Combining genotypic and phenotypic analyses on single mutant zebraﬁsh larvae

MethodsX, 2018 · DOI: https://doi.org/10.1016/j.mex.2018.03.002 · Published: March 14, 2018

Simple Explanation

This paper presents a method for analyzing both the genetic makeup and physical characteristics of individual zebraﬁsh larvae. The method is useful when studying mutations that are lethal at the embryonic or larval stages. The method involves taking a small sample from the tail of the larva for genetic testing (genotyping) and then examining the rest of the larva for any physical differences (phenotype). The procedure includes protocols for genotyping, histological analyses, protein and histone extraction, RNA extraction, and studying spinal cord regeneration, all optimized for single zebraﬁsh embryos and larvae.

Study Duration

Not specified

Participants

Zebrafish embryos or larvae

Evidence Level

Not specified

Key Findings

1
The method allows for both genotyping and phenotype analyses of mutant zebraﬁsh larvae from heterozygous zebraﬁsh incrosses.
2
The larval tail is used for genotyping after transection, while phenotypic studies are performed on the anterior part of the zebraﬁsh larvae.
3
The method includes protocols for DNA extraction, RFLP assay, paraffin embedding, histological analyses, protein and histone extraction, Western blot, RNA extraction, RT-PCR, and caudal spinal cord regeneration studies.

Research Summary

The presented method combines genotyping and phenotype analyses of mutant zebraﬁsh larvae from heterozygous incrosses. The procedure involves genotyping the larval tail after transection and performing phenotypic studies on the anterior part of the larvae. The method includes protocols for various analyses such as histology, protein extraction, and RNA extraction, optimized for single zebraﬁsh embryos and larvae.

Practical Implications

Improved mutant analysis

Enables the study of lethal mutations by allowing both genotype and phenotype analysis on single larvae.

Optimized protocols

Provides detailed protocols for various molecular and histological analyses optimized for single zebraﬁsh larvae.

Spinal cord regeneration studies

Includes a protocol for studying caudal spinal cord regeneration in mutant zebraﬁsh larvae.

Study Limitations

1
The Western blot results strongly rely on the quality of the antibodies.
2
The limited number of zebraﬁsh antibodies available often makes protein-based analyses more difﬁcult.
3
Setting PCR conditions may need modifications depending on primer annealing temperatures.

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