The research presented in this article was supported by a grant from the National Institutes of Health/National Institute on Alcohol Abuse (NIH/NIAAA) R00AA023560 to C.B.L.

All zebrafish embryos used in this procedure were raised and bred following established IACUC protocols12. These protocols were approved by the University of Louisville.
NOTE: The wild-type zebrafish strain, AB, and the bmp4st72;smad5b1100 double mutant line were used in this study. All the water used in this procedure was sterile reverse osmosis water. Confocal images were taken under a laser-scanning confocal microscope. The endoderm mea...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+alcohol+spectrum+disorders:+Characteristics,+complications,+and+treatment.">Fetal alcohol spectrum disorders: Characteristics, complications, and treatment.</a> Community Mental Health Journal. 53, 711-718 (2017).</li><li>Wozniak, J. R., Riley, E. P., Charness, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+presentation,+diagnosis,+and+management+of+fetal+alcohol+spectrum+disorder.">Clinical presentation, diagnosis, and management of fetal alcohol spectrum disorder.</a> The Lancet Neurology. 18 (8), 760-770 (2019).</li><li>Patten, A. R., Fontaine, C. J., Christie, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+the+different+animal+models+of+fetal+alcohol+spectrum+disorders+and+their+use+in+studying+complex+behaviors.">A Comparison of the different animal models of fetal alcohol spectrum disorders and their use in studying complex behaviors.</a> Frontiers in Pediatrics. 2, 93 (2014).</li><li>Lovely, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+gene-alcohol+interactions.">Animal models of gene-alcohol interactions.</a> Birth Defects Research. 112 (4), 367-379 (2020).</li><li>Fernandes, Y., Lovely, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+of+fetal+alcohol+spectrum+disorders.">Zebrafish models of fetal alcohol spectrum disorders.</a> Genesis. 59 (11), 23460 (2021).</li><li>Fernandes, Y., Buckley, D. M., Eberhart, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diving+into+the+world+of+alcohol+teratogenesis:+a+review+of+zebrafish+models+of+fetal+alcohol+spectrum+disorder.">Diving into the world of alcohol teratogenesis: a review of zebrafish models of fetal alcohol spectrum disorder.</a> Biochemistry and Cell Biology. 96 (2), 88-97 (2018).</li><li>Choe, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+fluorescent+zebrafish+lines+that+have+revolutionized+biomedical+research.">Transgenic fluorescent zebrafish lines that have revolutionized biomedical research.</a> Lab Animal Research. 37 (1), 26 (2021).</li><li>Kwan, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+A+multisite+gateway-based+construction+kit+forTol2+transposon+transgenesis+constructs.">The Tol2kit: A multisite gateway-based construction kit forTol2 transposon transgenesis constructs.</a> Developmental Dynamics. 236 (11), 3088-3099 (2007).</li><li>Don, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tol2+gateway-compatible+toolbox+for+the+study+of+the+nervous+system+and+neurodegenerative+disease.">A Tol2 gateway-compatible toolbox for the study of the nervous system and neurodegenerative disease.</a> Zebrafish. 14 (1), 69-72 (2017).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio). , (2000).</li><li>Protocols. UMass Chan Medical School Available from: <a target="_blank" href="https://www.umassmed.edu/lawson-lab/reagents/lawson-lab-protocols">https://www.umassmed.edu/lawson-lab/reagents/lawson-lab-protocols</a> (2017)</li><li>Mosimann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisite+gateway+calculations:+Excel+spreadsheet.">Multisite gateway calculations: Excel spreadsheet.</a> protocols.io. , (2022).</li><li>Chung, W. -. S., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-endodermal+interactions+are+required+for+pancreatic+&#946;+cell+induction.">Intra-endodermal interactions are required for pancreatic &#946; cell induction.</a> Developmental Cell. 14 (4), 582-593 (2008).</li><li>Grevellec, A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharyngeal+pouches+and+clefts:+Development,+evolution,+structure+and+derivatives.">The pharyngeal pouches and clefts: Development, evolution, structure and derivatives.</a> Seminars in Cell &amp; Developmental Biology. 21 (3), 325-332 (2010).</li><li>Lovely, C. B., Swartz, M. E., McCarthy, N., Norrie, J. L., Eberhart, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp+signaling+mediates+endoderm+pouch+morphogenesis+by+regulating+Fgf+signaling+in+zebrafish.">Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish.</a> Development. 143 (11), 2000-2011 (2016).</li><li>Silva Brito, R., Canedo, A., Farias, D., Rocha, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+zebrafish+(Danio+rerio)+as+an+emerging+model+system+in+ecotoxicology+and+toxicology:+Historical+review,+recent+advances,+and+trends.">Transgenic zebrafish (Danio rerio) as an emerging model system in ecotoxicology and toxicology: Historical review, recent advances, and trends.</a> Science of The Total Environment. 848, 157665 (2022).</li><li>Lai, K. P., Gong, Z., Tse, W. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+the+toxicant+screening+model:+Transgenic+and+omics+approaches.">Zebrafish as the toxicant screening model: Transgenic and omics approaches.</a> Aquatic Toxicology. 234, 105813 (2021).</li><li>Stuart, G. W., McMurray, J. V., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+lines+of+transgenic+zebrafish+exhibit+reproducible+patterns+of+transgene+expression.">Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression.</a> Development. 109 (3), 577-584 (1988).</li><li>Stuart, G. W., McMurray, J. V., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication,+integration+and+stable+germ-line+transmission+of+foreign+sequences+injected+into+early+zebrafish+embryos.">Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos.</a> Development. 103 (2), 403-412 (1990).</li><li>Thermes, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish.">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mechanisms of Development. 118 (1-2), 91-98 (2002).</li><li>Kawakami, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transposon-mediated+gene+trap+approach+identifies+developmentally+regulated+genes+in+zebrafish.">A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish.</a> Developmental Cell. 7 (1), 133-144 (2004).</li><li>Kawakami, K., Asakawa, K., Muto, A., Wada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tol2-mediated+transgenesis,+gene+trapping,+enhancer+trapping,+and+Gal4-UAS+system.">Tol2-mediated transgenesis, gene trapping, enhancer trapping, and Gal4-UAS system.</a> Methods in Cell Biology. 135, 19-37 (2016).</li></ol>

Zebrafish are ideally suited for studying the impact of ethanol exposure on development and disease states7,8. Zebrafish produce large numbers of translucent, externally fertilized, genetically tractable embryos, which allows for the live imaging of several transgene-labeled tissues and cell types simultaneously in multiple environmental contexts19,20. These strengths, combined with the strong development...

Prenatal ethanol exposure gives rise to a continuum of structural deficits and cognitive impairments termed fetal alcohol spectrum disorders (FASD)1,2,3,4. The complex relationships between multiple factors make studying and understanding the etiology of FASD in humans challenging. To resolve this challenge, a wide variety of animal models have been used. The biological and experimental tools available in these models have proven crucial in developing our understanding of the mechanistic basis of ethanol teratogenicity, and the results from these model systems have been remarkably consistent with what is found in human ethanol studies5,6. Among these, zebrafish have emerged as a powerful model to study ethanol teratogenesis7,8, in part due to their external fertilization, high fecundity, genetic tractability, and translucent embryos. These strengths combine to make zebrafish ideal for real-time live imaging studies of FASD using transgenic zebrafish lines.
Transgenic zebrafish have been extensively used to study multiple aspects of embryonic development9. However, creating transgenic constructs and subsequent transgenic lines can be exceedingly difficult. A standard transgene requires an active promoter element to drive the transgene and a poly A signal or &#34;tail&#34;, all in a stable bacterial vector for general vector maintenance. The traditional generation of a multi-component transgenic construct requires multiple time-consuming sub-cloning steps10. PCR-based approaches, such as Gibson assembly, can circumvent some of the issues associated with sub-cloning. However, unique primers must be designed and tested for the generation of every unique transgenic construct10. Beyond transgene construction, genomic integration, germline transmission, and screening for proper transgene integration have been difficult as well. Here, we describe a protocol for using the transposon-based Tol2 transgenesis system (Tol2Kit)10,11. This modular system uses multisite Gateway cloning to quickly generate multiple transgenic constructs from an ever-expanding library of &#34;entry&#34; and &#34;destination&#34; vectors. Integrated Tol2 transposable elements greatly increase the rate of transgenesis, allowing for the rapid construction and genomic integration of multiple transgenes. Using this system, we show how the generation of an endoderm transgenic zebrafish line can be used to study the tissue-specific structural defects underlying FASD. Ultimately, in this protocol, we show that the modular setup and the construction of transgenic constructs will greatly aid zebrafish-based FASD research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Addgene Tol2 toolbox</td>
			<td>https://www.addgene.org/kits/cole-tol2-neuro-toolbox/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Air</td>
			<td></td>
			<td></td>
			<td>Provided directly by the university</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>VWR</td>
			<td>10204-962</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosil 1.0 mm OD x 0.75 mm ID Capillary</td>
			<td>FHC</td>
			<td>30-30-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>97062-590</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>BioVision</td>
			<td>2486</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP118-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Olympus</td>
			<td>Fluoview FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lawsone Lab Donor Plasmid Prep</td>
			<td>https://www.umassmed.edu/lawson-lab/reagents/lawson-lab-protocols/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher Scientific</td>
			<td>BP9724</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-EEO/Multi-Purpose/Molecular Biology Grade Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LR Clonase II Plus Enzyme</td>
			<td>Fisher Scientific</td>
			<td>12538200</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (Heptahydrate)</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Pipette holder</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MIMPH-M-PIP</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 0.5 mL&#160;</td>
			<td>VWR</td>
			<td>10025-724</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL&#160;</td>
			<td>VWR</td>
			<td>10025-716</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 10 &#956;L&#160;</td>
			<td>Fisher Scientific</td>
			<td>13611106</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 1000 &#956;L&#160;</td>
			<td>Fisher Scientific</td>
			<td>13611127</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 200 &#956;L&#160;</td>
			<td>Fisher Scientific</td>
			<td>13611112</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Fisher Scientific</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Mosimann Lab Tol2 Calculation Worksheet</td>
			<td>https://www.protocols.io/view/multisite-gateway-calculations-excel-spreadsheet-8epv599p4g1b/v1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>NanoDrop</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>NcoI</td>
			<td>NEB</td>
			<td>R0189S</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>NEB</td>
			<td>R0189S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 100 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>FB012924</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>P4758-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p1000L Micropipette</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p200L Micropipette</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p2L Micropipette</td>
			<td>Gilson</td>
			<td>FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate (Dibasic)</td>
			<td>VWR</td>
			<td>BDH9266-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Injector</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>VWR</td>
			<td>BDH9280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Dibasic)</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup .22 &#181;m vacuum filtration system&#160;</td>
			<td>Millipore</td>
			<td>SCGPU11RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Tol2 Wiki Page</td>
			<td>http://tol2kit.genetics.utah.edu/index.php/Main_Page</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Top10 Chemically Competent E. coli</td>
			<td>Fisher Scientific</td>
			<td>C404010</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Pipetter Puller</td>
			<td>David Kopf Instruments</td>
			<td>720</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebrafish microinjection mold</td>
			<td>Adaptive Science Tools</td>
			<td>i34</td>
			<td></td>
		</tr>
	</tbody>
</table>

the zebrafish tol2 system: a modular and flexible gateway-based transgenesis approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

To generate the transgenic constructs, we used the Tol2 transgenesis system. Three entry vectors, including p5E, which holds the gene promoter/enhancer elements, pME, which holds the gene to be expressed by the promoter/enhancer elements, and p3E which, at minimum, holds the polyA tail, were used to generate the transgenic construct via multisite gateway LR cloning. The destination vector, pDest, provides the Tol2 repeats for the genomic insertion of the transgenic construct in zebrafish embryos and contains all...

Watch this Scientific Journal Video about The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach at JoVE.com

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

Transgenic zebrafish have proven critical to our understanding of embryo development. The Tol2 system is a versatile tool that enables researchers to quickly and systematically generate multiple transgenic zebrafish for FASD studies. The modular design and ease of generating new kit components make the Tol2 system a versatile tool for generating new transgenics without having to redesign any components of the kit.My advice to researchers performing this technique for the first time is to make several trial runs to train on the precision of generating and injecting transgenic constructs. To begin, linearize the Tol2 kit plasmid containing transposase with a nought one restriction endonuclease by combining 10 microliters of transposase plasmid, 1.5 microliters of restriction endonuclease reaction buffer, and 0.3 microliters of nought one endonuclease in a 500 microliter microcentrifuge tube. Fill the reaction up to 20 microliters with RNAse-free water.Mix and digest at 37 degrees Celsius overnight. The next day, stop the digestion by adding one microliter of 0.5 molar EDTA, two microliters of five molar ammonium acetate, and 80 microliters of 100%ethanol to the reaction mixture. Mix and chill at minus 20 degrees Celsius overnight.The following day, centrifuge and aspirate the supernatant before resuspending the DNA pellet in RNAse-free water. Then determine the linear plasmid concentration in nanograms per microliter on a fluorometer by running two microliters of the sample. Set up the SP6 mRNA production by combining 10 microliters of 2X NTP/CAP, two microliters of 10X reaction buffer, 0.1 to one microgram of linear DNA template in two microliters of SP6 enzyme mix in a 500 microliter tube.Fill the reaction up to 20 microliters with RNAse-free water and mix before incubating at 37 degrees Celsius. After two hours, add one microliter of DNAse, mix well, and incubate for an additional 15 minutes. Stop the reaction by adding 30 microliters of lithium chloride precipitation solution.Mix and chill at minus 20 degrees Celsius overnight. Next day, centrifuge the solution to pellet the mRNA and aspirate the supernatant before washing the pellet with one milliliter of 80%ethanol. Air dry the pellet and resuspend it in 20 microliters of RNAse-free water.Determine the mRNA concentration using a fluorometer, then aliquot 100 nanograms of mRNA per tube for single use and store at minus 80 degrees Celsius. To perform the reaction, gather 10 femtomole each of P5E, PME, and P3E and 20 femtomole of the destination vector PDEST. Identify the size of all four vectors and base pairs from the plasmid source.After determining the concentration of all four vectors, using the weight of DNA as 660 grams per mole and plasmid size and base pairs, calculate the total nanograms of plasmid needed to reach either 10 femtomole for entry vectors or 20 femtomole for destination vector. Next, generate the construct sox17:EGFP-CAAX using the components described in the manuscript. Using the plasmid concentration, calculate the total microliters of each plasmid needed to reach either 10 femtomole or 20 femtomole, and then divide this by two for the use in the five microliters half LR reactions.Generate 10 femtomole of P5E sox17, PME EGFP-CAAX, P3E Poly(A)and 20 femtomole of destination vector. Set up five microliters half LR reaction by combining the volumes of the P5E, PME, P3E, and PDEST in a 500 microliter tube. Then add sterile water to make the volume four microliters.Vortex the LR enzyme mix twice for one minute each before adding one microliter to the reaction and mix thoroughly before incubating at 25 degrees Celsius. The following day, stop the LR reaction by adding 0.5 microliters of proteinase K.Incubate at 37 degrees Celsius for 10 minutes and then cool to room temperature. Next, transform three to four microliters of the plasmid into thawed chemically competent cells by adding the plasmid and letting the cells sit on ice for 25 to 30 minutes.Then heat shock the cells in a 42 degree Celsius water bath for 30 seconds. After the heat shock, add 250 microliters of antibiotic-free rich liquid media to the cells and incubate with shaking at 37 degrees Celsius for 1.5 hours. Spread 300 microliters of bacterial suspension on one ampicillin Luria-Bertani plate and incubate overnight.The following day, screen the colonies for the presence of two phenotypes:clear and opaque. Pick the clear colonies one at a time, inoculate the liquid culture, and shake at 37 degrees Celsius overnight. Combine 150 nanograms of plasmid, 100 nanograms of transposase mRNA, and 2%phenol red on ice.Then add RNA-free water to make the volume three microliters. Transfer one cell stage embryo using transfer pipettes to the injection plate filled with EM completely covering the agar and then gently press approximately 50 to 75 embryos per slot of the injection plate. Add the mRNA plasmid phenol red mixture to the open end of a capillary injection needle.Place the capillary needle in the injection rig armature and turn on the injection rig. Lower the needle into the injection plate and break the tip using forceps to allow only a small amount of mixture to be pumped out by the injection rig. Inject the embryos with a three nanoliter bolus of the mRNA plasmid phenol red mixture into the cell body of the embryo.When finished, remove the embryos from the injection plate by gently popping them out of the slot and transfer pipetting them to a 100 millimeter Petri dish with fresh EM.Incubate at 28.5 degrees Celsius. Pick the appropriate developmental stage for fluorescent transgene expression and screen under a fluorescent dissecting microscope. Screen for non-fluorescent transgenes by screening for fluorescent transgenic markers such as GFP driven by the cardiac-specific promoter for the gene Cmlc2.When the embryos positive for transgenic insertion develop into adults and reach the breeding age, screen them individually for germline transmission and transgene expressivity by breeding them to wild type zebrafish. Example bacterial colonies obtained from the transformation of the LR recombination are shown. Clear colonies contained a correct LR recombination product more than 85%of the time, whereas opaque colonies never contained a correct recombination product.Diagnostic digestion of the three colonies from the transformation of the LR recombination products showed that the single opaque colony did not contain any plasmid, while the two clear colonies contained a single band at 9, 544 base pair. Transposase mRNA at 1, 950 base pair is shown here. Mosaic endoderm EGFP-CAAX expression was observed in 75%of the injected embryos.Transgenic marker expression of Cmlc2 EGFP in the developing heart was observed 24 hours post-fertilization. Adult zebrafish had germline transmission of sox17:EGFP-CAAX generated fluorescent embryos with the endoderm fully labeled with EGFP-CAAX. Both the anterior-posterior length of the endoderm and the dorsal-ventral length of each pouch from pouches one to five was measured in control and ethanol-treated wild type BMP mutant embryos.The overall length of the endoderm was not impacted by genotype or treatment. However, pouches one and three showed significant increases in pouch length between untreated and ethanol-treated wild type embryos, but significant decreases in length between untreated and ethanol-treated BMP mutants. Pouch two showed significant increases in pouch size between the untreated and ethanol-treated wild type and BMP mutant groups respectively.When attempting this procedure, it is critical to be precise in pipetting and diluting of the Tol2 system, as well as hitting the cell body during embryo injection.

the-zebrafish-tol2-system-modular-flexible-gateway-based-transgenesis

Research

JoVE Journal

Developmental Biology

2.5K vistas.  University of Louisville. El pez cebra transgénico ha demostrado ser fundamental para nuestra comprensión del desarrollo embrionario. El sistema Tol2 es una herramienta versátil que permite a los investigadores generar rápida y sistemáticamente múltiples peces cebra transgénicos para estudios de TEAF. El diseño modular y la facilidad de generación de nuevos componentes del kit hacen del sistema Tol2 una herramienta versátil para generar nuevos transgénicos sin tener que rediseñar ningún componente del kit...