongqing Key Laboratory of Vector Insects, Institute of Entomology and Molecular Biology, College of Life Sciences, Chongqing Normal University, Chongqing, 401331, China.
Insect Biochem Mol Biol. 2022 Dec;151:103861. doi: 10.1016/j.ibmb.2022.103861. Epub 2022 Nov 1.
Spatial or temporal specific gene knockout system is a valuable tool for studying the molecular mechanisms underlying developmental processes. The integument is essential for insect fitness and survival, but tools for dissecting function of genes in this tissue are lacking. In this study, we firstly identified an epidermis specifically expressed gene of the domesticated silkworm, BmCPG25, by comparative transcriptomic analysis. Furthermore, a transgenic silkworm expressing the RNA dependent CRISPR-Cas9 protein driven by the regulatory region of the BmCPG25 was established. Immunochemistry analysis showed the endonuclease was specifically expressed in the nuclear of epidermal cells. We also validated the efficiency of this system by disrupting the function of an epidermis specifically expressed cuticular protein gene (Cpr21) and a ubiquitously expressed cuticular gene (Cph18), respectively. In summary, we successfully constructed a conditional knockout toolkit to manipulate the gene editing in epidermal cells, which provides a valuable approach to study the molecular mechanism of integument development.
时空特异性基因敲除系统是研究发育过程中分子机制的有用工具。表皮对于昆虫的适应性和生存至关重要，但缺乏用于剖析该组织中基因功能的工具。在这项研究中，我们首先通过比较转录组分析鉴定了驯化家蚕 BmCPG25 的表皮特异性表达基因。此外，还构建了一种表达 RNA 依赖性 CRISPR-Cas9 蛋白的转基因家蚕，该蛋白由 BmCPG25 的调控区驱动表达。免疫化学分析表明，内切酶特异性表达在表皮细胞的核内。我们还通过分别破坏表皮特异性表达的表皮蛋白基因（Cpr21）和广泛表达的表皮基因（Cph18）的功能，验证了该系统的效率。总之，我们成功构建了一个条件性基因敲除工具包，用于操纵表皮细胞中的基因编辑，为研究表皮发育的分子机制提供了一种有价值的方法。
