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: 2.8K 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 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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

2.8K 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

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

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

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 ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

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 ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...