Name: The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach (Video) | JoVE Journal
Uploaded: 2022-11-30T14:50:00.000+00:00
Duration: 10 min
Description: Learn the modular Tol2 transgenesis system for generating transgenic zebrafish using gateway-based cloning. This protocol enables efficient creation of constructs for developmental biology and FASD research.

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5 minutes

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 measurements were made using the measure tool in ImageJ. All the statistical analyses were performed using statistical software.
1. Making the solutions and media
<ol>
	<li>Bacterial media: Dissolve LB broth or agar as per the manufacturer's instructions, and autoclave. Before pouring the media into 100 mm Petri dishes, add either ampicillin (50 µg/mL), kanamycin (50 µg/mL), or an ampicillin/chloramphenicol combination (50 µg/mL and 30 µg/mL, respectively).</li>
	<li>To make 20x embryo media stock, dissolve the following in 1 L of water: 17.5 g of NaCl, 0.75 g of KCl, 2.9 g of CaCl2, 0.41 g of K2HPO4, 0.142 g of Na2HPO4, and 4.9 g of MgSO4·7H2O. Filter-sterilize the stock solution using a 0.22 µm vacuum filtration system, and store at 4 °C. 
	NOTE: Ignore the white precipitate that forms; this will not impact the media.</li>
	<li>In 19 L of water, dissolve 1.2 g of NaHCO3 and add 1 L of the 20x embryo media stock to generate the working embryo media solution, and maintain this solution at 28 °C.</li>
	<li>For 2% phenol red solution, dissolve 20 mg of phenol red sodium salt in 1 mL of RNase-free water, and store at room temperature.</li>
</ol>
2. Embryo injection molds
<ol>
	<li>To make the agarose injection plate, dissolve agarose in the embryo media to a final concentration of 3% by heating to a boil.</li>
	<li>Pour 50 mL of agarose into 100 mm Petri dishes, and while the agarose is still liquid, gently place the injection mold in the agarose to prevent bubble formation under the mold.</li>
	<li>Let the agarose plates cool. Once the agarose is solid, remove the injection mold, and store at 4 °C until ready to use.</li>
</ol>
3. Injection pipettes
<ol>
	<li>Pull 1.0 mm (OD) capillaries containing a filament into needles using a vertical pipette puller with the solenoid set to 2.5 and the heating element set to 14.6. 
	NOTE: Any heated puller will work if it pulls the capillaries into two needles cleanly and evenly, but the injection needles need to be pulled within 1 week of injecting the zebrafish embryos for consistent injection results.
	<ol>
		<li>Place the capillary in the puller, tighten the clamp at each end, and then activate the heating element.</li>
		<li>Pull the capillaries to create two injection needles.</li>
		<li>Remove the capillaries from the puller, and store them in an empty Petri dish with a strip of modeling clay that acts as the needle holder.</li>
	</ol>
	</li>
</ol>
4. Transposase mRNA preparation
<ol>
	<li>Linearize the Tol2Kit plasmid containing transposase with a NotI restriction endonuclease by combining the following in a 0.5 mL microcentrifuge tube: 10 µL of transposase plasmid (150 ng/µL), 1.5 µL of restriction endonuclease reaction buffer, 0.3 µL of NotI endonuclease.</li>
	<li>Fill the reaction up to 20 µL with RNase-free water, and then mix and digest at 37 °C overnight.</li>
	<li>Stop the transposase digestion by adding 1 µL of 0.5 M EDTA (pH 8.0), 2 µL of 5 M NH4OAc, and 80 µL of 100% EtOH to the digestion reaction, and then mix and chill at −20 °C overnight.</li>
	<li>Centrifuge the solution at 14,000x g for 20 min at 4 °C, and then aspirate the supernatant and resuspend the DNA pellet in RNase-free water.</li>
	<li>Determine the linear plasmid concentration in nanograms per microliter (ng/µL) using a fluorometer by first running 2 µL of an RNase-free water blank and then running 2 µL of the linearized plasmid. 
	NOTE: The plasmid concentrations typically range between 100-200 ng/µL</li>
	<li>Set up the SP6 mRNA production by combining the following components in order in a 0.5 mL microcentrifuge tube: 10 µL of 2x NTP/CAP, 2 µL of 10x reaction buffer, 0.1-1 µg of linear DNA template, and 2 µL of SP6 enzyme mix.</li>
	<li>Fill the reaction up to 20 µL with RNase-free water, and then mix and incubate at 37 °C for 2 h.</li>
	<li>After incubating for 2 h, add 1 µL of DNase, mix well, and incubate for an additional 15 min at 37 °C.</li>
	<li>To stop the SP6 reaction, add 30 µL of LiCl precipitation solution to the reaction tube, and then mix and chill at −20 °C overnight.</li>
	<li>Centrifuge the solution at 14,000 x g for 20 min at 4 °C to pellet the mRNA, and then aspirate the supernatant and wash the mRNA pellet with 1 mL of 80% EtOH (diluted with RNase-free water).</li>
	<li>Centrifuge the solution at 14,000 x g for 20 min at 4 °C to pellet the mRNA, and then aspirate the supernatant. Allow the pellet to air dry.</li>
	<li>Resuspend the mRNA pellet in 20 µL of RNase-free water, determine the mRNA concentration in nanograms per microliter (ng/µL) using a fluorometer as described in step 4.5 (should be at least 100 ng/µL), aliquot 100 ng of mRNA per tube for single use, and store at −80 °C. 
	​NOTE: To make sure the mRNA is not degraded, 2 µL of mRNA can be quickly run on a DNA electrophoresis gel to confirm a single band at 1,950 bps.</li>
</ol>
5. Multisite Gateway cloning to create entry vectors for transgenesis
NOTE: This protocol is modified from Kwan et al.10, with the LR reaction written as a half-LR reaction and with a total volume of 5 µL. To generate new entry elements, BP reactions using 5', middle, and 3' donor vectors need to be used10,13.
<ol>
	<li>To perform the reaction, gather 10 fmol each of p5E, pME, and p3E and 20 fmol of the destination vector (pDest). 
	NOTE: Multisite LR recombination uses four different plasmid vectors: a 5' entry vector (p5E), a middle entry vector (pME), a 3' entry vector (p3E), and a destination vector (pDest), to generate a unique transgenic construct flanked by Tol2 repeats ready to be injected into zebrafish embryos.
	<ol>
		<li>Identify the size of all four vectors in base pairs from the plasmid source (Table 1, Tol2Kit Wiki page14 or Addgene11; see the Table of Materials). 
		NOTE: For novel vectors, full plasmid sequencing via commercial sequencing companies can provide the exact plasmid size in bps.</li>
		<li>Determine the concentration of all four vectors in nanograms per microliter (ng/µL) using a fluorometer, as described in step 4.5.</li>
		<li>Using the weight of DNA as 660 g/mol and the plasmid size (in bps), calculate the total nanograms (ng) of plasmid needed to reach either 10 fmol (entry vectors) or 20 fmol (destination vector). 
		<img alt="Vector calculation formula, \(Vector Size [bps] \times 10\: fmol\), for nucleic acid quantification." src="/files/ftp_upload/64679/64679eq01.jpg" style="margin: auto;" data-altupdatedat="1788304762000" data-alt="Equation 1"> 
		NOTE: The Mosimann lab at the University of Colorado, Anschutz Medical Campus, has generated a freely available spreadsheet to calculate the amount of plasmid needed (in ng) and the volume (in µL) of the vectors needed in the LR reaction15.</li>
	</ol>
	</li>
	<li>To generate the construct described below, sox17:EGFPCAAX, use the following vectors: a p5E vector containing the zebrafish sox17 promoter, which is 6,918 bp in size16; a pME vector containing the prenylated EGFP, which is 3,345 bp in size10; a p3E vector containing the SV40 late poly A signal sequence, which is 2,838 bp in size10; and the pDest, which is 5,883 bp in size10.</li>
	<li>Using the plasmid concentration determined in step 5.1.2 and the amount in nanograms (ng) for each vector (step 5.1.3), calculate the total microliters (µL) of each plasmid needed to reach either 10 fmol (entry vectors) or 20 fmol (destination vector), and then divide this by two for use in the 5 µL half-LR reactions (all the values listed below are listed in Table 2). 
	<img alt="Vector calculation formula for LR reaction; equation for volume vector concentration in μL." src="/files/ftp_upload/64679/64679eq02v2.jpg" style="margin: auto;" data-altupdatedat="1788304762000" data-alt="Equation 2">
	<ol>
		<li>To generate 10 fmol of p5E-sox17, use 45.66 ng (at a concentration of 140.3 ng/µL) and dilute with sterile water by a factor of 4 to get 0.65 µL.</li>
		<li>To generate 10 fmol of pME-EGFPCAAX, use 22.08 ng (at a concentration of 213.5 ng/µL) and dilute with sterile water by a factor of 10 to get 0.52 µL.</li>
		<li>To generate 10 fmol of p3E-poly A, use 15.73 ng (at a concentration of 276.1 ng/µL) and dilute with sterile water by a factor of 20 to get 0.57 µL.</li>
		<li>For 20 fmol of the destination vector, use 77.66 ng of pDest (at a concentration of 307.3 ng/µL) and dilute with sterile water by a factor of 5 to get 1.63 µL.</li>
	</ol>
	</li>
	<li>To set up a 5 µL half-LR reaction, combine the volumes of the p5E, pME, p3E, and pDest determined in step 5.3 in a 0.5 mL microcentrifuge tube, and then add sterile water to reach a total reaction volume of 4 µL.</li>
	<li>Vigorously vortex the LR enzyme mix 2x for 1 min each to ensure proper mixing, and then add 1 µL to the reaction and mix thoroughly.</li>
	<li>Incubate at 25 °C overnight (16+ h).</li>
	<li>Stop the LR reaction by adding 0.5 µL of proteinase K (2 µg/µL), incubate at 37 °C for 10 min, and then cool to room temperature.</li>
	<li>Transform 3-4 µL of plasmid into thawed, chemically competent cells by adding the plasmid and letting the cells sit on ice for 25-30 min.</li>
	<li>While the cells sit on ice, warm two LB-ampicillin agar plates to room temperature.</li>
	<li>Heat-shock the cells in a 42°C water bath for precisely 30 s.</li>
	<li>After the heat shock, add 250 µL of antibiotic-free, rich liquid media to the cells, and incubate with shaking at 37 °C for 1.5 h.</li>
	<li>Spread 300 µL of bacteria on one ampicillin plate, and incubate at 37°C overnight.</li>
	<li>The next morning, screen the colonies for the presence of two phenotypes, clear and opaque, pick the clear colonies (as described in Kwan et al.10) one at a time, inoculate the liquid culture, and then shake at 37 °C overnight. 
	NOTE: Clear colonies almost always contain the correct LR colony (&gt;85% of the time).</li>
	<li>Using a commercial kit as per the manufacturer's instructions, perform a plasmid prep on each liquid culture, and diagnostically digest each plasmid to confirm the appropriate size of the transgenic construct, which indicates that the LR gateway reaction has worked properly.</li>
	<li>Re-transform the correct transgenic constructs using the standard bacterial transformation protocol as described in steps 5.4-5.12 using 0.5-1 µL of the plasmid, and only incubate at 37 °C for 1 h.</li>
	<li>Re-streak the colonies, and confirm that each has the LR transgenic construct as in step 5.14.</li>
	<li>Determine the plasmid concentration as described in step 4.5 (at least 150 ng/µL is needed for the embryo injection).</li>
</ol>
6. Injection of the transgene into the embryos
<ol>
	<li>Thaw a single-use transposase mRNA sample and Tol2 transgenic construct on ice and prewarn an agarose injection plate to room temperature.</li>
	<li>Combine the following on ice: 150 ng of plasmid, 100 ng of transposase mRNA, and 2% phenol red (0.3 µL). Then, add RNA-free water to make up the volume to 3 µL.</li>
	<li>Transfer one cell-stage embryos using transfer pipettes to the injection plate (described in step 2) filled with EM completely covering the agar, and then gently press approximately 50-75 embryos per slot of the injection plate.</li>
	<li>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. 
	NOTE:The injection rig uses compressed gas, such as air, to pulse the desired volume (described in step 6.5) of the mRNA/plasmid/phenol red mixture into the embryo when activated.</li>
	<li>Lower the capillary needle into the EM of the injection plate, and break the tip using forceps to allow only a small amount of mRNA/plasmid/phenol red mixture to be pumped out by the injection rig.</li>
	<li>Inject the embryos with a small 3 nL bolus of the mRNA/plasmid/phenol red mixture into the cell body of the embryo. 
	NOTE: A 3 nL bolus is a volume of mRNA/plasmid/phenol red mixture at which seven boluses can theoretically fit equally across the midline of the embryo.</li>
	<li>When finished injecting the embryos, remove them from the injection plate by gently popping them out of the slot and transfer-pipetting them to a 100 mm Petri dish with fresh EM (approximately 40 mL), and then incubate at 28.5 °C to allow the embryos to develop.</li>
</ol>
7. Screening embryos for transgenic insertion
<ol>
	<li>For the expression of fluorescent proteins, pick the appropriate developmental stage when the fluorescent transgene should be expressed, and screen for the presence of fluorescence under a fluorescent dissecting microscope. 
	NOTE: These embryos will be mosaic in their expression and show a genomic insertion of the transgenic construct.</li>
	<li>Screen for non-fluorescent transgenes by screening for a fluorescent transgenic marker (as described in step 7.1), such as GFP driven by the cardiac-specific promoter for the gene cmlc2 or mCherry driven by the lens-specific promoter of αcyrstallin, which are contained in distinct destination vectors.</li>
	<li>Allow the embryos positive for transgenic insertion to fully develop to the adult zebrafish stages.</li>
	<li>Once potential transgenic fish reach breeding age, screen them individually for both germline transmission and transgene expressivity by breeding them to wild-type zebrafish.</li>
	<li>Those adults whose embryos develop normally and give the strongest and most appropriate transgene expression are kept as unique transgenic zebrafish lines for future work and analyses. 
	NOTE: As an alternative approach to screening for the genomic insertion of transgenic constructs, design PCR primers that only amplify the transgenic construct and not wild-type DNA.</li>
</ol>

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</ol>

The authors have nothing to disclose.

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><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>Lawson 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&amp;nbsp;</td><td>VWR</td><td>10025-724</td><td></td></tr><tr><td>Microcentrifuge tube 1.5 mL&amp;nbsp;</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 &amp;mu;L&amp;nbsp;</td><td>Fisher Scientific</td><td>13611106</td><td></td></tr><tr><td>Micropipette tips 1000 &amp;mu;L&amp;nbsp;</td><td>Fisher Scientific</td><td>13611127</td><td></td></tr><tr><td>Micropipette tips 200 &amp;mu;L&amp;nbsp;</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&amp;nbsp;</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 &amp;micro;m vacuum filtration system&amp;nbsp;</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 &lt;em&gt;E. coli&lt;/em&gt;</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

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...

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 ...

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

Research

JoVE Journal

Developmental Biology

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Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

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Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos

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Construction of an Affordable and Easy-to-Build Zebrafish Facility

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Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

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Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

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Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

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Imaging Subcellular Structures in the Living Zebrafish Embryo

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Manipulation of Gene Function in Mexican Cavefish

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Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

Learn the modular Tol2 transgenesis system for generating transgenic zebrafish using gateway-based cloning. This protocol enables efficient creation of constructs for developmental biology and FASD research.

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

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