The authors thank Sunishka Thakur for her assistance in genotyping and imaging the oca2 mutant fish depicted in Figure 2. This work was supported by National Science Foundation (NSF) award 1656574 to A.C.K., NSF award 1754321 to J.K. and A.C.K., and National Institutes of Health (NIH) award R21NS105071 to A.C.K. and E.R.D.

The authors have nothing to disclose.

Here, we provided a methodology for manipulating gene function using morpholinos, CRISPR/Cas9 gene editing, and transgenesis methodology. The wealth of genetic technology and the optimization of these systems in zebrafish will likely allow for the transfer of these tools into A. mexicanus with ease52. Recent findings have used these approaches in A. mexicanus, but they remain underutilized in the investigation of diverse morphological, developmental, and behavioral traits in this...

Since Darwin&#8217;s Origin of Species1, scientists have gained profound insights into how traits are shaped evolutionarily in response to defined environmental and ecological pressures, thanks to cave organisms2. The Mexican tetra, A. mexicanus, consists of eyed ancestral &#8216;surface&#8217; populations that inhabit rivers throughout Mexico and southern Texas and of at least 29 geographically isolated populations of derived cave morphs inhabiting the Sierra del Abra and other areas of Northeast Mexico3. A number of cave-associated traits have been identified in A. mexicanus, including altered oxygen consumption, depigmentation, loss of eyes, and altered feeding and foraging behavior4,5,6,7,8,9. A. mexicanus presents a powerful model for investigating mechanisms of convergent evolution due to a well-defined evolutionary history, a detailed characterization of ecological environment, and the presence of independently evolved cave populations10,11. Many of the cave-derived traits that are present in cavefish, including eye loss, sleep loss, increased feeding, loss of schooling, reduced aggression, and reduced stress responses, have evolved multiple times through independent origins, often utilizing different genetic pathways between caves8,12,13,14,15. This repeated evolution is a powerful aspect of the A. mexicanus system and can provide insight into the more general question of how genetic systems may be perturbed to generate similar phenotypes.&#160;
While the application of genetic technology for the mechanistic investigation of gene function has been limited in many fish species (including A. mexicanus), recent advances in the zebrafish provide a basis for genetic technology development in fish16,17,18,19,20. Numerous tools are widely used in zebrafish to manipulate gene expression, and the implementation of these procedures have long been standardized. For example, the injection of morpholino oligos (MOs) at the single-cell stage selectively blocks RNA and prevents translation for a brief temporal window during development21,22. In addition, gene-editing approaches, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and transcription activator-like effector nuclease (TALEN), allow for the generation of defined deletions or, in some cases, insertions through a recombination in genomes19,20,23,24. Transgenesis is used to manipulate stable gene expression or function in a cell-type specific manner. The Tol2 system is used effectively to generate transgenic animals by coinjecting transposase mRNA with a Tol2 DNA plasmid containing a transgene25,26. The Tol2 system utilizes the Tol2 transposase of medaka to generate stable germline insertions of transgenic construct17. Generating Tol2 transgenics involves coinjecting a plasmid containing a transgene flanked by Tol2 integration sites and mRNA for Tol2 transposase17. This system has been used to generate an array of transgenic lines in zebrafish and its use has recently expanded to additional emergent model systems, including cichlids, killifish, the stickleback, and, more recently, the Mexican cavefish27,28,29,30.&#160;
While the cavefish is a fascinating biological system for elucidating mechanisms of trait evolution, its full capability as an evolutionary model has not been fully harnessed. This has partially been due to an inability to manipulate genetic and cellular function directly31. Candidate genes regulating complex traits have been identified using quantitative trait loci (QTL) studies, but the validation of these candidate genes has been difficult32,33,34. Recently, transient knockdown using morpholinos, gene editing using CRISPR and TALEN systems, and the use of Tol2-mediated transgenesis have been used to investigate the genetic basis underlying a number of traits35,36,37,38. The implementation and standardization of these techniques will allow for manipulations that interrogate the molecular and neural underpinnings of biological traits, including the manipulation of gene function, the labeling of defined cell populations, and the expression of functional reporters. Whereas the successful implementation of these genetic tools to manipulate gene or cellular function has been demonstrated in emergent model systems, detailed protocols are still lacking in A. mexicanus.&#160;
A. mexicanus provide critical insight into the mechanisms of evolution in response to a changing environment and present the opportunity to identify novel genes regulating diverse traits. A number of factors suggest that A. mexicanus is an extremely tractable model for applying established genomic tools currently available in established genetic models, including the ability to easily maintain fish in the laboratories, large brood size, transparency, a sequenced genome, and defined behavioral assays39. Here, we describe a methodology for the use of morpholinos, transgenesis, and gene editing in surface and cave populations of A. mexicanus. The broader application of these tools in A. mexicanus will allow for a mechanistic investigation into the molecular processes underlying the evolution of developmental, physiological, and behavioral differences between cavefish and surface fish.&#160;

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Fish breeding &amp;amp; egg supplies&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Fine mesh fish net</td><td>Penn Plax</td><td>BN4</td><td></td></tr><tr><td>Fish tank heater</td><td>Aqueon</td><td>100106108</td><td></td></tr><tr><td>Egg traps</td><td>Custom made</td><td>NA</td><td>Design and create plastic grate to place at bottom of tank to protect eggs</td></tr><tr><td>Glass pipettes</td><td>Fisher Scientific</td><td>13-678-20C</td><td></td></tr><tr><td>Pipette bulbs</td><td>Fisher Scientific</td><td>03-448-21</td><td></td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP160-500</td><td></td></tr><tr><td>Egg molds</td><td>Adaptive Science Tools</td><td>TU-1</td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Morpholino supplies&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Control Morpholino</td><td>Gene Tools, LLC</td><td>Standard control olio</td><td></td></tr><tr><td>Custom Morpholino</td><td>Gene Tools, LLC</td><td>NA</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma Aldrich</td><td>P0290-100ML</td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;CRISPR supplies&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Cas9 Plasmid</td><td>AddGene</td><td>46757</td><td></td></tr><tr><td>GoTaq DNA Polymerase</td><td>Promega</td><td>M3001</td><td></td></tr><tr><td>KOD Hot Start Taq</td><td>EMD Millipore</td><td>71-842-3</td><td></td></tr><tr><td>Primers</td><td>Integrated DNA Technologies</td><td>Custom</td><td></td></tr><tr><td>T7 Megascript Kit</td><td>Ambion/Thermofisher</td><td>AM1333</td><td></td></tr><tr><td>miRNeasy Kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>mMessage mMachine T3 kit</td><td>Ambion/Thermofisher</td><td>AM1348</td><td></td></tr><tr><td>MinElute Kit</td><td>Qiagen</td><td>28204</td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Tol2 transgenesis supplies&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>pCS-zT2TP plasmid</td><td>Kawakami et al., 2004</td><td>Request from senior author</td><td></td></tr><tr><td>CutSmart Buffer</td><td>New England Biolabs</td><td>B7204</td><td></td></tr><tr><td>NotI-HF Restriction Enzyme</td><td>New England Biolabs</td><td>R3189</td><td></td></tr><tr><td>PCR purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>SP6 mMessenger Kit</td><td>Ambion/Thermofisher</td><td>AM1340</td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Microinjection supplies&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Glass Capillary Tubes</td><td>Sutter Instruments</td><td>BF100-58-10</td><td></td></tr><tr><td>Pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Picoinjector</td><td>Warner Instruments</td><td>PLI-100A</td><td></td></tr><tr><td>Micromanipulator</td><td>World Precision Instruments</td><td>M3301R</td><td></td></tr><tr><td>Micromanipulator Stand</td><td>World Precision Instruments</td><td>M10</td><td></td></tr><tr><td>Micmanipulator Base</td><td>World Precision Instruments</td><td>Steel Plate Base, 10 lbs</td><td></td></tr></tbody></table>

manipulation of gene function in mexican cavefish

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

Multiple populations of cave-dwelling A. mexicanus show reduced sleep and increased wakefulness/activity relative to their surface-dwelling conspecifics14. Hypocretin/orexin (HCRT) is a highly conserved neuropeptide, which acts to increase wakefulness, and aberrations in the HCRT pathway cause narcolepsy in humans and other mammals47,48. We have previously demonstrated that cave A. mexicanus have increased expression of H...

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Manipulacja funkcją genów u meksykańskich ryb jaskiniowych | Genetyka | Wideo

Manipulation of Gene Function in Mexican Cavefish

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

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

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9.0K Views.  Florida Atlantic University. We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Video: Manipulation of Gene Function in Mexican Cavefish

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Developmental Biology

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 ...

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 ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

Behavior

Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep deprivation, and consolidated periods of behavioral immobility. The Mexican cavefish, Astyanax mexicanus (A. mexicanus), is a model for studying trait evolution in response to environmental perturbation. A. mexicanus exist as in eyed surface-dwelling forms and multiple blind cave-dwelling populations that have robust morphological and behavioral differences. Sleep loss has occurred in multiple, independently-evolved cavefish populations. This protocol describes a methodology for quantifying sleep and locomotor activity in A. mexicanus cave and surface fish. A cost-effective video monitoring system allows for behavioral imaging of individually-housed larval or adult fish for periods of a week or longer. The system can be applied to fish aged 4 days post fertilization through adulthood. The approach can also be adapted for measuring the effects of social interactions on sleep by recording multiple fish in a single arena. Following behavioral recordings, data is analyzed using automated tracking software and sleep analysis is processed using customized scripts that quantify multiple sleep variables including duration, bout length, and bout number. This system can be applied to measure sleep, circadian behavior, and locomotor activity in almost any fish species including zebrafish and sticklebacks.

This protocol details the methodology for quantifying locomotor behavior and sleep in the Mexican cavefish. Previous analyses are extended to measure these behaviors in socially-housed fish. This system can be widely applied to study sleep and activity in other fish species.

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) and blind cave (cave fish) morphs in this species presents an opportunity to interrogate the mechanisms underlying morphological and behavioral evolution. Cave fish have evolved novel constructive and regressive traits. The constructive changes include increases in taste buds and jaws, lateral line sensory organs, and body fat. The regressive changes include loss or reduction of eyes. melanin pigmentation, schooling behavior, aggression, and sleep. To experimentally interrogate these changes, it is crucial to obtain large numbers of spawned embryos. Since the original A. mexicanus surface fish and cave fish were collected in Texas and Mexico in the 1990s, their descendants have been routinely stimulated to breed and spawn large numbers of embryos bimonthly in the Jeffery laboratory. Although breeding is controlled by food abundance and quality, light-dark cycles, and temperature, we have found that incremental temperature changes play a key role in stimulating maximal spawning. The gradual increase of temperature from 72 &#176;F to 78 &#176;F in the first three days of a breeding week provides two-three consecutive spawning days with maximal numbers of high-quality embryos, which is then followed by a gradual decrease of temperature from 78 &#176;F to 72 &#176;F during the last three days of the spawning week. The procedures shown in this video outline the workflow before and during a laboratory breeding week for incremental temperature stimulated spawning.

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

This article outlines the basic laboratory conditions and protocols for an incremental temperature regime to stimulate maximal spawning in the Mexican tetra Astyanax mexicanus, which is an emerging model for developmental and evolutionary studies.

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) ...

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 ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Manipulation of Gene Function in Mexican Cavefish

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Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

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

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish

Wholemount In Situ Hybridization for Astyanax Embryos

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

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

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus