Name: microinjection for transgenesis and genome editing in threespine sticklebacks
Uploaded: 2016-05-13T15:00:00
Description: Watch this Scientific Journal Video about Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks at JoVE.com

This work was funded in part by NIH R01 #DE021475 (CTM), an NIH Predoctoral Training Grant 5T32GM007127 (PAE), and an NSF Graduate Research Fellowship (NAE). We thank Kevin Schwalbach for performing BAC recombineering and injections, Nick Donde for generating CRISPR Sanger sequencing data, and Katherine Lipari for helpful feedback on the injection protocol.

All fish work was approved by the Institutional Animal Care and Use Committee of the University of California-Berkeley (protocol number R330).
1. Prepare Nucleic Acids for Injection
<ol>
	<li>Tol2 Plasmid Transgenesis (Adapted from Fisher26).
	<ol>
		<li>Cut 10 &#181;g transposase plasmid (pCS-Tp)39 with 10 U NotI in supplied buffer for 1 hr at 37 &#176;C to linearize. 
		Note: Material Transfer Agreements may be required to obtain Tol2 plasmids.</li>
		<li>Extract the cut plasmid with...

Injecting one-cell stickleback embryos for transgenesis or genome editing presents three main challenges. First, relative to zebrafish embryos, the stickleback embryonic chorion is tough and will often break needles. This problem can be partially overcome by using thicker and stronger glass micropipettes and injecting perpendicular to the chorion (see Protocol, Figure 2). Ensuring that as little water as possible is added to the embryos (just enough to cause the chorion to swell and lift away from the ce...

One fundamental component of understanding how biodiversity arises is determining the genetic and developmental bases of evolved phenotypic changes in nature. The threespine stickleback fish, Gasterosteus aculeatus, has emerged as an excellent model for studying the genetic basis of evolution. Sticklebacks have undergone many adaptive evolutionary changes as marine fish have colonized countless freshwater environments around the northern hemisphere, resulting in dramatic morphological, physiological, and behavioral changes1. The genomes of individuals from twenty-one stickleback populations have been sequenced and assembled, and a high density linkage map has been generated to further improve the assembly2,3. Genetic mapping experiments have identified genomic regions underlying evolved phenotypes4-6, and in a few cases, the functional roles of specific candidate genes have been tested7,8. A number of genomic regions underlying morphological changes have been identified with promising candidate genes, but these candidates have not yet been functionally tested9-12. In addition, sticklebacks are common models for studies of population genetics/genomics13,14, speciation15, behavior1, endocrinology16, ecotoxicology17, immunology18 and parasitology19. Future studies in each of these fields will benefit from the ability to perform functional genetic manipulations in sticklebacks. In addition to manipulating their coding sequences, the roles of candidate genes can be assessed by studying their cis-regulatory sequences and by functionally increasing, decreasing, or eliminating expression of the candidate gene. Microinjection and transgenesis methods in sticklebacks are well established7,8,20 and were initially developed using a meganuclease-mediated method21 first described in medaka22. The modified microinjection method presented here has been optimized for both Tol2-mediated transgenesis and recently developed genome editing reagents including TALENs and CRISPRs.
Changes to cis-regulatory elements are thought to be critical to morphological evolution, as cis-regulatory changes can avoid the negative pleiotropic consequences of coding mutations23. Therefore, testing and comparing putative cis-regulatory sequences has become a central goal of an increasing number of evolutionary studies. In addition, most human disease variants are regulatory variants24,25, and model vertebrate systems are sorely needed to study cis-regulatory element function and logic. Fish that fertilize their embryos externally in large numbers offer powerful vertebrate systems to study cis-regulation. The Tol2 transposon system, in which foreign DNA to be integrated in the genome is flanked by Tol2 transposase binding sites and co-injected with Tol2 transposase mRNA, works with high efficiency for successfully integrating plasmid constructs into fish genomes26-28. Typically, a potential enhancer is cloned upstream of a basal promoter (such as hsp70l29) and fluorescent reporter gene such as EGFP (enhanced green fluorescent protein) or mCherry in a Tol2 backbone and injected with transposase mRNA26. Observation of expression of the fluorescent reporter, either in injected embryos or offspring with stably integrated transgenes, provides information about the spatiotemporal regulation of gene expression driven by the putative enhancer. In further experiments, validated enhancers can be used to drive tissue-specific overexpression of genes of interest.
For analysis of larger cis-regulatory regions, high quality large-insert genomic libraries using bacterial artificial chromosomes (BACs) have been constructed for both marine and freshwater sticklebacks30. These BACs can be recombineered to replace a gene with a fluorescent reporter gene in the context of a large (150-200 kb) genomic region31. The fluorescent reporter is then expressed in a spatiotemporal pattern as determined by regulatory sequences within the BAC. For studies in fish, Tol2 sites can be added to the BAC to facilitate genomic integration32,33. In later stages of development when in situ hybridization is technically challenging, the fluorescent readout of the BAC can be used to study patterns of gene expression, as has been shown for stickleback Bone morphogenetic protein 6 (Bmp6)20. Additionally, fluorescent expression patterns in an individual can be tracked over time, which cannot be accomplished with in situ hybridization. BACs can also be used to add an additional copy of a genomic region to increase dosage of a gene of interest.
For the study of gene function, genome editing is an explosively expanding field that can be used to produce targeted changes to genomic sequences in a wide variety of organisms34. Transcription activator-like effector nucleases (TALENs) are modular, sequence-specific nucleases originally isolated from plant pathogens that can be precisely engineered to bind directly to a genomic sequence of choice and generate a double strand break35,36. Clustered regularly interspaced short palindromic repeats (CRISPR)/CAS systems were originally found in bacteria and use a guide RNA and the Cas9&#160;protein to generate a break in a target DNA sequence complementary to the guide37. The subsequent repair of the double strand break created by both TALENs and CRISPRs often leaves behind a small insertion or deletion, which can disrupt the function of the target sequence35-37. In sticklebacks, TALENs have been used to disrupt gene expression by targeting an enhancer20, and both TALENs and CRISPRs have successfully produced mutations in coding sequences (unpublished data). A detailed protocol for the generation of CRISPRs for use in zebrafish can be used as a guideline to develop CRISPRs for sticklebacks38.
Transgenic and genome editing experiments require introduction of nucleic acids into a newly fertilized one-cell embryo. By introducing the transgene or genome-editing tool early in development, the number of genetically manipulated daughter cells in the embryo is maximized. Injected embryos are then visually screened for fluorescence or molecularly screened for genome modifications. If cells contributing to the germline are successfully targeted, the transgene or mutation can be passed on to a subset of offspring, even when post-injection lethality is high. The mosaic fish can be outcrossed or intercrossed and their offspring screened to recover the mutant alleles or a stably integrated transgene of interest. This protocol describes methods for introducing transgenes and genome editing reagents into one-cell stickleback embryos and monitoring for successful genomic modifications.

<table border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stereomicroscope with transillumination</td>
			<td style="width:145px;">Leica</td>
			<td style="width:119px;">S6e/ KL300 LED</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Manual micromanipulator</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">MM33</td>
			<td style="width:167px;">Marzhauser M33 Micromanipulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Injecion system</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">MPPI-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Back pressure unit</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">BPU</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette holder kit</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">MPIP</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic base holder</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">Magnetic base</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Foot switch</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">FSW</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iron plate (magnetic base)</td>
			<td style="width:145px;">Narishige</td>
			<td style="width:119px;">IP</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:145px;">Sutter Instrument</td>
			<td style="width:119px;">P-97</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable transfer pipettes</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">13-711-7M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5% phenol red in DPBS</td>
			<td style="width:145px;">Sigma&#160;</td>
			<td style="width:119px;">P0290</td>
			<td style="width:167px;">injection tracer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">#5 forceps, biologie dumoxel&#160;</td>
			<td style="width:145px;">Fine Science Tools</td>
			<td style="width:119px;">11252-30</td>
			<td style="width:167px;">for needle breaking</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Storage Jar</td>
			<td style="width:145px;">World Precision Instruments</td>
			<td style="width:119px;">E210</td>
			<td>holds needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">6&#34;, 6 teeth per inch plaster drywall saw</td>
			<td style="width:145px;">Lenox</td>
			<td>20571 (S636RP)</td>
			<td>hold eggs for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">13 cm x 13 cm glass plate</td>
			<td style="width:145px;">any hardware store</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Borosilicate glass capillaries, 1.0 mm OD/0.58 mm ID&#160;</td>
			<td style="width:145px;">World Precision Instruments</td>
			<td style="width:119px;">1B100-F4</td>
			<td style="width:167px;">*harder glass than zebrafish injection capillaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">150 x 15mm petri dish</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">FB0875714</td>
			<td>raise stickleback embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 x 10mm petri dish</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">08-757-100A</td>
			<td>store eggs pre-injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant Ocean Salt</td>
			<td style="width:145px;">Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:145px;">Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricaine methanesulfonate/MS-222</td>
			<td style="width:145px;">Western Chemical Inc</td>
			<td style="width:119px;">MS222</td>
			<td style="width:167px;">fish anaesthesia/euthanasia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sp6 transcription kit</td>
			<td style="width:145px;">Ambion</td>
			<td style="width:119px;">AM1340</td>
			<td style="width:167px;">For transcription of TALENs and transposase mRNA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RNeasy cleanup kit</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td style="width:167px;">purify transposase or TALEN RNA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QiaQuick PCR cleanup kit</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">28104</td>
			<td style="width:167px;">clean up plasmids for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K 20 mg/ml</td>
			<td style="width:145px;">Ambion</td>
			<td style="width:119px;">AM2546</td>
			<td style="width:167px;">for DNA preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nucleobond BAC 100 kit</td>
			<td style="width:145px;">Clontech</td>
			<td style="width:119px;">740579</td>
			<td>for BAC DNA preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NotI</td>
			<td style="width:145px;">NEB</td>
			<td style="width:119px;">R0189L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion polymerase</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">F-530L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen PlasmidPlus Midi kit</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">12943</td>
			<td style="width:167px;">contains endotoxin rinse buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QIAQuick Gel Extraction</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">28704&#160;</td>
			<td>for sequencing induced mutations</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phenol:chloroform:Isoamyl alcohol</td>
			<td style="width:145px;">Sigma</td>
			<td>P2069-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium acetate</td>
			<td style="width:145px;">Sigma</td>
			<td>S2889-250G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol (molecular biology grade)</td>
			<td style="width:145px;">Sigma</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50X Tris-acetate-EDTA buffer</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:119px;">B49</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5-10KbRNA ladder</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:119px;">15623-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop&#160; Spectrophotometer</td>
			<td style="width:145px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:145px;">Sigma</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10X PBS</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1kb Plus DNA Ladder</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>10787-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Chloride</td>
			<td style="width:145px;">Sigma</td>
			<td>P9541-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:145px;">Sigma</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NP-40</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20</td>
			<td style="width:145px;">Sigma</td>
			<td>P1379-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris pH 8.3</td>
			<td style="width:145px;">Teknova</td>
			<td>T1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">12-strip PCR tube</td>
			<td style="width:145px;">Thermo Scientific</td>
			<td style="width:119px;">AB-1113</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

For reporter gene transgenes that have enhancer activity, successful injection will result in specific, cellular expression of the transgene (Figure 4A, 4C). Injected fish can then be outcrossed to produce stable lines (example of a BAC stable line shown in Figure 4B). Injecting DNA into stickleback embryos typically results in far higher lethality than RNA alone. It is typical to see up to 50% (sometimes even more) lethality or malformat...

Watch this Scientific Journal Video about Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks at JoVE.com

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

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 ...

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