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.

The authors have nothing to disclose.

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

JoVE Journal

Developmental Biology

13.9K Views.  University of California, Berkeley.  Transgene manipulaties en genoom editing zijn van cruciaal belang voor het functioneel testen van de rol van genen en cis van regelgevende elementen. Hier kunt u een gedetailleerde micro-injectie protocol voor het genereren van genomische modificaties (inclusief Tol2-gemedieerde fluorescerende reporter transgene constructen, Talens en CRISPRs) wordt gepresenteerd voor de opkomende model vis, de driedoornige stekelbaars. 

Video: Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Genetics

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Biology

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Neuroscience

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

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