W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Life Sciences, Imperial College London, London, UK.
Cell Rep Methods. 2024 Feb 26;4(2):100714. doi: 10.1016/j.crmeth.2024.100714.
Anopheles gambiae uses its sense of smell to hunt humans. We report a two-step method yielding cell-type-specific driver lines for enhanced neuroanatomical and functional studies of its olfactory system. We first integrated a driver-responder-marker (DRM) system cassette consisting of a linked T2A-QF2 driver, QUAS-GFP responder, and a gut-specific transgenesis marker into four chemoreceptor genes (Ir25a, Ir76b, Gr22, and orco) using CRISPR-Cas9-mediated homology-directed repair. The DRM system facilitated rapid selection of in-frame integrations via screening for GFP+ olfactory sensory neurons (OSNs) in G larval progeny, even at genomic loci such as orco where we found the transgenesis marker was not visible. Next, we converted these DRM integrations into T2A-QF2 driver-marker lines by Cre-loxP excision of the GFP responder, making them suitable for binary use in transcuticular calcium imaging. These cell-type-specific driver lines tiling key OSN subsets will support systematic efforts to decode olfaction in this prolific malaria vector.
冈比亚按蚊利用嗅觉捕猎人类。我们报告了一种两步法，可产生细胞类型特异性驱动线，用于增强其嗅觉系统的神经解剖学和功能研究。我们首先使用 CRISPR-Cas9 介导的同源定向修复将一个由连接的 T2A-QF2 驱动子、QUAS-GFP 应答子和肠道特异性转座酶标记组成的驱动-应答-标记（DRM）系统盒整合到四个化学感受器基因（Ir25a、Ir76b、Gr22 和 orco）中。DRM 系统通过筛选 G 幼虫后代中的 GFP+嗅觉感觉神经元（OSN），促进了框架内整合的快速选择，即使在基因组基因座（如 orco）中，我们发现转座酶标记不可见。接下来，我们通过 Cre-loxP 切除 GFP 应答子，将这些 DRM 整合转化为 T2A-QF2 驱动子-标记线，使其适合于经皮钙成像的二进制使用。这些针对关键 OSN 亚群的细胞类型特异性驱动线将支持在这种多产疟疾媒介中解码嗅觉的系统努力。
