显微注射转基因的基因组，并在编辑Threespine刺鱼
Summary
转基因操作和基因组编辑是功能测试基因和顺 -regulatory元素的作用是至关重要的。此处的基因组修饰（包括TOL2介导的荧光报道基因构建体，TALENS和CRISPRs）的生成的详细显微注射协议提出了紧急模型鱼的三刺。
Abstract
该三刺鱼已成为一个强大的系统来研究各种各样的形态，生理的遗传基础和行为的表型。海洋种群适应无数淡水环境已经放出的非常多样化的表型，用交叉的海洋和淡水的形式的能力相结合，提供了难得脊椎动物系统，其中遗传学可用于映射的基因组区域控制演变特征。优秀的基因资源，现已有售，促进发展变化的分子遗传学解剖。而映射实验产生性的候选基因列表，官能基因操作都必须测试这些基因的作用。基因调控可以集成到使用TOL2转座系统的转基因报告质粒和BAC进行研究。具体的候选基因与顺 -regulatory元素功能可以通过诱导目标进行评估突变的TALEN和CRISPR / Cas9基因组编辑试剂。所有的方法都需要将核酸引入受精单细胞刺胚胎中，任务由刺胚胎的厚绒毛膜和相对小而薄的卵裂球制成具有挑战性。这里，一个详细的协议用于核酸显微注射到刺胚胎为转基因和基因组编辑应用程序来研究基因表达和功能，以及技术来评估转基因的成功和恢复稳定线说明。
Introduction
了解生物多样性是如何产生是确定的在自然界进化表型变化的遗传和发育基础的一个基本组成部分。该三刺鱼，Gasterosteus叶树 ，先后涌现作为研究进化的遗传基础的优秀典范。刺鱼已经发生了许多适应性进化改变海鱼都围绕殖民地北半球无数的淡水环境，造成巨大的形态，生理和行为的变化1。个人从21刺种群的基因组已经被测序，组装，和高密度图谱已经产生，进一步提高了装配2,3。遗传作图实验已经确定的基因组区域4底层演变表型– 6，和在一些情况下，具体的候选基因的功能角色已经测试-7,8-。一些潜在的形态改变的基因组区域的已确定与希望的候选基因，但这些候选人尚未功能测试9 – 12。此外，刺鱼是群体遗传学/基因组13,14，形态15，行为1，内分泌学16，生态毒理学17，免疫18和19寄生虫研究常见型号。在这些领域未来的研究将受益于在刺鱼进行功能基因操作的能力。除了 操纵他们的编码序列，候选基因的作用可以通过研究其顺 -regulatory序列和由在功能上增加，减少或消除了候选基因的表达进行评估。在刺鱼显微注射和转基因方法已经非常成熟7,8,20和最初开发利用大范围核酸酶介导方法21在青鳉22首先说明。这里提出的修改显微注射法已为TOL2介导的转基因进行了优化和最近开发的基因组编辑试剂，包括TALENS和CRISPRs。
到顺 -regulatory元件变化被认为是对形态演变关键，因为顺 -regulatory变化能够避免编码突变23的负多效性的后果。因此，检测和比较的顺 -regulatory序列已经成为越来越多的进化研究的中心目标。此外，大多数人疾病变体是调控变体24,25，和模型脊椎动物系统急需研究顺调控元件的功能和逻辑。鱼在外部受精及其胚胎大量提供强大的脊椎动物系统研究顺 -regulation。该TOL2座子系统，其中forei被整合在基因组中GN DNA通过TOL2转座结合位点和侧翼共注射与TOL2转座的mRNA，以高效率工作为成功整合质粒构建成鱼的基因组26 – 28。通常情况下，一个潜在增强剂克隆一个基本启动子（如hsp70l 29）和荧光报告基因，如绿色荧光蛋白（增强型绿色荧光蛋白）或mCherry在TOL2骨干的上游，并与转座基因26注入。的荧光报道的，无论是在注射的胚胎或具有稳定整合的转基因后代表达的观察，提供了关于由推定增强剂驱动的基因表达的时空管制信息。在进一步的实验中，验证了增强剂可以用来驱动目的基因的组织特异性表达。
对于较大的顺 -regulatory地区分析，高品质的大插入GENOM用细菌人工染色体（BAC）IC库已建成为海洋和淡水棘鱼30。这些BAC的可recombineered在一个大的（150-200 kb的）的环境中的荧光报道基因的基因组区31，以取代一个基因。的荧光报道然后在时空图案表示为通过BAC内调节序列来确定。在鱼的研究中，TOL2站点可以加入到BAC，促进基因组整合32,33。在发育的后期阶段，当在原位杂交在技术上是挑战性的，所述的BAC的荧光读出可用于研究基因表达的模式，如已经示出了刺骨形态发生蛋白6（BMP6）20。此外，在一个单独的荧光表达模式可随着时间的推移，它不能与原位杂交来实现跟踪。 BAC的还可以用来增加一个additiona一个基因组区l的副本，以增加的感兴趣的基因的剂量。
基因功能的研究，基因组编辑是一种爆发扩展字段可用于生产各种各样的生物体34的基因组序列的有针对性的变化。转录活化剂-样效应核酸（TALENS）是最初从能够精确设计以直接结合到所选择的基因组序列，并产生双链断裂35,36植物病原体分离模块化，序列特异性核酸酶。群集定期相互间隔短回文重复序列（CRISPR）/ CAS系统中的细菌最初发现并使用一个导的RNA和Cas9蛋白以生成到导向37的互补靶DNA序列中断。由两个TALENS和CRISPRs创建往往双链断裂的后续修复留下一个小的插入或缺失，其可以破坏靶序列的功能35-37。在刺鱼，TALENS已被用于针对增强20破坏基因的表达，TALENS和CRISPRs无论是在编码序列（未发表资料）已成功生产突变。对于CRISPRs在斑马鱼使用的生成详细的协议可以作为一个准则制定刺鱼38 CRISPRs。
转基因和基因组编辑实验需要引入核酸到一个新受精的单细胞胚胎。通过引入在发育早期的转基因或基因组编辑工具，在胚胎基因操纵子细胞的数目被最大化。注射胚胎，然后目测筛选荧光或分子筛选基因组修改。如果有助于生殖系细胞成功靶向，转基因或突变可以传递给后代的一个子集，即使当后喷射杀伤力是高的。马赛克鱼可还是远交互交和他们的后代中筛选到恢复突变等位基因或感兴趣的稳定整合的转基因。本协议描述引入转基因和基因组编辑试剂成单细胞胚胎刺和监测成功的基因修改方法。
Protocol
Representative Results
Discussion
注射单细胞胚胎刺为转基因或基因组编辑提出了三大挑战。首先，相对于斑马鱼胚胎中，胚胎刺蛋壳坚韧往往会打破针。这个问题可以通过使用较厚的和更强的玻璃微并垂直于绒毛膜注入部分地克服（参见协议， 图2）。确保尽可能少水尽可能加入到胚胎（刚好足以引起绒毛膜膨胀并解除从细胞以外）有助于减少绒毛膜硬度。绒毛膜变硬随着时间的推移，所以润燥胚胎后迅速的工…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院R01＃DE021475（CTM），美国国立卫生研究院博士前培训资助5T32GM007127（PAE）和美国国家科学基金会研究生研究奖学金（NAE）的部分资助。我们感谢凯文施瓦尔巴赫用于产生CRISPR Sanger测序数据进行BAC重组工程和注射，尼克东德和凯瑟琳利帕里关于注射方案有帮助的反馈。
Materials
| Stereomicroscope with transillumination | Leica | S6e/ KL300 LED | |
| Manual micromanipulator | Applied Scientific Instrumentation | MM33 | Marzhauser M33 Micromanipulator |
| Pressure Injecion system | Applied Scientific Instrumentation | MPPI-3 | |
| Back pressure unit | Applied Scientific Instrumentation | BPU | |
| Micropipette holder kit | Applied Scientific Instrumentation | MPIP | |
| Magnetic base holder | Applied Scientific Instrumentation | Magnetic base | |
| Foot switch | Applied Scientific Instrumentation | FSW | |
| Iron plate (magnetic base) | Narishige | IP | |
| Flaming/Brown Micropipette Puller | Sutter Instrument | P-97 | |
| Disposable transfer pipettes | Fisher | 13-711-7M | |
| 0.5% phenol red in DPBS | Sigma | P0290 | injection tracer |
| #5 forceps, biologie dumoxel | Fine Science Tools | 11252-30 | for needle breaking |
| Micropipette Storage Jar | World Precision Instruments | E210 | holds needles |
| 6", 6 teeth per inch plaster drywall saw | Lenox | 20571 (S636RP) | hold eggs for injection |
| 13 cm x 13 cm glass plate | any hardware store | – | |
| Borosilicate glass capillaries, 1.0 mm OD/0.58 mm ID | World Precision Instruments | 1B100-F4 | *harder glass than zebrafish injection capillaries |
| 150 x 15mm petri dish | Fisher | FB0875714 | raise stickleback embryos |
| 35 x 10mm petri dish | Fisher | 08-757-100A | store eggs pre-injection |
| Instant Ocean Salt | Instant Ocean | SS15-10 | |
| Sodium Bicarbonate | Sigma | S5761-500G | |
| Tricaine methanesulfonate/MS-222 | Western Chemical Inc | MS222 | fish anaesthesia/euthanasia |
| Sp6 transcription kit | Ambion | AM1340 | For transcription of TALENs and transposase mRNA |
| RNeasy cleanup kit | Qiagen | 74104 | purify transposase or TALEN RNA |
| QiaQuick PCR cleanup kit | Qiagen | 28104 | clean up plasmids for injection |
| Proteinase K 20 mg/ml | Ambion | AM2546 | for DNA preparation |
| Nucleobond BAC 100 kit | Clontech | 740579 | for BAC DNA preparation |
| NotI | NEB | R0189L | |
| Phusion polymerase | Fisher | F-530L | |
| Qiagen PlasmidPlus Midi kit | Qiagen | 12943 | contains endotoxin rinse buffer |
| QIAQuick Gel Extraction | Qiagen | 28704 | for sequencing induced mutations |
| Phenol:chloroform:Isoamyl alcohol | Sigma | P2069-100ML | |
| Sodium acetate | Sigma | S2889-250G | |
| Ethanol (molecular biology grade) | Sigma | E7023-500ML | |
| Agarose | Sigma | A9539 | |
| 50X Tris-acetate-EDTA buffer | ThermoFisher | B49 | |
| 0.5-10KbRNA ladder | ThermoFisher | 15623-200 | |
| Nanodrop Spectrophotometer | Thermo Scientific | Nanodrop 2000 | |
| Paraformaldehyde | Sigma | 158127-500G | |
| 10X PBS | ThermoFisher | 70011-044 | |
| 1kb Plus DNA Ladder | ThermoFisher | 10787-018 | |
| Potassium Chloride | Sigma | P9541-500G | |
| Magnesium Chloride | Sigma | M8266-100G | |
| NP-40 | ThermoFisher | 28324 | |
| Tween 20 | Sigma | P1379-500ML | |
| Tris pH 8.3 | Teknova | T1083 | |
| 12-strip PCR tube | Thermo Scientific | AB-1113 |
Referenzen
- Bell, M. A., Foster, S. A. . The Evolutionary Biology of the Threespine Stickleback. , (1994).
- Jones, F. C., et al. The genomic basis of adaptive evolution in threespine sticklebacks. Nature. 484 (7392), 55-61 (2012).
- Glazer, A. M., Killingbeck, E. E., Mitros, T., Rokhsar, D. S., Miller, C. T. Genome assembly improvement and mapping convergently evolved skeletal traits in sticklebacks with Genotyping-by-Sequencing. G3. 5 (7), 1463-1472 (2015).
- Miller, C. T., et al. Modular skeletal evolution in sticklebacks is controlled by additive and clustered quantitative trait Loci. Genetik. 197 (1), 405-420 (2014).
- Shapiro, M. D., et al. Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks. Nature. 428 (6984), 717-723 (2004).
- Colosimo, P. F., et al. Widespread parallel evolution in sticklebacks by repeated fixation of Ectodysplasin alleles. Science. 307 (5717), 1928-1933 (2005).
- Chan, Y. F., et al. Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer. Science. 327 (5963), 302-305 (2010).
- O’Brown, N. M., Summers, B. R., Jones, F. C., Brady, S. D., Kingsley, D. M. A recurrent regulatory change underlying altered expression and Wnt response of the stickleback armor plates gene EDA. eLife. , e05290 (2015).
- Cleves, P. A., et al. Evolved tooth gain in sticklebacks is associated with a cis-regulatory allele of Bmp6. Proc. Natl. Acad. Sci. 111 (38), 13912-13917 (2014).
- Erickson, P. A., Glazer, A. M., Cleves, P. A., Smith, A. S., Miller, C. T. Two developmentally temporal quantitative trait loci underlie convergent evolution of increased branchial bone length in sticklebacks. Proc. R. Soc. Lond. B Biol. Sci. 281 (1788), 20140822 (2014).
- Glazer, A. M., Cleves, P. A., Erickson, P. A., Lam, A. Y., Miller, C. T. Parallel developmental genetic features underlie stickleback gill raker evolution. EvoDevo. 5 (1), (2014).
- Ellis, N. A., et al. Distinct developmental and genetic mechanisms underlie convergently evolved tooth gain in sticklebacks. Development. 142, 2442-2451 (2015).
- Hohenlohe, P. A., Bassham, S., Currey, M., Cresko, W. A. Extensive linkage disequilibrium and parallel adaptive divergence across threespine stickleback genomes. Philos. Trans. R. Soc. Lond. B Biol. Sci. 367 (1587), 395-408 (2012).
- Hohenlohe, P. A., et al. Population genomics of parallel adaptation in threespine stickleback using sequenced RAD tags. PLoS Genet. 6 (2), e1000862 (2010).
- McKinnon, J. S., Rundle, H. D. Speciation in nature: the threespine stickleback model systems. Trends Ecol. Evol. 17 (10), 480-488 (2002).
- Shao, Y. T., et al. Androgen feedback effects on LH and FSH, and photoperiodic control of reproduction in male three-spined sticklebacks, Gasterosteus aculeatus. Gen. Comp. Endocrinol. 182, 16-23 (2013).
- Katsiadaki, I., et al. Three-spined stickleback: an emerging model in environmental endocrine disruption. Environ. Sci. Int. J. Environ. Physiol. Toxicol. 14 (5), 263-283 (2007).
- Lenz, T. L., Eizaguirre, C., Rotter, B., Kalbe, M., Milinski, M. Exploring local immunological adaptation of two stickleback ecotypes by experimental infection and transcriptome-wide digital gene expression analysis. Mol. Ecol. 22 (3), 774-786 (2013).
- Barber, I. Sticklebacks as model hosts in ecological and evolutionary parasitology. Trends Parasitol. 29 (11), 556-566 (2013).
- Erickson, P. A., et al. A 190 base pair, TGF-β responsive tooth and fin enhancer is required for stickleback Bmp6 expression. Dev. Biol. 401 (2), 310-323 (2015).
- Hosemann, K. E., Colosimo, P. F., Summers, B. R., Kingsley, D. M. A simple and efficient microinjection protocol for making transgenic sticklebacks. Behaviour. 141 (11/12), 1345-1355 (2004).
- Thermes, V., et al. I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118 (1-2), 91-98 (2002).
- Carroll, S. B. Evo-Devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell. 134 (1), 25-36 (2008).
- Maurano, M. T., et al. Systematic localization of common disease-associated variation in regulatory DNA. Science. 337 (6099), 1190-1195 (2012).
- Hindorff, L. A., et al. Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc. Natl. Acad. Sci. U. S. A. 106 (23), 9362-9367 (2009).
- Fisher, S., et al. Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nat. Protoc. 1 (3), 1297-1305 (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. Proc. Natl. Acad. Sci. U.S.A. 97 (21), 11403-11408 (2000).
- Kikuta, H., Kawakami, K. Transient and stable transgenesis using Tol2 transposon vectors. Methods in Moleculario Biology: Zebrafish. , 69-84 (2009).
- Halloran, M. C., et al. Laser-induced gene expression in specific cells of transgenic zebrafish. Dev. Camb. Engl. 127 (9), 1953-1960 (2000).
- Kingsley, D. M., et al. New genomic tools for molecular studies of evolutionary change in threespine sticklebacks. Behaviour. 141 (11/12), 1331-1344 (2004).
- Mortlock, D. P., Guenther, C., Kingsley, D. M. A general approach for identifying distant regulatory elements applied to the Gdf6 gene. Genome Res. 13 (9), 2069-2081 (2003).
- Suster, M. L., Abe, G., Schouw, A., Kawakami, K. Transposon-mediated BAC transgenesis in zebrafish. Nat. Protoc. 6 (12), 1998-2021 (2011).
- Suster, M. L., Sumiyama, K., Kawakami, K. Transposon-mediated BAC transgenesis in zebrafish and mice. BMC Genomics. 10 (1), 477 (2009).
- Kim, H., Kim, J. S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15 (5), 321-334 (2014).
- Cermak, T., et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39 (17), (2011).
- Miller, J. C., et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29 (2), 143-148 (2011).
- Jinek, M., et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337 (6096), 816-821 (2012).
- Talbot, J. C., Amacher, S. L. A streamlined CRISPR pipeline to reliably generate zebrafish frameshifting alleles. Zebrafish. 11 (6), 583-585 (2014).
- Kawakami, K., et al. A Transposon-Mediated Gene Trap Approach Identifies Developmentally Regulated Genes in Zebrafish. Dev. Cell. 7 (1), 133-144 (2004).
- Green, M. R., Sambrook, J. . Molecular cloning a laboratory manual. , (2012).
- Villefranc, J. A., Amigo, J., Lawson, N. D. Gateway compatible vectors for analysis of gene function in the zebrafish. Dev. Dyn. 236 (11), 3077-3087 (2007).
- Doyle, E. L., et al. TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res. 40 (W1), W117-W122 (2012).
- Untergasser, A., et al. Primer3-new capabilities and interfaces. Nucleic Acids Res. 40 (15), e115 (2012).
- Westerfield, M. . The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio) . , (2007).
- Dale, R. M., Topczewski, J. Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a. Dev. Biol. 357 (2), 518-531 (2011).
- Ran, F. A., et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8 (11), 2281-2308 (2013).
- Qi, L. S., et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of Gene expression. Cell. 152 (5), 1173-1183 (2013).
