Mayo Clinic, Rochester, Minnesota, USA.
Nat Methods. 2011 Jun;8(6):506-15. doi: 10.1038/nmeth.1606. Epub 2011 May 8.
We describe a conditional in vivo protein-trap mutagenesis system that reveals spatiotemporal protein expression dynamics and can be used to assess gene function in the vertebrate Danio rerio. Integration of pGBT-RP2.1 (RP2), a gene-breaking transposon containing a protein trap, efficiently disrupts gene expression with >97% knockdown of normal transcript amounts and simultaneously reports protein expression for each locus. The mutant alleles are revertible in somatic tissues via Cre recombinase or splice-site-blocking morpholinos and are thus to our knowledge the first systematic conditional mutant alleles outside the mouse model. We report a collection of 350 zebrafish lines that include diverse molecular loci. RP2 integrations reveal the complexity of genomic architecture and gene function in a living organism and can provide information on protein subcellular localization. The RP2 mutagenesis system is a step toward a unified 'codex' of protein expression and direct functional annotation of the vertebrate genome.
我们描述了一种条件性体内蛋白捕获突变系统，该系统可揭示时空蛋白质表达动态，并可用于评估脊椎动物斑马鱼中的基因功能。pGBT-RP2.1（RP2）的整合，一种含有蛋白捕获的基因破坏转座子，高效地破坏了基因表达，使正常转录本数量的表达降低了>97%，同时报告了每个基因座的蛋白质表达。突变等位基因可以通过 Cre 重组酶或剪接位点阻断性 morpholino 在体组织中可逆，因此据我们所知，这是除了小鼠模型之外的第一个系统的条件性突变等位基因。我们报告了一个包含 350 个斑马鱼系的集合，其中包括多种分子基因座。RP2 整合揭示了基因组结构和活体生物中基因功能的复杂性，可提供有关蛋白质亚细胞定位的信息。RP2 诱变系统是迈向统一的“蛋白质表达编码”和脊椎动物基因组的直接功能注释的一步。
