PUBLICATION

Efficient transgenesis mediated by pigmentation rescue in zebrafish

Authors
Harrold, I., Carbonneau, S., Moore, B.M., Nguyen, G., Anderson, N.M., Saini, A.S., Kanki, J.P., Jette, C.A., Feng, H.
ID
ZDB-PUB-160114-10
Date
2016
Source
Biotechniques   60: 13-20 (Journal)
Registered Authors
Anderson, Nicole M., Feng, Hui, Harrold, Itrat, Jette, Cicely A., Kanki, John, Moore, Bethany
Keywords
pigmentation rescue, transgenesis, zebrafish
MeSH Terms
  • Animals
  • Animals, Genetically Modified
  • Gene Transfer Techniques*
  • Genotype
  • Green Fluorescent Proteins/genetics
  • Microphthalmia-Associated Transcription Factor/genetics*
  • Microscopy, Fluorescence
  • Pigmentation/genetics*
  • Promoter Regions, Genetic*
  • Zebrafish/genetics
  • Zebrafish/physiology
  • Zebrafish Proteins/genetics*
PubMed
26757807 Full text @ Biotechniques
Abstract
The zebrafish represents a revolutionary tool in large-scale genetic and small-molecule screens for gene and drug discovery. Transgenic zebrafish are often utilized in these screens. Many transgenic fish lines are maintained in the heterozygous state due to the lethality associated with homozygosity; thus, their progeny must be sorted to ensure a population expressing the transgene of interest for use in screens. Sorting transgenic embryos under a fluorescence microscope is very labor-intensive and demands fine-tuned motor skills. Here we report an efficient transgenic method of utilizing pigmentation rescue of nacre mutant fish for accurate naked-eye identification of both mosaic founders and stable transgenic zebrafish. This was accomplished by co-injecting two constructs with the I-SceI meganuclease enzyme into pigmentless nacre embryos: I-SceI-mitfa:mitfa-I-SceI to rescue the pigmentation and I-SceI-zpromoter:gene-of-interest-I-SceI to express the gene of interest under a zebrafish promoter (zpromoter). Pigmentation rescue reliably predicted transgene integration. Compared with other transgenic techniques, our approach significantly increases the overall percentage of founders and facilitates accurate naked-eye identification of stable transgenic fish, greatly reducing laborious fluorescence microscope sorting and PCR genotyping. Thus, this approach is ideal for generating transgenic fish for large-scale screens.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes