Name: manipulation of gene function in mexican cavefish
Uploaded: 2019-04-22T13:00:00.000+00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Manipulation of Gene Function in Mexican Cavefish at JoVE.com

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 system30,36,42,53.&#160;
Morpholinos have been widely used in zebrafish research to knockdown the expression of genes. The approach is facile and results in a robust knockdown of expression. However, off-target effects have been widely documented22,53; thus, animals in which morpholinos have been injected to should be carefully monitored for any unexpected phenotypes22,55. When possible, results obtained from morpholino knockdown should be validated through the use of other methods, such as CRISPR/Cas9-mediated knockout approaches. While morpholinos allow for the robust knockdown of gene expression, morpholino-mediated knockdown is transient. Thus, analyzing adult phenotypes is not possible when using this method.
CRISPR/Cas9-mediated mutagenesis offers direct manipulation of specific genes. Further, unlike morpholino-mediated knockdown, CRISPR/Cas9 mutagenesis allows for the analysis of mutant phenotypes into adulthood. To prevent off-target effects of CRISPR/Cas9, mutant lines should be outcrossed several generations, and when possible, more than one allele should be obtained and tested. The CRISPR/Cas9 system also provides the potential for utilizing gene-editing approaches to knock-in alleles or to produce specific genetic changes. The CRISPR/Cas9 system has been used in zebrafish to produce precise integrations of exogenous DNA and to generate precise point mutations19,20,56,57,58,59. With the sequencing of the cavefish genome, it is now possible to identify single nucleotide polymorphisms (SNPs) or other subtle genetic changes between surface fish and cavefish populations33. The application of CRISPR/Cas9 gene editing provides the opportunity to exchange alleles between surface fish and cavefish, or between different populations of cavefish, to examine the role of these genetic changes in different developmental processes.
The transgenesis approaches described in this protocol provide a simple and powerful method for gain-of-function studies and for generating tools to alter biological processes genetically. The Tol2 system is widely used in zebrafish research, and we have shown that it is similarly powerful in A. mexicanus. Moreover, we demonstrated a transgenic construct generated in zebrafish that utilizes a zebrafish promoter and recapitulates endogenous expression in A. mexicanus. We have found four other promoters isolated from zebrafish that drive tissue-specific expression as expected in A. mexicanus (data not shown). Since zebrafish promoters recapitulate their conserved expression patterns in A. mexicanus, this suggests that many of the genetic tools can be transferred directly from zebrafish to A. mexicanus without the need for modification with A. mexicanus promotors. Moreover, with the advance in sequencing technologies in A. mexicanus33, the transgenic approaches described here will permit a powerful future for the investigation of enhancers and promoters that may play a role in the variation between surface and cave forms. Lastly, the powerful tools for the genetic manipulation of biological processes that have made zebrafish valuable are equally important in A. mexicanus60,61,62,63. Differences in diverse behavioral traits, such as sleep, feeding, stress, and aggression, between A. mexicanus surface- and cave-dwelling forms have been extensively documented12,14,15,38,64, yet the underlying neuronal correlates are not well understood. Tools such as Tg(elavl3:GCaMP6s) will permit a dissection of how differences in neuronal activity brain-wide correlate with differences in behavior and offer a unique insight into how the brain has be modified evolutionarily.
Taken together, A. mexicanus is poised to become a leading model for investigating the evolution of a variety of morphological and behavioral traits. The diverse differences in complex biological processes in A. mexicanus provide a platform for investigating genetic mechanisms of trait evolution. The application of tools for manipulating gene function may help develop this organism into a model that can be applied to investigate biological diseases related to eye degeneration, neurodevelopmental abnormalities, and insomnia.

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 HCRT peptide, suggesting that an increased expression of this peptide may underlie the loss of sleep in cavefish38. The MO knockdown of hcrt expression provides a powerful approach for directly examining the effect of increased hcrt expression mediating the loss of sleep in cavefish.
To examine the relationship between hcrt expression and sleep, we designed a translation-blocking morpholino. The MO targets the first 25 bp of exon one, including the ATG start site (Figure 1A,B). As a control, we utilized a commercially available scrambled MO control (Figure 1B). Using the BLAST algorithm on NCBI, we confirmed that there are no off-target effects for either MO throughout the genome (data not shown). A. mexicanus surface fish and Pach&#243;n cavefish were bred and their eggs were collected and then injected with 400 pg of MO in a 1 nL-volume at the one-cell stage (Figure 1C,D). The fish were raised to 4 dpf and then measured for activity and sleep behavior.&#160;
Cave A. mexicanus injected with the control MO exhibited significantly more locomotor activity and reduced sleep over a 24 h period compared to surface fish also injected with the scrambled MO (Figure 1E,F), suggesting no effect of the control morpholino injections as the results are consistent with previously published activity and sleep patterns in each morphotype (t = 5.021, df = 88, p &#60; 0.0001). The injection of the HCRT-MO had little effect on sleep in surface fish compared to control-injected fish (t = 0.17, df = 88, p &#62; 0.99). In contrast, hcrt knockdown via MO injection had a significant effect on sleep in cave-dwelling fish. Pach&#243;n cavefish larvae showed less than a fourfold reduction in locomotor activity, and sleep nearly two times more than control Pach&#243;n larvae (Figure 1E,F; t = 2.694, df = 88, p &#60; 0.05). A comparison of locomotor activity and sleep in MO-knockdown surface- and cave-dwelling fish revealed comparable locomotion and sleep amounts (Figure 1E,F). These data provide a direct link between hcrt expression and sleep loss and provide a method to interrogate the biological mechanisms for loss of sleep in the evolutionarily derived cave-dwelling morphs.
Pigmentation loss is a hallmark of cave organisms, and multiple cave-dwelling Astyanax populations demonstrate loss of pigmentation. Albinism in Molino and Pach&#243;n cavefish has been mapped using QTL mapping to a genomic region containing the gene, ocular albinism 2 (oca2), suggesting mutations in oca2 underlie albinism in cavefish6.
To validate oca2 as the causative locus for albinism in Pach&#243;n cave morphs, we utilized CRISPR/Cas9 gene editing to mutate this region in surface fish populations. Since exon 21 is deleted in Molino fish6, we designed a gRNA to target this region of the gene (Figure 2A). The genomic sequence, including the gRNA target sequence and the PAM (bolded), is 5&#39;-GGTCATGTGGGTCTCAGCTTTGG-3&#39;. This target sequence (without the PAM sequence) was used to generate a gRNA for targeted mutagenesis. Surface breeders were made to mate, and the resulting embryos were collected at the one-cell stage. One-cell stage embryos were injected with Cas9 mRNA and gRNA-targeting oca2, and the injected animals were raised to adulthood43. The injected adults were made to mate to wild-type surface fish, and the embryos from these crosses were genotyped to determine germ-line transmission, using primers from step 6.3.1. We sequenced a mutant oca2 allele and identified a germ-line-transmitted 2 bp deletion in oca2 (Figure 2B,C). For ease of genotyping, we designed allele-specific primers to identify the wild-type and mutant alleles (Figure 2D) and genotyped fish, using PCR followed by gel electrophoresis (Figure 2E). We incrossed surface fish heterozygous for this oca2 mutant allele. The resulting progeny were pigmented or albino (Figure 2F-I). Pigmented individuals were wild-type or heterozygous at the oca2 locus, while albino individuals were homozygous mutant (Figure 2E). These data provide a direct link between the oca2 gene locus and albinism in A. mexicanus cavefish.
Myriad behaviors such as sleep, feeding, and stress differ in A. mexicanus cavefish relative to surface conspecifics, yet the underlying neuronal determinants between morphs remain unclear. Whole-brain calcium imaging provides a powerful unbiased approach for examining correlations between altered neural activity and modified behavior. We generated surface fish and cavefish with a near-ubiquitous neuronal expression of the genetically encoded calcium indicator, GCaMP6s, using reagents widely used in zebrafish research. ELAV-like neuron-specific RNA-binding protein 3 (elavl3) is expressed endogenously in newly differentiated neurons throughout the central nervous system49 and has been used in zebrafish to drive the expression of proteins throughout the majority of the nervous system50. We obtained a Tol2 construct containing ~2.8 kb zebrafish elavl3 promoter transcription upstream of the genetically encoded calcium indicator GCaMP6s (a fusion of green fluorescent protein [GFP], calmodulin, and M13, a peptide sequence from myosin light-chain kinase), flanked at the 5&#39; and 3&#39; ends with Tol2 sites (Tol2-elavl3:GCaMP6s-Tol2)51.&#160;
A. mexicanus surface fish and Molino cavefish were bred, and the resulting embryos were coinjected at the single-cell stage with 25 ng/&#956;L of the Tol2-elavl3:GCaMP6s-Tol2 construct and 25 ng/&#956;L Tol2 transposase mRNA (Figure 3A). At 24&#8211;48 dpf, larvae were screened for a transient neuronal expression of GCaMP6s. Those injected (F0) embryos with an expression of GCaMP6s were raised to adulthood and backcrossed with wild-type adults derived from the same lineage of surface fish or cavefish. The resulting F1 adults were screened for a stable expression of GCaMP6s expression, and those larvae were maintained to generate stable lines (Figure 3B,C). Because each F1 adult with a stable expression has likely integrated Tol2-elavl3:GCaMP6s-Tol2 at different genomic sites, each F1 is designated a different allele. Using this approach, we have generated stable F1s for surface fish and Molino cavefish populations (Figure 3B,C), thus enabling live calcium imaging to uncover differences in neuronal activity mediating behavioral changes in the cave environment (Figure 3C,D). Further, this approach lays the groundwork for the expression of many additional transgenes to characterize and manipulate the gene function in A. mexicanus.&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59093/59093fig1.jpg" /> 
Figure 1: Morpholino knockdown of Hcrt reduces activity and increases sleep in cavefish. (A) Translation-blocking morpholino targets the first 25 bp of the Hcrt coding sequence, including the ATG start site. (B) Hcrt morpholino oligo (MO) and control sequences. (C) Alignment of ~200 single-cell eggs on agarose egg molds for injection. (D) Microinjection of 1.0 nL of injection mixture with phenol red indicator for visualization. The scale bar = 0.5 mm. (E) Morpholino knockdown of Hcrt reduces the activity (total distance traveled) of Pach&#243;n cavefish (t = 5.021, df = 88, p &#60; 0.0001) but not of surface fish (t = 1.318, df = 88, p &#62; 0.72). (F) Knockdown increases sleep in Pach&#243;n cavefish (t = 2.694, df = 88, p &#60; 0.05) but has no effect on surface fish (t = 0.17, df = 88, p &#62; 0.99). The scale bar = 1 mm. The error bars in panels E and F denote the standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59093/59093fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59093/59093fig2.jpg" /> 
Figure 2: CRISPR gene-editing of oca2 introduces albinism in surface-dwelling A. mexicanus. (A) Schematic of the oca2 coding regions, and guide RNA (gRNA) targeting exon 21 for CRISPR-mediated gene editing. (B) Sequencing chromatogram of wild-type Oca2+ and (C) mutant Oca2- alleles. The red box in panel B indicates the 2 bp sequence, which is missing in the mutant Oca2- allele depicted in panel C. (D) Targeted CRISPR introduces a 2 bp deletion, disrupting Oca2 function. Primers are designed for the genotypic screening of 2 bp deletion in F0 offspring. (E) PCR and gel electrophoresis of Oca2 in wild-type surface fish (band size = 134 bp) and F0 CRISPR-injected offspring (band size = 133 bp). Individuals homozygous or heterozygous for the wild-type variant of Oca2 (Oca2+) are pigmented, while F0s homozygous for the 2 bp deletion (Oca2-) harbor albinism. (F) Whole-body images of wild-type surface fish and (G) of surface fish with CRISPR-mediated albinism. The scale bar = 5 mm. (H and I) Magnified view of the heads of individuals depicted in panels F and G, respectively. The scale bar = 2 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59093/59093fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59093/59093fig3.jpg" /> 
Figure 3: Tol2 transgenesis of pan-neuronal GCAMP6s enables the live imaging of brain activity in A. mexicanus. (A) Tol2-mediated transgenesis in A. mexicanus. Coinjection of Tol2 mRNA and Tol2-elav3l:GCAMP6s-Tol2 plasmid integrates GCAMP6s transgene in F0 founders. Backcrossing to the original stock of wild-type fish yields F1 individuals with a stable pan-neuronal expression of GCAMP6s. (B and C) The resulting offspring are screened for a stable expression using dissection and confocal microscopy. Stable TgAsty(elav3l:GCaMP6s) F1 individuals have been established for both surface- (depicted in panel B) and cave-dwelling morphotypes (depicted in panel C). The scale bar = 200 &#956;m.&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59093/59093fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume or Amount</td>
		</tr>
		<tr>
			<td>10.0 &#956;g of pCS-zT2TP plasmid DNA</td>
			<td>X &#956;l</td>
		</tr>
		<tr>
			<td>NEB CutSmart Buffer</td>
			<td>10.0 &#956;l</td>
		</tr>
		<tr>
			<td>NotI-HF Enzyme</td>
			<td>2.0 &#956;l</td>
		</tr>
		<tr>
			<td>Nuclease-free H2O</td>
			<td>X &#956;l</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>1.0 &#956;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>100 &#956;l</td>
		</tr>
	</tbody>
</table>
Table 1: Restriction digest of Tol2 plasmid.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume or Amount</td>
		</tr>
		<tr>
			<td>1.0 &#956;g of linearized pCS-zT2TP plasmid DNA</td>
			<td>X &#956;l</td>
		</tr>
		<tr>
			<td>10x Reaction buffer</td>
			<td>2.0 &#956;l</td>
		</tr>
		<tr>
			<td>2x NTP/CAP</td>
			<td>10.0 &#956;l</td>
		</tr>
		<tr>
			<td>SP6 enzyme mix</td>
			<td>2.0 &#956;l</td>
		</tr>
		<tr>
			<td>RNase-free H2O&#160;</td>
			<td>X &#956;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>&#8804;20 &#956;l</td>
		</tr>
	</tbody>
</table>
Table 2: In vitro synthesis of Tol2 mRNA.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Setting name</td>
			<td>Setting value</td>
		</tr>
		<tr>
			<td>Heat</td>
			<td>510</td>
		</tr>
		<tr>
			<td>Pull</td>
			<td>55</td>
		</tr>
		<tr>
			<td>Velocity</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Time</td>
			<td>40</td>
		</tr>
		<tr>
			<td>Pressure</td>
			<td>500</td>
		</tr>
		<tr>
			<td>Ramp</td>
			<td>534</td>
		</tr>
	</tbody>
</table>
Table 3: Sample pipette-pulling protocol.&#160;
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume or Amount</td>
		</tr>
		<tr>
			<td>Morpholino (Freezer stock @ 4mM)</td>
			<td>1.0 &#956;l</td>
		</tr>
		<tr>
			<td>Nuclease-free H2O or Danieau&#8217;s solution</td>
			<td>17.0 &#956;l</td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>2.0 &#956;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>20 &#956;l</td>
		</tr>
	</tbody>
</table>
Table 4: Morpholino injection mixture.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume of Amount</td>
		</tr>
		<tr>
			<td>gRNA (final working concentration @ 100ng/&#956;l)</td>
			<td>1 &#956;l</td>
		</tr>
		<tr>
			<td>Cas9 mRNA (final working concentration @ 1200ng/&#956;l)</td>
			<td>1 &#956;l</td>
		</tr>
		<tr>
			<td>RNase-free H2O</td>
			<td>2 &#956;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>4 &#956;l</td>
		</tr>
	</tbody>
</table>
Table 5: Sample CRISPR/Cas9 injection mixture.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume of Amount</td>
		</tr>
		<tr>
			<td>Tol2 plasmid (final working concentration @ 25ng/&#956;l)</td>
			<td>X &#956;l</td>
		</tr>
		<tr>
			<td>Tol2 mRNA (final working concentration @ 25ng/&#956;l)</td>
			<td>X &#956;l</td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>1.0 &#956;l</td>
		</tr>
		<tr>
			<td>RNase-free H2O</td>
			<td>X &#956;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>15 &#956;l</td>
		</tr>
	</tbody>
</table>
Table 6: Sample Tol2 transgenesis injection mixture

Watch this Scientific Journal Video about Manipulation of Gene Function in Mexican Cavefish at JoVE.com

Manipulation of Gene Function in Mexican Cavefish | Genetics | Video

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, ...

manipulation-of-gene-function-in-mexican-cavefish

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Genetics

Evolution

Evolutionary History

SCIENTISTS IN ACTION

9.0K Views.  Florida Atlantic University. Grotdieren bieden een overtuigend systeem voor het onderzoeken van diverse morfologische, ontwikkelings- en gedragskenmerken. Maar een van de beperkende factoren bij het opzetten van dit modelsysteem is een gebrek aan genetische hulpmiddelen die ons in staat zouden stellen om de genfunctie te manipuleren. Hier tonen we drie verschillende benaderingen om de genfunctie te manipuleren in de Mexicaanse grotvis, Astyanax mexicanus.Deze instrumenten zullen het onderzoek mogelijk maken van de...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video