PUBLICATION

Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish

Authors
Horstick, E.J., Jordan, D.C., Bergeron, S.A., Tabor, K.M., Serpe, M., Feldman, B., Burgess, H.A.
ID
ZDB-PUB-150130-7
Date
2015
Source
Nucleic acids research   43(7): e48 (Journal)
Registered Authors
Bergeron, Sadie, Burgess, Harold, Feldman, Benjamin, Horstick, Eric, Tabor, Kathryn
Keywords
none
MeSH Terms
  • Animals
  • Animals, Genetically Modified
  • Blotting, Western
  • Codon
  • Computational Biology
  • Gene Expression Profiling*
  • Microinjections
  • Molecular Sequence Data
  • Protein Biosynthesis
  • Zebrafish/genetics*
  • Zebrafish Proteins/genetics*
PubMed
25628360 Full text @ Nucleic Acids Res.
Abstract
Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.
Genes / Markers
Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes