Tromp Alisha, Wang Haitao, Hall Thomas E, Mowry Bryan, Giacomotto Jean
Queensland Brain Institute, University of Queensland, St Lucia, QLD, Australia.
Institute for Molecular Biosciences, University of Queensland, St Lucia, QLD, Australia.
Front Physiol. 2023 Aug 2;14:1221310. doi: 10.3389/fphys.2023.1221310. eCollection 2023.
We recently introduced the Cre/Lox technology in our laboratory for both transient (mRNA injections) and stable/transgenic experiments. We experienced significant issues such as silencing, mosaicism, and partial recombination using both approaches. Reviewing the literature gave us the impression that these issues are common among the zebrafish community using the Cre/Lox system. While some researchers took advantage of these problems for specific applications, such as cell and lineage tracing using the Zebrabow construct, we tried here to improve the efficiency and reliability of this system by constituting and testing a new set of tools for zebrafish genetics. First, we implemented a codon-improved Cre version (iCre) designed for rodent studies to counteract some of the aforementioned problems. This eukaryotic-like iCre version was engineered to i) reduce silencing, ii) increase mRNA stability, iii) enhance translational efficiency, and iv) improve nuclear translocation. Second, we established a new set of tol2-kit compatible vectors to facilitate the generation of either iCre-mRNA or iCre-transgenes for transient and transgenic experiments, respectively. We then validated the use of this material and are providing tips for users. Interestingly, during the validation steps, we found that maternal iCRE-mRNA and/or protein deposition from female transgenics systematically led to complete/homogeneous conversion of all tested Lox-responder-transgenes, as opposed to some residual imperfect conversion when using males-drivers or mRNA injections. Considering that we did not find any evidence of Cre-protein soaking and injections in the literature as it is usually conducted with cells, we tested these approaches. While soaking of cell-permeant CRE-protein did not lead to any detectable Lox-conversion, 1ng-10 ng protein injections led to robust and homogeneous Lox-recombination, suggesting that the use of protein could be a robust option for exogenous delivery. This approach may be particularly useful to manipulate housekeeping genes involved in development, sex determination and reproduction which are difficult to investigate with traditional knockout approaches. All in all, we are providing here a new set of tools that should be useful in the field.
我们最近在实验室中引入了Cre/Lox技术，用于瞬时（mRNA注射）和稳定/转基因实验。我们在使用这两种方法时都遇到了诸如沉默、嵌合现象和部分重组等重大问题。查阅文献给我们的印象是，在使用Cre/Lox系统的斑马鱼研究群体中，这些问题很常见。虽然一些研究人员利用这些问题进行特定应用，例如使用Zebrabow构建体进行细胞和谱系追踪，但我们在此尝试通过构建和测试一套新的斑马鱼遗传学工具来提高该系统的效率和可靠性。首先，我们采用了一种为啮齿动物研究设计的密码子优化的Cre版本（iCre），以应对上述一些问题。这种类似真核生物的iCre版本经过设计，能够：i）减少沉默；ii）提高mRNA稳定性；iii）增强翻译效率；iv）改善核转运。其次，我们建立了一套新的与tol2试剂盒兼容的载体，分别便于为瞬时和转基因实验生成iCre-mRNA或iCre转基因。然后，我们验证了这种材料的用途，并为用户提供了一些提示。有趣的是，在验证步骤中，我们发现来自雌性转基因动物的母源iCRE-mRNA和/或蛋白质沉积会系统性地导致所有测试的Lox反应转基因完全/均匀转化，而使用雄性驱动者或mRNA注射时则会出现一些残留的不完全转化。鉴于我们在文献中未发现任何关于Cre蛋白浸泡和注射的证据（通常是在细胞中进行），我们对这些方法进行了测试。虽然细胞渗透性CRE蛋白浸泡未导致任何可检测到的Lox转化，但1 ng - 10 ng蛋白注射导致了强烈且均匀的Lox重组，这表明使用蛋白可能是一种强大的外源递送选择。这种方法对于操纵参与发育、性别决定和繁殖的管家基因可能特别有用，而这些基因用传统的敲除方法很难研究。总而言之，我们在此提供了一套新的工具，应该会在该领域发挥作用。
