Name: Video: The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach
Uploaded: 2022-11-30T14:50:00.000+00:00
Duration: 10 min
Description: 2.9K Views. University of Louisville. 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.

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&#39;s instructions, and autoclave. Before pouring the media into 100 mm Petri dishes, add either ampicillin (50 &#181;g/mL), kanamycin (50 &#181;g/mL), or an ampicillin/chloramphenicol combination (50 &#181;g/mL and 30 &#181;g/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&#183;7H2O. Filter-sterilize the stock solution using a 0.22 &#181;m vacuum filtration system, and store at 4 &#176;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 &#176;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 &#176;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 &#181;L of transposase plasmid (150 ng/&#181;L), 1.5 &#181;L of restriction endonuclease reaction buffer, 0.3 &#181;L of NotI endonuclease.</li>
	<li>Fill the reaction up to 20 &#181;L with RNase-free water, and then mix and digest at 37 &#176;C overnight.</li>
	<li>Stop the transposase digestion by adding 1 &#181;L of 0.5 M EDTA (pH 8.0), 2 &#181;L of 5 M NH4OAc, and 80 &#181;L of 100% EtOH to the digestion reaction, and then mix and chill at &#8722;20 &#176;C overnight.</li>
	<li>Centrifuge the solution at 14,000x g for 20 min at 4 &#176;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/&#181;L) using a fluorometer by first running 2 &#181;L of an RNase-free water blank and then running 2 &#181;L of the linearized plasmid. 
	NOTE: The plasmid concentrations typically range between 100-200 ng/&#181;L</li>
	<li>Set up the SP6 mRNA production by combining the following components in order in a 0.5 mL microcentrifuge tube: 10 &#181;L of 2x NTP/CAP, 2 &#181;L of 10x reaction buffer, 0.1-1 &#181;g of linear DNA template, and 2 &#181;L of SP6 enzyme mix.</li>
	<li>Fill the reaction up to 20 &#181;L with RNase-free water, and then mix and incubate at 37 &#176;C for 2 h.</li>
	<li>After incubating for 2 h, add 1 &#181;L of DNase, mix well, and incubate for an additional 15 min at 37 &#176;C.</li>
	<li>To stop the SP6 reaction, add 30 &#181;L of LiCl precipitation solution to the reaction tube, and then mix and chill at &#8722;20 &#176;C overnight.</li>
	<li>Centrifuge the solution at 14,000 x g for 20 min at 4 &#176;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 &#176;C to pellet the mRNA, and then aspirate the supernatant. Allow the pellet to air dry.</li>
	<li>Resuspend the mRNA pellet in 20 &#181;L of RNase-free water, determine the mRNA concentration in nanograms per microliter (ng/&#181;L) using a fluorometer as described in step 4.5 (should be at least 100 ng/&#181;L), aliquot 100 ng of mRNA per tube for single use, and store at &#8722;80 &#176;C. 
	&#8203;NOTE: To make sure the mRNA is not degraded, 2 &#181;L 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 &#181;L. To generate new entry elements, BP reactions using 5&#39;, middle, and 3&#39; 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&#39; entry vector (p5E), a middle entry vector (pME), a 3&#39; 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/&#181;L) 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="Equation 1" src="/files/ftp_upload/64679/64679eq01.jpg" style="margin: auto;" /> 
		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 &#181;L) of the vectors needed in the LR reaction15.</li>
	</ol>
	</li>
	<li>To generate the construct described below, sox17:EGFPCAAX,&#160;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 (&#181;L) 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 &#181;L half-LR reactions (all the values listed below are listed in Table 2). 
	<img alt="Equation 2" src="/files/ftp_upload/64679/64679eq02v2.jpg" style="margin: auto;" />
	<ol>
		<li>To generate 10 fmol of p5E-sox17, use 45.66 ng (at a concentration of 140.3 ng/&#181;L) and dilute with sterile water by a factor of 4 to get 0.65 &#181;L.</li>
		<li>To generate 10 fmol of pME-EGFPCAAX, use 22.08 ng (at a concentration of 213.5 ng/&#181;L) and dilute with sterile water by a factor of 10 to get 0.52 &#181;L.</li>
		<li>To generate 10 fmol of p3E-poly A, use 15.73 ng (at a concentration of 276.1 ng/&#181;L) and dilute with sterile water by a factor of 20 to get 0.57 &#181;L.</li>
		<li>For 20 fmol of the destination vector, use 77.66 ng of pDest (at a concentration of 307.3 ng/&#181;L) and dilute with sterile water by a factor of 5 to get 1.63 &#181;L.</li>
	</ol>
	</li>
	<li>To set up a 5 &#181;L 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 &#181;L.</li>
	<li>Vigorously vortex the LR enzyme mix 2x for 1 min each to ensure proper mixing, and then add 1 &#181;L to the reaction and mix thoroughly.</li>
	<li>Incubate at 25 &#176;C overnight (16+ h).</li>
	<li>Stop the LR reaction by adding 0.5 &#181;L of proteinase K (2 &#181;g/&#181;L), incubate at 37 &#176;C for 10 min, and then cool to room temperature.</li>
	<li>Transform 3-4 &#181;L 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&#176;C water bath for precisely 30 s.</li>
	<li>After the heat shock, add 250 &#181;L of antibiotic-free, rich liquid media to the cells, and incubate with shaking at 37 &#176;C for 1.5 h.</li>
	<li>Spread 300 &#181;L of bacteria on one ampicillin plate, and incubate at 37&#176;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 &#176;C overnight. 
	NOTE: Clear colonies almost always contain the correct LR colony (&#62;85% of the time).</li>
	<li>Using a commercial kit as per the manufacturer&#39;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 &#181;L of the plasmid, and only incubate at 37 &#176;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/&#181;L 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 &#181;L). Then, add RNA-free water to make up the volume to 3 &#181;L.</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 &#176;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 &#945;cyrstallin, 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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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

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 ...

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Research

JoVE Journal

Developmental Biology

2.9K Views.  University of Louisville. 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.

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

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

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

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

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

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.

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Neuroscience

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

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.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Genetics

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

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.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Biology

Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional mutagenesis using fluorescent reporters to tag expression of mutated genes. Several laboratories have constructed and tested enhancer- and gene-trap vectors in zebrafish, using fluorescent proteins, Gal4- and lexA- based transcriptional activators as reporters 1-7. These vectors had two potential drawbacks: suboptimal stringency (e.g. lack of ability to differentiate between enhancer- and gene-trap events) and low mutagenicity (e.g. integrations into genes rarely produced null alleles). Gene Breaking Transposon (GBTs) were developed to address these drawbacks 8-10. We have modified one of the first GBT vectors, GBT-R15, for use with Gal4-VP16 as the primary gene trap reporter and added UAS:eGFP as the secondary reporter for direct detection of gene trap events. Application of Gal4-VP16 as the primary gene trap reporter provides two main advantages. First, it increases sensitivity for genes expressed at low expression levels. Second, it enables researchers to use gene trap lines as Gal4 drivers to direct expression of other transgenes in very specific tissues. This is especially pertinent for genes with non-essential or redundant functions, where gene trap integration may not result in overt phenotypes. The disadvantage of using Gal4-VP16 as the primary gene trap reporter is that genes coding for proteins with N-terminal signal sequences are not amenable to trapping, as the resulting Gal4-VP16 fusion proteins are unlikely to be able to enter the nucleus and activate transcription. Importantly, the use of Gal4-VP16 does not pre-select for nuclear proteins: we recovered gene trap mutations in genes encoding proteins which function in the nucleus, the cytoplasm and the plasma membrane.

This protocol describes the method of gene trap insertional mutagenesis using Gal4-VP16 as the primary reporter and GFP/RFP as secondary reporters in zebrafish. Approximately one in ten high-expressing F0 fish yield gene trap progeny co-expressing GFP and RFP. The screening procedure can be readily scaled to adapt to the size of the laboratory performing the insertional mutagenesis screen.

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...

Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between these features may be interrogated by controlling the phase transition of a disease-associated protein in a vulnerable cell type, although this experimental approach has been limited so far. Here, we describe a protocol to induce phase transition of the RNA/DNA-binding protein TDP-43 in spinal motor neurons of zebrafish larvae for modeling cytoplasmic aggregation of TDP-43 occurring in degenerating motor neurons in amyotrophic lateral sclerosis (ALS). We describe a bacterial artificial chromosome (BAC)-based genetic method to deliver an optogenetic TDP-43 variant selectively to spinal motor neurons of zebrafish. The high translucency of zebrafish larvae allows for the phase transition of the optogenetic TDP-43 in the spinal motor neurons by a simple external illumination using a light-emitting diode (LED) against unrestrained fish. We also present a basic workflow of live imaging of the zebrafish spinal motor neurons and image analysis with freely available Fiji/ImageJ software to characterize responses of the optogenetic TDP-43 to the light illumination. This protocol enables the characterization of TDP-43 phase transition and aggregate formation in an ALS-vulnerable cellular environment, which should facilitate an investigation of its cellular and behavioral consequences.

We describe a protocol to induce phase transition of TAR DNA-binding protein 43 (TDP-43) by light in the spinal motor neurons using zebrafish as a model.

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between ...

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

Summary

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Chapters in this video

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

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

Imaging Subcellular Structures in the Living Zebrafish Embryo

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

Manipulation of Gene Function in Mexican Cavefish

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Gene Trapping Using Gal4 in Zebrafish

Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae