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Genetic transformation of mosquitoes by microparticle bombardment
Insect Molecular Biology ( IF 2.3 ) Pub Date : 2020-10-03 , DOI: 10.1111/imb.12670 A N Lule-Chávez 1 , R Carballar-Lejarazú 1, 2 , J L Cabrera-Ponce 1 , H Lanz-Mendoza 2 , J E Ibarra 1
Insect Molecular Biology ( IF 2.3 ) Pub Date : 2020-10-03 , DOI: 10.1111/imb.12670 A N Lule-Chávez 1 , R Carballar-Lejarazú 1, 2 , J L Cabrera-Ponce 1 , H Lanz-Mendoza 2 , J E Ibarra 1
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Mosquitoes constitute the major living beings causing human deaths in the world. They are vectors of malaria, yellow fever, dengue, zika, filariases, chikungunya, among other diseases. New strategies to control/eradicate mosquito populations are based on newly developed genetic manipulation techniques. However, genetic transformation of mosquitoes is a major technical bottleneck due to low efficiency, the need of sophisticated equipment, and highly trained personnel. The present report shows the transgenerational genetic transformation of Aedes aegypti, using the particle inflow gun (PIG), by integrating the ecfp gene in the AAEL000582 mosquito gene with the CRISPR‐Cas9 technique, achieving a mean efficiency of 44.5% of bombarded individuals (G0) that showed ECFP expression in their tissues, and a mean of 28.5% transformation efficiency measured on G1 individuals. The same transformation technique was used to integrate the egfp/scorpine genes cloned in the Minos transposon pMinHygeGFP into the Anopheles albimanus genome, achieving a mean efficiency of 43.25% of bombarded individuals (G0) that showed EGFP expression in their tissues. Once the technique was standardized, transformation of Ae. aegypti neonate larvae and An. albimanus eggs was achieved when exposed to gold microparticle bombardment. Integration of genes and heterologous protein expression were confirmed by PCR, sequencing, fluorescent microscopy, mass spectrometry, Western blot and dot blot analyses. Transgenerational inheritance of the transgenes was observed only on Ae. aegypti, as all transformed An. albimanus individuals died at the pupal stage of the G0 generation.
中文翻译：
微粒轰击蚊子的遗传转化
蚊子是世界上造成人类死亡的主要生物。它们是疟疾、黄热病、登革热、寨卡病毒、丝虫病、基孔肯雅热等疾病的传播媒介。控制/根除蚊子种群的新策略基于新开发的基因操作技术。然而，由于效率低、需要复杂的设备和训练有素的人员，蚊子的遗传转化是一个主要的技术瓶颈。本报告显示了埃及伊蚊的跨代遗传转化，使用粒子流入枪 (PIG)，通过整合ecfpAAEL000582 蚊子基因中的基因与 CRISPR-Cas9 技术，实现了 44.5% 的轰击个体 (G0) 在其组织中显示 ECFP 表达的平均效率，以及在 G1 个体上测量的平均转化效率为 28.5%。使用相同的转化技术将克隆在Minos转座子 pMinHygeGFP 中的egfp/scorpine基因整合到白按蚊基因组中，实现了 43.25% 的被轰击个体 (G0) 在其组织中显示 EGFP 表达的平均效率。一旦技术标准化，Ae的转化。aegypti新生幼虫和An. 白化病当暴露于金微粒轰击时获得了卵。基因的整合和异源蛋白表达通过 PCR、测序、荧光显微镜检查、质谱、蛋白质印迹和斑点印迹分析得到证实。仅在Ae上观察到转基因的跨代遗传。aegypti，因为都转化了An。albimanus个体在 G0 代的蛹期死亡。
更新日期：2020-10-03
中文翻译：
微粒轰击蚊子的遗传转化
蚊子是世界上造成人类死亡的主要生物。它们是疟疾、黄热病、登革热、寨卡病毒、丝虫病、基孔肯雅热等疾病的传播媒介。控制/根除蚊子种群的新策略基于新开发的基因操作技术。然而，由于效率低、需要复杂的设备和训练有素的人员，蚊子的遗传转化是一个主要的技术瓶颈。本报告显示了埃及伊蚊的跨代遗传转化，使用粒子流入枪 (PIG)，通过整合ecfpAAEL000582 蚊子基因中的基因与 CRISPR-Cas9 技术，实现了 44.5% 的轰击个体 (G0) 在其组织中显示 ECFP 表达的平均效率，以及在 G1 个体上测量的平均转化效率为 28.5%。使用相同的转化技术将克隆在Minos转座子 pMinHygeGFP 中的egfp/scorpine基因整合到白按蚊基因组中，实现了 43.25% 的被轰击个体 (G0) 在其组织中显示 EGFP 表达的平均效率。一旦技术标准化，Ae的转化。aegypti新生幼虫和An. 白化病当暴露于金微粒轰击时获得了卵。基因的整合和异源蛋白表达通过 PCR、测序、荧光显微镜检查、质谱、蛋白质印迹和斑点印迹分析得到证实。仅在Ae上观察到转基因的跨代遗传。aegypti，因为都转化了An。albimanus个体在 G0 代的蛹期死亡。
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