We appreciate Dr. Jeong-Soo Lee for sharing the Tg(dbh:EGFP-MYCN) transgenic fish with us in our study. This work was supported by a grant 1K99CA178189-01 from the National Cancer Institute, a fellowship from the Pablove Foundation and the Friends for Life, and young investigator awards from the Alex's Lemonade Stand Foundation and the CureSearch for Children's Cancer Foundation.

NOTE: All zebrafish studies and maintenance of the animals were done in accord with Mayo Clinic Institute IACUC-approved protocol # A41213.
1. DNA Constructs for Transgenesis
<ol>
	<li>Amplify a 5.2-kb dopamine beta hydroxylase (d&#946;h) promoter region8 using the CH211-270H11 BAC clone (from BACPAC resources center (BPRC)) as a DNA template. Use a PCR system appropriate for long and accurate PCR amplification of long DNA templates and the following cycle progra...

<ol>
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In this representative study, we used transient coinjection and coexpression of activated ALK with the mCherry reporter gene in MYCN-expressing transgenic fish to show that these genes cooperate to markedly accelerate the onset of neuroblastoma, consistent with our previous finding in compound stable transgenic fish coexpressing both activated ALK and MYCN 8. This mosaic transgenic approach possesses several distinct advantages over the conventional method. Most imp...

Cancers are progressive diseases marked by the accumulation of pathologic mutations, deletions and chromosome gains over time. These genetic abnormalities can affect multiple cellular processes ranging from the cell cycle, cell death, energetic metabolism and assembly of the cytoskeleton to stress responses such as hypoxia. Hence, tumorigenesis reflects the collective actions of multiple genetic aberrations across a spectrum of biological processes. Recent integrative genomic research efforts, including whole genome sequencing, exome sequencing, targeted sequencing, deep sequencing and genome-wide association studies, have identified a growing number of novel genetic alterations in essentially all types of tumors 1-4. In many instances, the genetic lesions occur together in a nonrandom manner 5-8, suggesting their cooperation in disease pathogenesis. Dissecting the oncogenic roles of the large array of aberrantly expressed genes resulting from these genomic lesions is necessary to devise new therapeutic strategies and to understand the responses of tumor cells to these agents, but this has proved to be a daunting task, requiring very robust animal model systems for the conduct of high-throughput functional genomic analysis in vivo.
Although mammals, especially rodents, are favored models in cancer biology, the zebrafish has begun to attract considerable attention. The teleost zebrafish (Dario rerio) has been used as a model organism for development study since the 1960s and was first applied to the study of tumor pathogenesis in 1982 9-11. Ease of maintenance, small body size, and high fecundity make the zebrafish a robust model for large-scale forward genetic screens to identify mutations that confer abnormal and pathological phenotypes 10. The optical transparency of zebrafish embryos is another key feature supporting wider use of this cancer model, as it allows in vivo imaging to be conducted to locate tumor development in real time 9, an application that is relatively difficult in rodents 12. Recent comparative genomics analysis of the zebrafish reference genome (Zv9) revealed 26,206 protein-coding genes, with 71% having human orthologues, of which 82% are correlated with disease-associated genes in the Online Mendelian Inheritance in Man (OMIM) database 13,14. Consequently, the zebrafish has been used to model diverse types of human cancers, including neuroblastoma 8, T-cell acute lymphoblastic leukemia (T-ALL) 15,16, melanoma 17,18, Ewing&#8217;s sarcoma 19, rhabdomyosarcoma 20,21, pancreatic carcinoma 22, hepatocellular carcinoma 23 and myeloid malignancies 24,25, and has been selected as a cancer model for xenotransplantation studies 11,26.
A stable transgenic approach in zebrafish is commonly used to study the effect of gain-of-function of genes in normal development or disease pathogenesis 27,28. To develop such a model (Figure 1A), one injects a DNA construct containing the gene of interest driven by a tissue-specific promoter into one-cell wild-type embryos. Three to four months after injection, when the injected embryos reach sexual maturity, they are outcrossed with wild-type fish to screen for the ones showing integration of the DNA construct in their germline, which licenses them as founder fish. Many factors, such as the copy number and integration site of the transgene, affect expression of the transgene in stable transgenic lines. Thus, to develop a transgenic tumor model, multiple stable transgenic lines overexpressing a single oncogene have to be generated first and screened for the line expressing the transgene at a level that might lead to tumor induction. However, if overexpression of a candidate oncogene is toxic to germ cells, it is difficult to generate a stable transgenic line by directly overexpressing the transgene 29. Hence, this approach can be time-consuming, with a high risk of failure to generate a suitable cancer model.
Here, we illustrate an alternative strategy based on mosaic transient transgenesis (Figure 1B) that provides unique advantages over traditional stable transgenesis for functional genomic study in vivo. In this approach, one or more transgene constructs are injected into the one-cell stage of transgenic or wild-type embryos. The injected DNA constructs containing transgenes are then mosaically and randomly integrated into the primary injected fish, resulting in mixed genotypes within multiple cell populations in individual fish 30. Moreover, coinjection of multiple DNA constructs in one-cell embryos leads to co-integration into the same cell at random sites, allowing one to trace the cells with expression of transgenes and explore the interactions of different genes during disease pathogenesis in the mosaic animals 31. As proof of principle, we transiently overexpressed mutationally activated ALK (F1174L) with mCherry reporter gene in the peripheral sympathetic nervous system (PSNS) under control of the dopamine beta hydroxylase (d&#946;h) promoter in wild-type fish and transgenic fish overexpressing MYCN. ALK, which encodes a receptor tyrosine kinase, is the most frequently mutated gene in high-risk neuroblastoma 5-7,32,33. ALK (F1174L), as one of the most frequent and potent somatic activating mutations, is over-represented in MYCN-amplified high-risk neuroblastoma patients and synergizes with MYCN overexpression to accelerate neuroblastoma tumorigenesis in both stable transgenic mice and transgenic zebrafish models 8,34,35. By mosaic transient overexpression of ALK (F1174L) with mCherry in the MYCN transgenic fish, we recapitulated the acceleration of tumor onset observed in the stable transgenic fish overexpressing both ALK (F1174L) and MYCN, suggesting that the mosaic transgenesis strategy can be used to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="663">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Name of Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Expand Long Template PCR System&#160;</td>
			<td style="width:171px;">Roche Applied Science, IN</td>
			<td style="width:119px;">11681834001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCR-TOPO vector&#160;</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td style="width:119px;">451641</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">T4 DNA ligase</td>
			<td style="width:171px;">New England Biolabs, MA</td>
			<td style="width:119px;">M0202M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway LR Clonase II enzyme 
			Mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11791-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway&#174; BP Clonase&#174; II enzyme mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11789-020</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">GC-RICH PCR System&#160;</td>
			<td style="width:171px;">Roche Applied Science, IN</td>
			<td>12 140 306 001</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Meganuclease I-SceI&#160;</td>
			<td style="width:171px;">New England Biolabs, MA</td>
			<td>R0694S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon SMZ-1500 stereoscopic fluorescence microscope&#160;</td>
			<td style="width:171px;">Nikon, NY</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon digital sight DS-U1 camera</td>
			<td style="width:171px;">Nikon, NY</td>
			<td style="width:119px;"></td>
		</tr>
	</tbody>
</table>

mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

To investigate whether overexpression of mutationally activated ALKF1174L or wild-type ALK could collaborate with MYCN in neuroblastoma induction, we overexpressed either activated human ALK or wild-type human ALK under control of the d&#946;h promoter in the PSNS of transgenic fish overexpressing MYCN. Either of the following constructs, d&#946;h-ALKF1174L or d&#946;h-ALKWT, were coinjected with d&#94...

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis at JoVE.com

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

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11.0K ビュー.  Mayo Clinic College of Medicine, Center for Individualized Medicine. The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

ビデオ: Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in...

zebrafish model of neuroblastoma metastasis

Zebrafish Model of Neuroblastoma Metastasis

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

mosaic zebrafish transgenesis for evaluating enhancer sequences

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish...

minicoopr vector-based transgenesis: a technique to screen melanoma-inducing genes in zebrafish tumor model

This video describes a method to screen for the melanoma modifying genes using a transgenic zebrafish tumor model. When injected with a melanoma-inducing miniCoopR vector, the zebrafish develops melanomas and can therefore be used to study the genetic factors affecting tumor development in...

miniCoopR Vector-based Transgenesis: A Technique to Screen Melanoma-inducing Genes in Zebrafish Tumor Model

genetic manipulation in &delta;ku80 strains for functional genomic analysis of toxoplasma gondii

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

microinjection for transgenesis and genome editing in threespine sticklebacks

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...

the zebrafish tol2 system: a modular and flexible gateway-based transgenesis approach

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish...

lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (madm)

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

generation of zebrafish larval xenografts and tumor behavior analysis

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging...

a simple, rapid, and effective method for tumor xenotransplantation analysis in transparent zebrafish embryos

Author Spotlight: Advancing Cancer Therapeutics Using Tumor Xenotransplantation in Zebrafish

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow...

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

embryo microinjection for transgenesis in drosophila

Author Spotlight: Optimizing Embryo Microinjection for Transgenesis in &lt;i&gt;Drosophila&lt;/i&gt;

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic...

phiC31 Integrase-Mediated Embryo Transgenesis in &lt;i&gt;Drosophila&lt;/i&gt;

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transient expression of foreign genes in insect cells (sf9) for protein functional assay

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Embryo Microinjection for Transgenesis in Drosophila

screening for melanoma modifiers using a zebrafish autochthonous tumor model

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are...

use of a recombinant mosquito densovirus as a gene delivery vector for the functional analysis of genes in mosquito larvae

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

deciphering and imaging pathogenesis and cording of mycobacterium abscessus in zebrafish embryos

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Utilizing Larval Zebrafish for Efficient In Vivo Tumor Studies

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orthotopic xenografting of human luciferase-tagged malignant peripheral nerve sheath tumor cells for in vivo testing of candidate therapeutic agents

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described.  The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also...

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

tol2 transposon-mediated zebrafish transgenesis: a procedure to generate transgenic zebrafish following the coinjection of tol2 system into fertilized zebrafish embryos

In this video, we demonstrate a procedure to generate a stable zebrafish transgenic line constitutively expressing a fluorescent voltage indicator protein following the co-injection of the Tol2 transposon system comprising the transposase mRNA and Tol2 transposon construct encoding the fluorescent voltage...

Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos

an in vitro protocol for evaluating microrna levels, functions, and associated target genes in tumor cells

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of...

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

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 ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...