Pub Date : 2024-06-01Epub Date: 2024-02-20DOI: 10.1089/zeb.2023.0030
Katherine M Silvius, Genevieve C Kendall
The tp53M214K zebrafish mutant is a versatile platform with which to model a diverse spectrum of human diseases. However, currently available genotyping methods for this mutant require lengthy hands-on processes such as restriction digests and outsourced Sanger sequencing. To address this deficiency, we leveraged high-resolution melting analysis technology in conjunction with a parallel, in-tandem wild-type spike-in approach to develop a robust genotyping protocol capable of discriminating tp53M214K zygosity. In this study, we describe our method in detail. We anticipate that our genotyping protocol will benefit researchers utilizing the tp53M214K zebrafish mutant by offering reliable results with a shorter turnaround time, lower personnel involvement, and higher throughput than traditional methods, thereby decreasing the burden of genotyping and maximizing research efficiency.
{"title":"A Robust Closed-Tube Method for Resolving <i>tp53</i><sup>M214K</sup> Genotypes.","authors":"Katherine M Silvius, Genevieve C Kendall","doi":"10.1089/zeb.2023.0030","DOIUrl":"10.1089/zeb.2023.0030","url":null,"abstract":"<p><p>The <i>tp53</i><sup>M214K</sup> zebrafish mutant is a versatile platform with which to model a diverse spectrum of human diseases. However, currently available genotyping methods for this mutant require lengthy hands-on processes such as restriction digests and outsourced Sanger sequencing. To address this deficiency, we leveraged high-resolution melting analysis technology in conjunction with a parallel, in-tandem wild-type spike-in approach to develop a robust genotyping protocol capable of discriminating <i>tp53</i><sup>M214K</sup> zygosity. In this study, we describe our method in detail. We anticipate that our genotyping protocol will benefit researchers utilizing the <i>tp53</i><sup>M214K</sup> zebrafish mutant by offering reliable results with a shorter turnaround time, lower personnel involvement, and higher throughput than traditional methods, thereby decreasing the burden of genotyping and maximizing research efficiency.</p>","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":" ","pages":"250-254"},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11296207/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139935016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-02-20DOI: 10.1089/zeb.2023.0100
Ligia Carla Balini, Carlos Alexandre Fernandes, Ana Luiza de Brito Portela-Castro, Rafael Fernando de Melo, Cláudio Henrique Zawadzki, Luciana Andreia Borin-Carvalho
In fish species, heterochromatinization is one process that could trigger sex chromosome differentiation. The present article describes a nascent XX/XY sex chromosome system evidenced by heterochromatin accumulation and microsatellite (GATA)8 in Hypostomus albopunctatus from two populations of the Paraná River basin. The specimens of H. albopunctatus from the Campo and Bossi Rivers share the same karyotype. The species exhibits 74 chromosomes (8m+14sm +16st +36a, fundamental number = 112). The C-banding technique suggests male heterogamety in H. albopunctatus, where the Y-chromosome is morphologically like the X-chromosome but differs from it for having long arms that are entirely heterochromatic. Double fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes confirmed the Ag-nucleolus organizer region sites in a single pair for both populations, and minor rDNA clusters showed interpopulational variation. FISH with the microsatellite (GATA)8 probe showed a dispersed pattern in the karyotype, accumulating these sequences of sex chromosomes of both populations. FISH with microsatellite (CGC)10 probe showed interpopulational variation. The absence of differentiated sex chromosomes in H. albopunctatus is described previously, and a new variant is documented herein where XY chromosomes can be seen in an early stage of differentiation.
在鱼类物种中，异染色质化是引发性染色体分化的一个过程。本文描述了来自巴拉那河流域两个种群的白臀鲤的异染色质积累和微卫星（GATA）8所证明的新生XX/XY性染色体系统。来自 Campo 河和 Bossi 河的 H. albopunctatus 标本具有相同的核型。该物种有 74 条染色体（8m+14sm +16st +36a，基数 = 112）。C-banding 技术表明，H. albopunctatus 存在雄性异配现象，其中 Y 染色体在形态上与 X 染色体相似，但长臂完全异色，与 X 染色体不同。用 18S 和 5S rDNA 探针进行的双荧光原位杂交（FISH）证实，两个种群的 Ag 核组织者区域位点均为一对，而次要 rDNA 簇则显示出种群间的差异。用微卫星（GATA）8 探针进行的 FISH 显示，两个种群的核型中都有分散的模式，并积累了性染色体的这些序列。用微卫星（CGC）10探针进行的 FISH 显示了种群间的差异。以前曾描述过白头翁没有分化的性染色体，本文记录了一种新的变异，即在分化的早期阶段可以看到 XY 染色体。
{"title":"Initial Steps of XY Sex Chromosome Differentiation in the Armored Catfish <i>Hypostomus albopunctatus</i> (Siluriformes: Loricariidae) Revealed by Heterochromatin Accumulation.","authors":"Ligia Carla Balini, Carlos Alexandre Fernandes, Ana Luiza de Brito Portela-Castro, Rafael Fernando de Melo, Cláudio Henrique Zawadzki, Luciana Andreia Borin-Carvalho","doi":"10.1089/zeb.2023.0100","DOIUrl":"10.1089/zeb.2023.0100","url":null,"abstract":"<p><p>In fish species, heterochromatinization is one process that could trigger sex chromosome differentiation. The present article describes a nascent XX/XY sex chromosome system evidenced by heterochromatin accumulation and microsatellite (GATA)<sub>8</sub> in <i>Hypostomus albopunctatus</i> from two populations of the Paraná River basin. The specimens of <i>H. albopunctatus</i> from the Campo and Bossi Rivers share the same karyotype. The species exhibits 74 chromosomes (8m+14sm +16st +36a, fundamental number = 112). The C-banding technique suggests male heterogamety in <i>H. albopunctatus</i>, where the Y-chromosome is morphologically like the X-chromosome but differs from it for having long arms that are entirely heterochromatic. Double fluorescence <i>in situ</i> hybridization (FISH) with 18S and 5S rDNA probes confirmed the Ag-nucleolus organizer region sites in a single pair for both populations, and minor rDNA clusters showed interpopulational variation. FISH with the microsatellite (GATA)<sub>8</sub> probe showed a dispersed pattern in the karyotype, accumulating these sequences of sex chromosomes of both populations. FISH with microsatellite (CGC)<sub>10</sub> probe showed interpopulational variation. The absence of differentiated sex chromosomes in <i>H. albopunctatus</i> is described previously, and a new variant is documented herein where XY chromosomes can be seen in an early stage of differentiation.</p>","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":" ","pages":"265-273"},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139935017","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-02-14DOI: 10.1089/zeb.2023.0105
Aubrey E Manning, Hannah Grunwald, Rachel Moran, Roberto Rodriguez-Morales, Amanda K Powers, Suzanne McGaugh, Johanna E Kowalko
Astyanax mexicanus is an emerging model system used to study development, evolution, and behavior of multiple cavefish populations that have repeatedly evolved from conspecific surface fish. Although surface and cavefish live and breed in the laboratory, there are no rapid methods for distinguishing between different cavefish populations. We present 2 methods for genotyping fish for a total of 16 population-specific markers using methods that are easy and inexpensive to implement in a basic molecular biology laboratory. This resource will help researchers maintain independent stocks within the laboratory and distinguish between fish from different populations.
{"title":"Defining the Unseen: Population-Specific Markers for <i>Astyanax mexicanus</i> Blind Cavefish.","authors":"Aubrey E Manning, Hannah Grunwald, Rachel Moran, Roberto Rodriguez-Morales, Amanda K Powers, Suzanne McGaugh, Johanna E Kowalko","doi":"10.1089/zeb.2023.0105","DOIUrl":"10.1089/zeb.2023.0105","url":null,"abstract":"<p><p><i>Astyanax mexicanus</i> is an emerging model system used to study development, evolution, and behavior of multiple cavefish populations that have repeatedly evolved from conspecific surface fish. Although surface and cavefish live and breed in the laboratory, there are no rapid methods for distinguishing between different cavefish populations. We present 2 methods for genotyping fish for a total of 16 population-specific markers using methods that are easy and inexpensive to implement in a basic molecular biology laboratory. This resource will help researchers maintain independent stocks within the laboratory and distinguish between fish from different populations.</p>","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":" ","pages":"255-258"},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11301698/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139737046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-03-04DOI: 10.1089/zeb.2023.0102
Eric Paulissen, Benjamin L Martin
An effective method for tissue-specific ablation in zebrafish is the nitroreductase (NTR)/metronidazole (MTZ) system. Expressing bacterial NTR in the presence of nitroimidazole compounds causes apoptotic cell death, which can be useful for understanding many biological processes. However, this requires tissue-specific expression of the NTR enzyme, and many tissues have yet to be targeted with transgenic lines that express NTR. We generated a transgenic zebrafish line expressing NTR in differentiated skeletal muscle. Treatment of embryos with MTZ caused muscle specific cell ablation. We demonstrate this line can be used to monitor muscle regeneration in whole embryos and in transplanted transgenic cells.
{"title":"A Chemically Inducible Muscle Ablation System for the Zebrafish.","authors":"Eric Paulissen, Benjamin L Martin","doi":"10.1089/zeb.2023.0102","DOIUrl":"10.1089/zeb.2023.0102","url":null,"abstract":"<p><p>An effective method for tissue-specific ablation in zebrafish is the nitroreductase (NTR)/metronidazole (MTZ) system. Expressing bacterial NTR in the presence of nitroimidazole compounds causes apoptotic cell death, which can be useful for understanding many biological processes. However, this requires tissue-specific expression of the NTR enzyme, and many tissues have yet to be targeted with transgenic lines that express NTR. We generated a transgenic zebrafish line expressing NTR in differentiated skeletal muscle. Treatment of embryos with MTZ caused muscle specific cell ablation. We demonstrate this line can be used to monitor muscle regeneration in whole embryos and in transplanted transgenic cells.</p>","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":" ","pages":"243-249"},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11301710/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140023896","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-02-20DOI: 10.1089/zeb.2023.0101
Ana Paula Apolinário da Silva, João Xavier da Silva Neto, José Ytalo Gomes da Silva, Ana Lorena Pereira Bezerra, Bruno Bezerra da Silva, Maria Izabel Florindo Guedes
This study addresses the challenge of collecting blood samples from zebrafish for biochemical analysis. Traditional methods are cumbersome due to low blood flow and rapid coagulation. Based on a previously published technique, we simplified the process by applying an anticoagulant solution directly to the incision site. The modified protocol involves immersing the fish in an ice bath, making a cross-sectional incision, and immediately applying anticoagulant solution. Centrifugation of the specimens provides a streamlined and efficient approach to zebrafish fluid sample collection, compatible with classic biochemical marker analyses.
{"title":"Modified Procedure for Simple and Rapid Collection of Zebrafish Fluid Samples for Biochemical Analyses.","authors":"Ana Paula Apolinário da Silva, João Xavier da Silva Neto, José Ytalo Gomes da Silva, Ana Lorena Pereira Bezerra, Bruno Bezerra da Silva, Maria Izabel Florindo Guedes","doi":"10.1089/zeb.2023.0101","DOIUrl":"10.1089/zeb.2023.0101","url":null,"abstract":"<p><p>This study addresses the challenge of collecting blood samples from zebrafish for biochemical analysis. Traditional methods are cumbersome due to low blood flow and rapid coagulation. Based on a previously published technique, we simplified the process by applying an anticoagulant solution directly to the incision site. The modified protocol involves immersing the fish in an ice bath, making a cross-sectional incision, and immediately applying anticoagulant solution. Centrifugation of the specimens provides a streamlined and efficient approach to zebrafish fluid sample collection, compatible with classic biochemical marker analyses.</p>","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":" ","pages":"259-264"},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139935018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Adrian Romero, Armando Sanchez, Jocelyn D Jones, Kristel Ledesma, M. El-Halawany, Ayman K Hamouda, Brent R Bill
Parkinson's disease (PD) is a neurodegenerative disorder that is clinically assessed by motor symptoms associated with the loss of midbrain dopaminergic neurons affecting the quality of life for over 8.5 million people worldwide. The neurotoxin 6-hydroxydopamine (6-OHDA) has been used to chemically induce a PD-like state in zebrafish larvae by several laboratories; however, highly variable concentration, methodology, and reagents have resulted in conflicting results suggesting a need to investigate these issues of reproducibility. We propose a protocol that addresses the differences in methodology and induces changes in 6 days postfertilization (dpf) larvae utilizing a 24-h exposure at 3 dpf with 30 μM 6-OHDA. Despite ∼50% lethality, no morphological or development differences in surviving fish are observed. Definition of our model is defined by downregulation of the expression of th1 by reverse transcriptase-quantitative polymerase chain reaction, a marker for dopaminergic neurons and a reduction in movement. Additionally, we observed a downregulation of pink1 and an upregulation of sod1 and sod2, indicators of mitochondrial dysfunction and response to reactive oxygen species, respectively.
{"title":"Optimization of Zebrafish Larvae 6-OHDA Exposure for Neurotoxin Induced Dopaminergic Marker Reduction.","authors":"Adrian Romero, Armando Sanchez, Jocelyn D Jones, Kristel Ledesma, M. El-Halawany, Ayman K Hamouda, Brent R Bill","doi":"10.1089/zeb.2023.0098","DOIUrl":"https://doi.org/10.1089/zeb.2023.0098","url":null,"abstract":"Parkinson's disease (PD) is a neurodegenerative disorder that is clinically assessed by motor symptoms associated with the loss of midbrain dopaminergic neurons affecting the quality of life for over 8.5 million people worldwide. The neurotoxin 6-hydroxydopamine (6-OHDA) has been used to chemically induce a PD-like state in zebrafish larvae by several laboratories; however, highly variable concentration, methodology, and reagents have resulted in conflicting results suggesting a need to investigate these issues of reproducibility. We propose a protocol that addresses the differences in methodology and induces changes in 6 days postfertilization (dpf) larvae utilizing a 24-h exposure at 3 dpf with 30 μM 6-OHDA. Despite ∼50% lethality, no morphological or development differences in surviving fish are observed. Definition of our model is defined by downregulation of the expression of th1 by reverse transcriptase-quantitative polymerase chain reaction, a marker for dopaminergic neurons and a reduction in movement. Additionally, we observed a downregulation of pink1 and an upregulation of sod1 and sod2, indicators of mitochondrial dysfunction and response to reactive oxygen species, respectively.","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":"9 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140712071","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fajar Sofyantoro, Nur Indah Septriani, D. S. Yudha, Ega Adhi Wicaksono, D. Priyono, Wahyu Aristyaning Putri, Alfian Primahesa, Anita Restu Puji Raharjeng, Y. A. Purwestri, T. R. Nuringtyas
Animal venoms and toxins hold promise as sources of novel drug candidates, therapeutic agents, and biomolecules. To fully harness their potential, it is crucial to develop reliable testing methods that provide a comprehensive understanding of their effects and mechanisms of action. However, traditional rodent assays encounter difficulties in mimicking venom-induced effects in human due to the impractical venom dosage levels. The search for reliable testing methods has led to the emergence of zebrafish (Danio rerio) as a versatile model organism for evaluating animal venoms and toxins. Zebrafish possess genetic similarities to humans, rapid development, transparency, and amenability to high-throughput assays, making it ideal for assessing the effects of animal venoms and toxins. This review highlights unique attributes of zebrafish and explores their applications in studying venom- and toxin-induced effects from various species, including snakes, jellyfish, cuttlefish, anemones, spiders, and cone snails. Through zebrafish-based research, intricate physiological responses, developmental alterations, and potential therapeutic interventions induced by venoms are revealed. Novel techniques such as CRISPR/Cas9 gene editing, optogenetics, and high-throughput screening hold great promise for advancing venom research. As zebrafish-based insights converge with findings from other models, the comprehensive understanding of venom-induced effects continues to expand, guiding the development of targeted interventions and promoting both scientific knowledge and practical applications.
{"title":"Zebrafish as Versatile Model for Assessing Animal Venoms and Toxins: Current Applications and Future Prospects.","authors":"Fajar Sofyantoro, Nur Indah Septriani, D. S. Yudha, Ega Adhi Wicaksono, D. Priyono, Wahyu Aristyaning Putri, Alfian Primahesa, Anita Restu Puji Raharjeng, Y. A. Purwestri, T. R. Nuringtyas","doi":"10.1089/zeb.2023.0088","DOIUrl":"https://doi.org/10.1089/zeb.2023.0088","url":null,"abstract":"Animal venoms and toxins hold promise as sources of novel drug candidates, therapeutic agents, and biomolecules. To fully harness their potential, it is crucial to develop reliable testing methods that provide a comprehensive understanding of their effects and mechanisms of action. However, traditional rodent assays encounter difficulties in mimicking venom-induced effects in human due to the impractical venom dosage levels. The search for reliable testing methods has led to the emergence of zebrafish (Danio rerio) as a versatile model organism for evaluating animal venoms and toxins. Zebrafish possess genetic similarities to humans, rapid development, transparency, and amenability to high-throughput assays, making it ideal for assessing the effects of animal venoms and toxins. This review highlights unique attributes of zebrafish and explores their applications in studying venom- and toxin-induced effects from various species, including snakes, jellyfish, cuttlefish, anemones, spiders, and cone snails. Through zebrafish-based research, intricate physiological responses, developmental alterations, and potential therapeutic interventions induced by venoms are revealed. Novel techniques such as CRISPR/Cas9 gene editing, optogenetics, and high-throughput screening hold great promise for advancing venom research. As zebrafish-based insights converge with findings from other models, the comprehensive understanding of venom-induced effects continues to expand, guiding the development of targeted interventions and promoting both scientific knowledge and practical applications.","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":"126 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140708841","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Renu Srivastava, Connor W Davison, Abigail G Krull, Seth M Entriken, Amanda Zumbrock, Maria Daniela Cortes Hidalgo, Kiernan J Adair, Anna M Escherich, Jonathan N Lara, Emma C Neverman, Megan Hodnefield, Elyse McElligtot, Elizabeth J Sandquist, Craig Ogilvie, Pascal J Lafontant, J. Essner
We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.
{"title":"An Undergraduate Course in CRISPR/Cas9-Mediated Gene Editing in Zebrafish.","authors":"Renu Srivastava, Connor W Davison, Abigail G Krull, Seth M Entriken, Amanda Zumbrock, Maria Daniela Cortes Hidalgo, Kiernan J Adair, Anna M Escherich, Jonathan N Lara, Emma C Neverman, Megan Hodnefield, Elyse McElligtot, Elizabeth J Sandquist, Craig Ogilvie, Pascal J Lafontant, J. Essner","doi":"10.1089/zeb.2023.0091","DOIUrl":"https://doi.org/10.1089/zeb.2023.0091","url":null,"abstract":"We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":"17 6","pages":"162-170"},"PeriodicalIF":0.0,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140763587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Carvan, Thomas Hansen, Renee A. Hesselbach, Amy Zientek, Craig A. Berg, David H. Petering
The goal of the University of Wisconsin-Milwaukee WInSTEP SEPA program is to provide valuable and relevant research experiences to students and instructors in diverse secondary educational settings. Introducing an online experience allows the expansion of a proven instructional research program to a national scale and removes many common barriers. These can include lack of access to zebrafish embryos, laboratory equipment, and modern classroom facilities, which often deny disadvantaged and underrepresented students from urban and rural school districts valuable inquiry-based learning opportunities. An online repository of zebrafish embryo imagery was developed in the Carvan laboratory to assess the effects of environmental chemicals. The WInSTEP SEPA program expanded its use as an accessible online tool, complementing the existing classroom experience of our zebrafish module. This virtual laboratory environment contains images of zebrafish embryos grown in the presence of environmental toxicants (ethanol, caffeine, and nicotine), allowing students to collect data on 19 anatomical endpoints and generate significant amounts of data related to developmental toxicology and environmental health. This virtual laboratory offers students and instructors the choice of data sets that differ in the independent variables of chemical concentration and duration of postfertilization exposure. This enables students considerable flexibility in establishing their own experimental design to match the curriculum needs of each instructor.
{"title":"Bringing Real Inquiry-Based Science to Diverse Secondary Educational Environments: A Virtual Zebrafish Laboratory to Investigate Environmental Health.","authors":"M. Carvan, Thomas Hansen, Renee A. Hesselbach, Amy Zientek, Craig A. Berg, David H. Petering","doi":"10.1089/zeb.2023.0087","DOIUrl":"https://doi.org/10.1089/zeb.2023.0087","url":null,"abstract":"The goal of the University of Wisconsin-Milwaukee WInSTEP SEPA program is to provide valuable and relevant research experiences to students and instructors in diverse secondary educational settings. Introducing an online experience allows the expansion of a proven instructional research program to a national scale and removes many common barriers. These can include lack of access to zebrafish embryos, laboratory equipment, and modern classroom facilities, which often deny disadvantaged and underrepresented students from urban and rural school districts valuable inquiry-based learning opportunities. An online repository of zebrafish embryo imagery was developed in the Carvan laboratory to assess the effects of environmental chemicals. The WInSTEP SEPA program expanded its use as an accessible online tool, complementing the existing classroom experience of our zebrafish module. This virtual laboratory environment contains images of zebrafish embryos grown in the presence of environmental toxicants (ethanol, caffeine, and nicotine), allowing students to collect data on 19 anatomical endpoints and generate significant amounts of data related to developmental toxicology and environmental health. This virtual laboratory offers students and instructors the choice of data sets that differ in the independent variables of chemical concentration and duration of postfertilization exposure. This enables students considerable flexibility in establishing their own experimental design to match the curriculum needs of each instructor.","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":"90 ","pages":"73-79"},"PeriodicalIF":0.0,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140794206","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Connor W Davison, Hamelynn Harzman, Jessie Nicholson, Seth M Entriken, Kierinn Mobley, Abigail G Krull, Manik Singhal, Caleb Skow, Nathan Matthews, Lindsey Kopp, Benjamin Gillette, Tyler J Weide, Jana R Hukvari, Sofia C P Stumpf, Olivia M Feldmann, M. McGrail, Renu Srivastava, J. Essner
Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFPis86 line to examine the behavior and interactions between cells during vascular formation in zebrafish.
{"title":"Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms.","authors":"Connor W Davison, Hamelynn Harzman, Jessie Nicholson, Seth M Entriken, Kierinn Mobley, Abigail G Krull, Manik Singhal, Caleb Skow, Nathan Matthews, Lindsey Kopp, Benjamin Gillette, Tyler J Weide, Jana R Hukvari, Sofia C P Stumpf, Olivia M Feldmann, M. McGrail, Renu Srivastava, J. Essner","doi":"10.1089/zeb.2023.0096","DOIUrl":"https://doi.org/10.1089/zeb.2023.0096","url":null,"abstract":"Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFPis86 line to examine the behavior and interactions between cells during vascular formation in zebrafish.","PeriodicalId":94273,"journal":{"name":"Zebrafish","volume":"640 ","pages":"191-197"},"PeriodicalIF":0.0,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140791119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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