使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法
Summary
我们开发了一种新颖的功能丧失方法，该方法涉及通过使用 卵 电穿孔和Tol2转座子系统将人工微RNA序列引入和基因组整合到鸡胚胎中。该技术为发育过程中的基因功能研究提供了一种强大而稳定的基因敲低方法。
Abstract
长期以来，小鸡视网膜一直是发育神经生物学中重要的模型系统，其优点包括体积大、发育迅速以及可视化和实验操作的可访问性。然而，其主要技术局限性是缺乏用于基因功能分析的强有力的功能丧失方法。该协议描述了一种在发育中的鸡视网膜中进行基因沉默的方法，该方法涉及通过使用Tol2转座子系统进行人工microRNA（miRNA）的转基因表达。在这种方法中，将包含EmGFP（翡翠绿色荧光蛋白）标记物表达盒和针对靶基因的人工前miRNA序列的Tol2转座子质粒引入胚胎小鸡视网膜中，并通过 卵内 电穿孔构建Tol2转座酶表达。在转染的视网膜细胞中，转座酶催化从转座子载体中切除表达盒并将其整合到宿主染色体中，从而导致miRNA和EmGFP蛋白的稳定表达。在我们之前的研究中，我们已经证明，Nel（一种在神经发育中发挥多种功能的糖蛋白）的表达可以通过使用该技术在发育中的小鸡视网膜中显着抑制。我们的结果表明，这种方法诱导了对基因表达的稳定和强大的抑制，从而为视网膜发育的研究提供了一种有效的功能丧失方法。
Introduction
脊椎动物视网膜是研究神经发育的重要模型系统。尽管视网膜位于外围位置，但在解剖学和发育上是中枢神经系统的延伸，视神经由视网膜神经节细胞的轴突组成，代表中枢神经系统内的束。小鸡视网膜作为研究神经发育分子机制的模型系统具有显著优势：体型大，发育迅速;它与人类视网膜具有结构和功能相似性;它很容易用于可视化和实验操作。利用鸡视网膜广泛研究了神经发育过程中细胞增殖和分化、形态发生和轴突引导的分子机制。
在过去的二十年中，卵子电穿孔已成功用于将异位基因引入发育中的雏鸡胚胎中的细胞中。该技术允许标记发育中的细胞，细胞命运追踪，细胞迁移和轴突束的追踪，以及异位基因表达，用于基因功能的体内分析。卵内电穿孔在鸡胚胎中有效表达异位基因的条件已经确立1,2,3。
尽管有这些优点，但缺乏用于基因功能研究的稳定功能丧失技术一直是雏鸡胚胎的主要技术限制。虽然用小干扰RNA（siRNA）4或短发夹RNA（shRNA）5的表达载体电穿孔的鸡胚胎显示出靶基因的敲低，但这些方法中的基因抑制是短暂的，因为一旦细胞失去引入的RNA或DNA，这种影响就会消失。通过RCAS（R转换Competent A型肉瘤-白血病病毒（ASLV）长末端重复（LTR）与S板受体）逆转录病毒系统将siRNA递送到雏鸡胚胎中可以实现更稳定的基因抑制6。病毒载体整合到宿主基因组中，异位基因稳定表达。然而，逆转录病毒只能在细胞周期的有丝分裂（M）阶段整合到分裂细胞的基因组中，这可能会对可以应用这种功能丧失方法的发育阶段和/或细胞类型施加限制。此外，RCAS对转基因的表达似乎比卵电穿孔诱导的表达更慢且更不稳定7。
转座子是从基因组上的一个位置移动到另一个位置的遗传元件。Tol2元件是hAT转座元件家族的成员，包含一个编码转座酶的内部基因，该转座酶催化Tol2元件8的转座子反应。当携带基因表达盒的质粒载体被引入具有Tol2转座酶表达构建体的脊椎动物细胞中时，将携带基因表达盒的质粒载体，两侧是Tol2元件的左端和右端序列（分别为200 bp和150 bp），将表达盒从质粒中切除并整合到宿主基因组中，从而支持异位基因的稳定表达（图1).已经表明，Tol2转座元件可以非常有效地诱导不同脊椎动物物种的基因转位，包括斑马鱼9,10，青蛙11，小鸡12和小鼠13，因此是一种有用的转基因和插入诱变方法。Tol2转座子系统已成功用于通过从长双链RNA14加工的siRNA的基因组整合来条件敲低靶基因。
该协议描述了雏鸡胚胎中的功能丧失方法，该方法涉及通过Tol2转座子系统引入人工microRNA（miRNA）15,16。在这种方法中，将EmGFP（翡翠绿色荧光蛋白）标记物的表达盒和针对靶基因的人工miRNA克隆到Tol2转座子载体中。然后将Tol2转座子构建体通过卵电穿孔将Tol2转座酶表达构建体引入胚胎小鸡视网膜中。在转染的视网膜细胞中，转座酶催化从转座子载体中切除表达盒并将其整合到宿主染色体中，从而导致miRNA和EmGFP蛋白的稳定表达。在我们之前的研究中，我们成功地敲低了Nel的表达，Nel是一种主要在神经系统中表达的细胞外糖蛋白，在发育中的小鸡视网膜中（见代表性结果）。我们的结果表明，通过该技术可以在卵中实现稳定有效的基因抑制。
Protocol
Representative Results
Discussion
该协议提供了通过使用 卵 电穿孔和 Tol2 转座子系统中人工 miRNA 的转基因表达在发育中的鸡视网膜中基因沉默的详细指南。
以下因素对于成功执行此技术至关重要。首先，使用经证实具有强大敲低效果的miRNA序列至关重要。在将它们应用于 卵 电穿孔之前，在 体外 测定中测试单个前 miRNA 序列的基因抑制效率（步骤 1.4），并链上显示最高敲低活性的两个前…
Declarações
The authors have nothing to disclose.
Acknowledgements
pT2K-CAGGS和pCAGGS-T2TP载体分别由Yoshiko Takahashi（日本京都京都大学）和Koichi Kawakami（日本三岛国立遗传学研究所）提供。我们感谢Michael Berberoglu对手稿的重要阅读。这项工作得到了皇家学会和生物技术与生物科学研究委员会（BBSRC）（英国）对M.N.的资助。
Materials
| 18 G needle, 2" | VWR | 89219-320 | |
| AP-TAG kit A and AP-TAG kit B | GenHunter Corp | Q201 and Q202 | Plasmid vectors for making AP fusion proteins (https://www.genhunter.com/products/ap-tag-kit-a.html, https://www.genhunter.com/products/ap-tag-kit-b.html) |
| Block-iT RNAi Designer | Invitrogen | An online tool to choose target sequences and design pre-miRNA sequences (https://rnaidesigner.thermofisher.com/rnaiexpress/) | |
| BSA 10 mg | Sigma-Aldrich | A2153 | |
| C115CB cables | Sonidel | C115CB | https://www.sonidel.com/product_info.php?products_id¼254 |
| C117 cables | Sonidel | C117 | https://www.sonidel.com/product_info.php?products_id¼252 |
| Capillary tubes with omega dot fiber (Micropipette needles) | FHC | 30-30-1 | 1 mm O.D. 0.75 mm I.D |
| CUY21 square wave electroporator | Nepa Gene | CUY21 | |
| Diethanolamine (pH 9.8) | Sigma-Aldrich | D8885 | |
| Dissecting microscope | |||
| Egg incubator | Kurl | B-Lab-600-110 | https://www.flemingoutdoors.com/kuhl%2D%2D-600-egglaboratory-incubator%2D%2D-b-lab-600-110.html |
| Electrode holder | Sonidel | CUY580 | https://www.sonidel.com/product_info.php?products_id¼85 |
| Electrodes | Nepa Gene | CUY611P3-1 | https://www.sonidel.com/product_info.php?products_id¼94 |
| Electromax DH10B | Invitrogen | 18290-015 | Electrocompetent E. coli cells |
| Fast green FCF | Sigma-Aldrich | F7258 | |
| Fertilized chicken eggs (Gallus gallus) | Obtained from commercial vendors (e.g. Charles River) or local farmers | ||
| Gooseneck fiber light source | |||
| FuGene 6 transfection reagent | Promega | E2691 | |
| Hamilton syringe (50 μL) | Sigma-Aldrich | 20715 | Hamilton Cat No 80901 |
| Hanks' balanced salt solution | Sigma-Aldrich | H6648 | |
| Heavy mineral oil | Sigma-Aldrich | 330760 | |
| HEPES | GIBCO | 15630080 | |
| L-Homoarginine | Sigma-Aldrich | H10007 | |
| MgCl2 | Sigma-Aldrich | 13112 | |
| Micromanipulator | Narishige (Japan) | MM3 | http://products.narishige-group.com/group1/MM-3/electro/english.html |
| Micropipette puller | Shutter Instrument | P97 | |
| p-Nitrophenylphosphate | Sigma-Aldrich | 20-106 | |
| PBS | Sigma-Aldrich | D8662 | |
| pCAGGS-T2TP vector | Tol2 transposase expression plasmid. A generous kind gift of Koichi Kawakami (National Institute of Genetics, Japan). Also available from Addgene. | ||
| Pfu | ThermoFisher | F566S | |
| Picospritzer (Optional) | Parker | Pressure microinjection system | |
| Plasmid maxi kit | Qiagen | 12163 | Plasmid maxiprep kit |
| pT2K-CAGGS vector | Tol2 transposon vector. Kindly provided by Yoshiko Takahashi (Kyoto University, Japan) | ||
| PVC tubing | VWR (UK) | 228-3830 | |
| Spectinomycin | Sigma-Aldrich | S9007-5 | |
| T4 DNA ligase | Promega | M1801 | |
| The BLOCK-iT Pol II miR RNA expression kit with EmGFP | Invitrogen | K493600 | Contains the miRNA expression vector (pcDNA6.2-GW/EmGFP-miRNA), a control vector (pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid), accessory reagents, and instructions (https://www.thermofisher.com/order/catalog/product/K493600?SID.srch-hj-K4936-00) |
| Thermal cycler |
Referências
- Muramatsu, T., Mizutani, Y., Ohmori, Y., Okumura, J. Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo. Biochemical and Biophysical Research Communications. 230, 376-380 (1997).
- Funahashi, J., et al. Role of Pax-5 in the regulation of a mid-hindbrain organizer’s activity. Development, Growth & Differentiation. 41 (1), 59-72 (1999).
- Harada, H., Omi, M., Nakamura, H. In ovo electroporation methods in chick embryos. Methods in Molecular Biology. 1650, 167-176 (2017).
- Hu, W. Y., Myers, C. P., Kilzer, J. M., Pfaff, S. L., Bushman, F. D. Inhibition of retroviral pathogenesis by RNA interference. Current Biology. 12 (15), 1301-1311 (2002).
- Katahira, T., Nakamura, H. Gene silencing in chick embryos with a vector-based small interfering RNA system. Development, Growth & Differentiation. 45 (4), 361-367 (2003).
- Harpavat, S., Cepko, C. L. RCAS-RNAi: a loss-of-function method for the developing chick retina. BMC Developmental Biology. 6, 2 (2006).
- Nakamura, H., Funahashi, J. Introduction of DNA into chick embryos by in ovo electroporation. Methods. 24, 43-48 (2001).
- Koga, A., Iida, A., Hori, H., Shimada, A., Shima, A. Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces. Molecular Biology and Evolution. 23 (7), 1414-1419 (2006).
- Kawakami, K., Shima, A., Kawakami, N. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proceedings of the National Academy of Sciences of the United States of America. 97 (21), 11403-11408 (2000).
- Kawakami, K., et al. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Developmental Cell. 7 (1), 133-144 (2004).
- Kawakami, K., Imanaka, K., Itoh, M., Taira, M. Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis. Gene. 338 (1), 93-98 (2004).
- Sato, Y., et al. Stable integration and conditional expression of electroporated transgenes in chicken embryos. Biologia do Desenvolvimento. 2 (2), 616-624 (2007).
- Kawakami, K., Noda, T. Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells. Genética. 166 (2), 895-899 (2004).
- Hou, X., et al. Conditional knockdown of target gene expression by tetracycline regulated transcription of double strand RNA. Development, Growth & Differentiation. 53, 69-75 (2011).
- Nakamoto, C., et al. Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development. Molecular Biology of the Cell. 25 (2), 234-244 (2014).
- Nakamoto, M., Nakamoto, C., Mao, C. -. A. . iRetinal Development: Methods and Protocols. Vol. 2092 Methods in Molecular Biology. 8, 91-108 (2020).
- BLOCK-iT PolII miR RNAi Expression Vector Kits, User Manual Pol II miR RNAi Expression Vector Kits. Invitrogen Available from: https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets/LSG/manuals/blockit_miRNAexpressionvector_man.pdf&title=BLOCK-iT&trade (2021)
- Flanagan, J. G., et al. Alkaline phosphatase fusions of ligands or receptors as in situ probes for staining of cells, tissues, and embryos. Methods in Enzymology. 327, 19-35 (2000).
- Hamburger, V., Hamilton, H. I. A series of normal stages in the development of the chick embryo. Journal of Morphology. 88, 49-92 (1951).
- Matsuhashi, S., et al. New gene, nel, encoding a M(r) 93 K protein with EGF-like repeats is strongly expressed in neural tissues of early stage chick embryos. Developmental Dynamics. 203 (2), 212-222 (1995).
- Matsuhashi, S., et al. New gene, nel, encoding a Mr 91 K protein with EGF-like repeats is strongly expressed in neural tissues of early stage chick embryos. Developmental Dynamics. 207 (2), 233-234 (1996).
- Jiang, Y., et al. In vitro guidance of retinal axons by a tectal lamina-specific glycoprotein Nel. Molecular and Cellular Neuroscience. 41 (2), 113-119 (2009).
- Nakamura, R., Nakamoto, C., Obama, H., Durward, E., Nakamoto, M. Structure-function analysis of Nel, a Thrombospondin-1-like glycoprotein involved in neural development and functions. Journal of Biological Chemistry. 287 (5), 3282-3291 (2012).
- Nakamoto, C., Durward, E., Horie, M., Nakamoto, M. Nell2 regulates the contralateral-versus-ipsilateral visual projection as a domain-specific positional cue. Development. 146 (4), (2019).
- Yee, J. K., et al. Gene expression from transcriptionally disabled retroviral vectors. Proceedings of the National Academy of Sciences of the United States of America. 84 (15), 5197-5201 (1987).
