A Multicassette Gateway Vector Set for High Throughput and Comparative Analyses in Ciona and Vertebrate Embryos

Agnès Roure,Clément Lamy,Ute Rothbächer,Patrick Lemaire,Caterina Missero,Eva Kalmar,François Robin,Ferenc Mueller,Giustina Ferone,Jean-Nicolas Volff

doi:10.1371/journal.pone.0000916

Abstract

BackgroundThe past few years have seen a vast increase in the amount of genomic data available for a growing number of taxa, including sets of full length cDNA clones and cis-regulatory sequences. Large scale cross-species comparisons of protein function and cis-regulatory sequences may help to understand the emergence of specific traits during evolution.Principal FindingsTo facilitate such comparisons, we developed a Gateway compatible vector set, which can be used to systematically dissect cis-regulatory sequences, and overexpress wild type or tagged proteins in a variety of chordate systems. It was developed and first characterised in the embryos of the ascidian Ciona intestinalis, in which large scale analyses are easier to perform than in vertebrates, owing to the very efficient embryo electroporation protocol available in this organism. Its use was then extended to fish embryos and cultured mammalian cells.ConclusionThis versatile vector set opens the way to the mid- to large-scale comparative analyses of protein function and cis-regulatory sequences across chordate evolution. A complete user manual is provided as supplemental material.

Highlights

The massive increase in sequence and annotation data that can be collected over a short term on any given organism has had major consequences in developmental and evolutionary studies
Systematic comparison of the activity of ascidian and vertebrate regulatory sequences and protein function may help define the extent of conservation in the various chordate developmental GRNs that lead to the establishment of the same larval body plan
To enforce compatibility between entry clones generated by the various laboratories of a scientific community, all our vectors make use of the Reading Frame A cassette (RfA) cassette, which is compatible with the Invitrogen Gateway bacterial expression vectors and Proquest Yeast 2 hybrid system

Summary

Introduction

The massive increase in sequence and annotation data that can be collected over a short term on any given organism has had major consequences in developmental and evolutionary studies. Systematic comparison of the activity of ascidian and vertebrate regulatory sequences and protein function may help define the extent of conservation in the various chordate developmental GRNs that lead to the establishment of the same larval body plan. To facilitate such comparisons, we developed a Gateway compatible vector set, which can be used to systematically dissect cis-regulatory sequences, and overexpress wild type or tagged proteins in a variety of chordate systems. We developed a Gateway compatible vector set, which can be used to systematically dissect cis-regulatory sequences, and overexpress wild type or tagged proteins in a variety of chordate systems It was developed and first characterised in the embryos of the ascidian Ciona intestinalis, in which large scale analyses are easier to perform than in vertebrates, owing to the very efficient embryo electroporation protocol available in this organism.

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Conclusion