NEW YORK (GenomeWeb News) – In a paper appearing in the early, online version of Nature Methods this week, researchers from the US, Germany, and India reported that they have developed a system for flipping the expression of vertebrate genes on and off in the zebrafish model system, while concurrently getting clues about protein function and localization.
Using a conditional insertional transposon mutagenesis approach, the team created hundreds of zebrafish lines in which gene expression is knocked down by a vector that can be used to track typical expression patterns at the affected locus. Consequently, the lines provide an opportunity to look at the functional consequences of knocking down a gene while concurrently getting clues about when and where it is normally expressed.
"Integration of … a gene-breaking transposon containing a protein trap, efficiently disrupts gene expression with [more than] 97 percent knockdown of normal transcript amounts and simultaneously reports protein expression for each locus," senior author Stephen Ekker, a molecular biology researcher affiliated with the Mayo Clinic and the University of Minnesota, and co-authors wrote.
"This opens up the door to a segment of biology that has been impossible or impractical with existing genomics research methods," Ekker said in a statement, explaining that the approach "adds an additional level of complexity to the genome project."
The researchers used a conditional in vivo protein-trap mutagenesis method to create a collection of zebrafish lines, employing a vector containing gene-break transposons coupled with protein trap and exon trap domains.
This vector, known as RP2, contains a red fluorescent protein tagged protein trap domain and a green fluorescent protein tagged 3' exon trap, as well as gene-break transposons.
Transcription gets disrupted when the vector is plopped into gene sequences by the transposon, they explained. Expression of the red fluorescent protein reporter and green fluorescent protein then helped them figure out where transposons are integrated, how this impacts gene expression, and where and when proteins at the loci are normally expressed. For instance, researchers reported, roughly one third of the 350 zebrafish protein trap lines assessed so far show neuronal expression patterns.
"An RP2 protein-trap-mutagenized gene produces [monomeric red fluorescent protein] expression that mimics endogenous expression of the gene and quantitatively removes the normal full-length protein," researchers wrote.
Because the mutagenic vector is flanked by sequences that are cut by a recombinase known as Cre, the team could reverse gene knockdown by tossing in and inducing messenger RNA that codes for the enzyme.
Along with the loci examined in the current study, researchers reportedly intend to use their approach to study thousands more loci in the zebrafish genome.
"The RP2 mutagenesis system is a step toward a unified 'codex' of protein expression and direct functional annotation of the vertebrate genome," the team wrote. "As the complexity of the protein-trap expression library increases, each line will contribute to its own codex as well as enable morphological, developmental, and molecular annotation of the zebrafish."
Even so, they emphasized, getting a more complete view of this codex system will likely require different alleles targeting the same coding genes, along with additional information generated using vectors that integrate at other sites in the genome.
Information on the existing zebrafish protein traps lines and the vectors used to develop them is available through zfishbook and will eventually be included in the Zebrafish International Resource Center.