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Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Sharma, Babita and Subramaniam, Yogaspoorthi J. and Ayyappa Raja, Desingu and Aggarwal, Ayush and Sivasubbu, Sridhar and Natarajan, Vivek T. (2022) Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish. Journal of Visualized Experiments (181). Art. No. e62955. ISSN 1940-087X. doi:10.3791/62955. https://resolver.caltech.edu/CaltechAUTHORS:20220329-597130944

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Abstract

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.3791/62955DOIArticle
Additional Information:© 2022 JoVE Journal of Visualized Experiments. Date Published: March 1, 2022. We acknowledge the funding support from the Council for Scientific and Industrial Research vide project MLP2008 and the Department of Science and Technology for the project GAP165 for supporting the work presented in this manuscript. We thank Jeyashri Rengaraju and Chetan Mishra for their help with experiments. All authors declare no conflict of interest.
Funders:
Funding AgencyGrant Number
Council for Scientific and Industrial Research (CSIR)MLP2008
Department of Science and Technology (South Africa)GAP165
Issue or Number:181
DOI:10.3791/62955
Record Number:CaltechAUTHORS:20220329-597130944
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20220329-597130944
Official Citation:Sharma, B., Subramaniam, Y. J., Ayyappa Raja, D., Aggarwal, A., Sivasubbu, S., Natarajan, V. T. Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish. J. Vis. Exp. (181), e62955, doi:10.3791/62955 (2022).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:114120
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:29 Mar 2022 12:27
Last Modified:04 Apr 2022 21:48

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  • Sharma, Babita and Subramaniam, Yogaspoorthi J. and Ayyappa Raja, Desingu and Aggarwal, Ayush and Sivasubbu, Sridhar and Natarajan, Vivek T. Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish. (deposited 29 Mar 2022 12:27) [Currently Displayed]

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