Plodia 盘蛾的高效超活性 piggyBac 转基因。

IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Frontiers in genome editing Pub Date : 2022-12-23 eCollection Date: 2022-01-01 DOI:10.3389/fgeed.2022.1074888
Christa Heryanto, Anyi Mazo-Vargas, Arnaud Martin
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引用次数: 0

摘要

虽然基于piggyBac转座子的转基因技术在各种新兴模式生物中得到了广泛应用,但其在蝴蝶和蛾类中相对较低的转座率阻碍了其在鳞翅目昆虫中常规基因转化的应用。在这里,我们测试了经密码子优化的超活性piggyBac转座酶(hyPBase)是否适合以mRNA形式将转基因盒递送并整合到盘蛾Plodia interpunctella的基因组中。hyPBase mRNA 与供体质粒的联合注射成功地在眼睛和神经胶质中整合了 1.5-4.4 kb 的表达盒,这些表达盒通过 3xP3 启动子驱动荧光标记 EGFP、DsRed 或 EYFP。从 72 h 胚胎开始,到携带隐性白眼突变的幼虫、蛹和成虫,都能检测到转基因在 G0 注射代中的体细胞整合和表达。总体而言,2.5%的注射卵存活下来,成为带有马赛克荧光的转基因成虫。随后,荧光 G0 发现者的外交传递了 3xP3::EGFP 和 3xP3::EYFP 的单插入拷贝,并产生了稳定的同源系。表达 3xP3::DsRed的一小群 G0 基因的随机内交产生了一个稳定的转基因品系,该品系有一个以上的转基因插入位点。我们讨论了如何利用 hyPBase 在 Plodia 和其他蛾类中产生稳定的转基因资源。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Efficient hyperactive piggyBac transgenesis in Plodia pantry moths.

While piggyBac transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive piggyBac transposase (hyPBase) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth Plodia interpunctella. Co-injection of hyPBase mRNA with donor plasmids successfully integrated 1.5-4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene in the G0 injected generation was detectable from 72-h embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G0 founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable isogenic lines. Random in-crossing of a small cohort of G0 founders expressing 3xP3::DsRed yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how hyPBase can be used to generate stable transgenic resources in Plodia and other moths.

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7.00
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审稿时长
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