Codon optimized Tol2 transposase results in increased transient expression of a crystallin-GFP transgene in zebrafish
Codon optimized Tol2 transposase results in increased transient expression of a crystallin-GFP transgene in zebrafish
复制标题
DOI：
10.17912/micropub.biology.000268
复制
发表时间：
2020-06
影响因子：
--
通讯作者：
Allison S Mackey;Allison S Mackey;Priscilla S. Redd;A. DeLaurier;C. N. Hancock
中科院分区：
文献类型：
--
作者：
Allison S Mackey;Allison S Mackey;Priscilla S. Redd;A. DeLaurier;C. N. Hancock
图1：密码子优化提高了存活鱼a的转基因效果。预测的NLS序列以黄色突出显示，预测的NES序列以蓝色突出显示。B.注射pDestTol2pACryGFP和CO Tol2 TPase mRNA后受精后2天的斑马鱼胚胎图像。右胚眼定位eGFP表达阳性。C.四种不同mRNA处理的pDestTol2pACryGFP胚胎共注射结果。存活率是总注射量的一个子集中存活2 dpf的鱼的比例（n =测量存活率的注射次数）。总效率是通过将存活率乘以存活鱼的眼睛局部表达eGFP的百分比来计算的。II型转座因子（te）是一种DNA片段，可以通过转座酶（TPase）蛋白的作用在基因组内被调动（Craig 2002）。一般来说，TPase蛋白结合元件末端序列形成功能性转位复合物的速度决定了元件的相对迁移性（Mizuuchi et al. 1992; Zayed et al. 2004; Zhang et al. 2001）。因此，转座复合体的形成速率取决于功能性TPase蛋白的浓度、存在的TE序列的数量以及TPase蛋白在细胞内的定位。一些研究表明，转位复合物的形成受进入细胞核的调控，因为核定位信号（NLS）和核输出信号（NES）的改变会影响转位（Hancock et al. 2010; Payero et al. 2016; Ramakrishnan et al. 2019）。斑马鱼研究通常涉及将DNA序列（即过表达磁带）整合到胚胎中，导致短暂或种系表达（Nusslein-Volhard and Dahm 2002）。在TE序列之间插入转基因并提供TPase蛋白来源已被证明可提高跨多种模式生物的转基因效率（Ding et al. 2005; Ivics and Izsvák 2004; Munoz-Lopez and Garcia-Perez 2010; Zayed et al. 2004）。来自Medaka鱼（Oryzias latipes）的Tol2 TE已被开发作为改善斑马鱼转基因的手段，包括瞬时和种系整合（Kawakami 2007； Koga等人1996；Kwan等人2007；Ni等人2016）。通过向1-4细胞期的斑马鱼胚胎共注射Tol2 TPase mRNA和一个Tol2末端序列侧翼的构建体，可以诱导Tol2介导的转基因（Kawakami 2007; Kwan et al. 2007; Ni et al. 2016）。先前的报告表明，与对照组相比，添加Tol2 TPase mRNA（由pCS2FA表达）可使Tol2侧翼转基因表达增加约3倍（Kwan et al. 2007）。另一项研究表明，2020年6月11日，斑马鱼密码子优化版本的Tol2 TPase导致了成功的种系传播，但没有直接比较效率的报道（Suster et al. 2011）。本研究的目的是探索通过对斑马鱼的Tol2 TPase基因进行密码子优化或改变Tol2 TPase中检测到的NES和NLS，可以在多大程度上提高Tol2介导的转基因表达效率（图1A）。我们假设，提高翻译效率（Gustafsson et al. 2004）和进入细胞核可能会在短暂时间内改善转位复合物的形成。通过对Tol2 TPase密码子使用情况的分析，发现其含有斑马鱼很少使用的19个密码子（TTA、CTA和TCG） （Nakamura et al. 2000）。密码子优化导致CO Tol2 TPase结构的发展，更合适地匹配斑马鱼特异性密码子偏见，而不改变氨基酸序列（Zhou et al. 2016）。在CO Tol2 TPase N结构中，通过改变L641A和L643A来去除Tol2 TPase中的NES（图1A），并通过将其改变为PKKKRKV （NLS评分=13）来加强现有的NLS (PKRARLD， NLS评分=8.5)（Dingwall and Laskey 1991; Kosugi et al. 2009）。将1-4个细胞期的斑马鱼胚胎注射pDestTol2pACryGFP后，与未注射野生型Tol2 TPase mRNA相比，眼部eGFP表达频率显著增加（见图1B）（图1C， p=0.0024）。这低于先前报道的使用非密码子优化的Tol2的表达频率增加3倍（Kwan et al. 2007）。当我们使用密码子优化的（CO Tol2 TPase） mRNA时，我们观察到眼睛eGFP表达频率显著增加（图1C，与无mRNA相比p=<0.0001，与WT TPase相比p=0.0008）。这些结果表明，密码子优化版本的mRNA在胚胎中更有效地翻译，导致Tol2 TPase蛋白的总体浓度更高。相比之下，NES和NLS改变版本（CO Tol2 TPase N）的表达频率比其他所有组都低（相对于无mRNA p=0.0013；相对于WT Tol2 TPase p<0.0001；相对于CO Tol2 TPase p<0.0001）。这表明对NES和NLS的改变可能破坏了CO Tol2 TPase N蛋白的整体蛋白质功能（图1C）。当使用表达频率和存活率来计算总体转基因表达效率时，我们观察到所有处理导致大约20%的注射卵产生表达egfp的胚胎存活到2 dpf（图1C）。存活频率与2 dpf时eGFP表达频率呈反比关系。我们观察到，与不携带CO Tol2 TPase mRNA （p= 0.0002）和WT Tol2 TPase mRNA （p=0.0033）相比，携带较高活性CO Tol2 TPase mRNA的鱼发育正常的数量明显减少（图1C）。这表明转基因插入可能引起的基因组破坏可能是转基因率的重要限制。本研究在无mRNA对照中观察到eGFP的高频率表达，表明观察到的高百分比的荧光不是由TPase介导的。此外，我们的研究结果表明，添加Tol2 mRNA并没有显著提高转基因瞬时表达的总体率（注射的转基因鱼/胚胎的存活总数）。然而，Tol2系统有很大的好处，因为存活的鱼中有更大一部分表现出瞬时转基因表达，这可能会使筛选表达胚胎变得更容易。我们的研究结果表明，与CO Tol2 TPase mRNA（可通过Addgene #133032获得）共注射构建物是对现有技术的重大改进。该密码子优化后的Tol2具有提高瞬时表达量的潜力，应该对其诱导遗传转基因的能力进行测试。虽然这项研究没有检测遗传转基因，但这项研究的下一步将是检测pDestTol2pACryGFP转基因在注射了每种mRNA的鱼体内的整合率。方法请求详细的协议
Figure 1: Codon-optimization improves transgenesis in surviving fish A. Alignment of the C-terminal region of the Tol2 TPase protein and its homologs. Predicted NLS sequences are highlighted in yellow, predicted NES sequences are highlighted in blue. B. Image of zebrafish embryos 2 days post fertilization (dpf) after injection with pDestTol2pACryGFP and CO Tol2 TPase mRNA. Right embryo is positive for eye-localized eGFP expression. C. Results from embryo coinjection of pDestTol2pACryGFP with four different mRNA treatments. Survival rate is the proportion of fish that survived 2 dpf for a subset of the total injections (n = number of injections for which survival was measured). Overall efficiency was calculated by multiplying the survival rate by the percent of surviving fish that showed eye localized eGFP expression. Description Type II transposable elements (TEs) are segments of DNA that can be mobilized within the genome through the action of transposase (TPase) proteins (Craig 2002). In general, the rate at which TPase proteins bind the terminal sequences of the elements to form a functional transposition complex determines the relative mobility of the element (Mizuuchi et al. 1992; Zayed et al. 2004; Zhang et al. 2001). The rate of transposition complex formation is thus determined by the concentration of functional TPase protein, the number of TE sequences present, and the localization of the TPase proteins within the cell. Several studies suggest that transposition complex formation is regulated by access to the nucleus, as alteration of nuclear localization signals (NLS) and nuclear export signals (NES) influences transposition (Hancock et al. 2010; Payero et al. 2016; Ramakrishnan et al. 2019). Zebrafish studies often involve the integration of DNA sequences (i.e. overexpression cassettes) into embryos, leading to transient or germ line expression (Nusslein-Volhard and Dahm 2002). Inserting transgenes between TE sequences and providing a TPase protein source has been shown to increase transgenesis efficiency across multiple model organisms (Ding et al. 2005; Ivics and Izsvák 2004; Munoz-Lopez and Garcia-Perez 2010; Zayed et al. 2004). The Tol2 TE from Medaka fish (Oryzias latipes) has been developed as a means to improve zebrafish transgenesis, including both transient and germline integration (Kawakami 2007; Koga et al. 1996; Kwan et al. 2007; Ni et al. 2016). Tol2-mediated transgenesis is induced by co-injecting Tol2 TPase mRNA together with a Tol2 terminal sequence-flanked construct into 1-4 cell stage zebrafish embryos (Kawakami 2007; Kwan et al. 2007; Ni et al. 2016). Previous reports indicated that addition of the Tol2 TPase mRNA (expressed from pCS2FA) produced about a 3fold increase in Tol2 flanked transgene expression compared to control (Kwan et al. 2007). Another study demonstrated 6/11/2020 Open Access that a zebrafish codon optimized version of Tol2 TPase led to successful germline transmission, but no direct comparison of efficiency was reported (Suster et al. 2011). The goal of this study was to explore the extent that efficiency of Tol2mediated transgene expression could be improved by codon-optimizing the Tol2 TPase gene for zebrafish or altering a detected NES and NLS in the Tol2 TPase (Figure 1A). We hypothesized that increasing the translation efficiency (Gustafsson et al. 2004) and access to the nucleus would potentially improve transposition complex formation in transience. Analysis of the Tol2 TPase codon usage showed that it contained 19 codons (TTA, CTA, and TCG) that are rarely used by zebrafish (Nakamura et al. 2000). Codon optimization resulted in the development of a CO Tol2 TPase construct that more appropriately matched the zebrafish-specific codon bias, without altering the amino acid sequence (Zhou et al. 2016). In the CO Tol2 TPase N construct, the NES in the Tol2 TPase (Figure 1A) was removed by changing L641A and L643A and the existing NLS (PKRARLD, NLS score=8.5) was strengthened by changing it to PKKKRKV (NLS score=13) (Dingwall and Laskey 1991; Kosugi et al. 2009). Injection of 1-4 cell-stage zebrafish embryos with pDestTol2pACryGFP showed a significantly increased frequency of eGFP expression in the eye (i.e. Figure 1B) when co-injected with wild type Tol2 TPase mRNA compared to no mRNA (Figure 1C, p=0.0024). This is below the reported 3-fold increased frequency of expression previously reported using noncodon optimized Tol2 (Kwan et al. 2007). We observed a significantly increased frequency of eye eGFP expression when we used the codon optimized (CO Tol2 TPase) mRNA (Figure 1C, vs. no mRNA p=<0.0001, vs. WT TPase p=0.0008). These results suggest that the codon optimized version of the mRNA is translated more effectively in embryos, leading to an overall higher concentration of Tol2 TPase protein. In contrast, the NES and NLS altered version (CO Tol2 TPase N) showed decreased expression frequency compared to all other groups (vs. no mRNA p=0.0013; vs. WT Tol2 TPase p<0.0001; vs. CO Tol2 TPase p<0.0001). This suggests that the changes made to the NES and NLS may have disrupted overall protein function of the CO Tol2 TPase N protein (Figure 1C). When both the expression frequencies and survival rates were used to calculate overall transgene expression efficiency, we observed that all treatments resulted in about 20% of the injected eggs producing eGFP-expressing embryos surviving to 2 dpf (Figure 1C). An inverse relationship was observed between the survival frequency and the frequency of eGFP expression at 2 dpf. We observed that significantly less fish developed properly with the higher activity CO Tol2 TPase mRNA compared to no mRNA (p = 0.0002) and the WT Tol2 TPase mRNA (p=0.0033) (Figure 1C). This suggests that genome disruption potentially caused by transgene insertion may be an important limit on transgenesis rates. The high frequency of eGFP expression observed in this study for the no mRNA control suggests that a high percentage of fluorescence observed was not TPase mediated. In addition, our results suggest that the addition of Tol2 mRNA does not drastically increase the overall rate of transient expression of transgenes (total number of surviving transgenic fish/ embryos injected). However, there are significant benefits to the Tol2 system, as a greater fraction of fish that survive show transient transgene expression, potentially leading to easier screening for expressing embryos. Our results suggest that co-injecting constructs with the CO Tol2 TPase mRNA (available through Addgene #133032) is a significant improvement over the existing technology. This codon optimized Tol2 has the potential to increase the yield of transient expression and should be tested for its ability to induce heritable transgenesis. Although this study did not examine heritable transgenesis, the next step in this investigation would be to examine the rates of integration of the pDestTol2pACryGFP transgene in fish injected with each mRNA. Methods Request a detailed protocol