A multicassette Gateway vector set for high throughput and comparative analyses in ciona and vertebrate embryos

Abstract

Background: The 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 findings: 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. 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.

Conclusion: This 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.

Figure 1. Overview of the vectors described in this article.

Figure 2. Destination vectors and ORF entry clones used to test the pSP72-swa1::RfA vector series.

A) Destination vectors for the overexpresion of WT or tagged ORFs under the control of the animal-specific cis-regulatory regions of Ciona Friend of GATA (Ci-FOG). B) Generated ORF entry clones with the obeserved subcellular localisation of the corresponding tagged proteins.

Figure 3. Subcellular localisation of tagged proteins expressed from pSP72-pFOG::RfA vector series.

A, B, F, G epifluorescence pictures of embryos expressing GAP-43 venus (A), Histone2B-venus (B), venus-GATAa (F) and GATAa-HA (G). F and G show the same embryo co-electroporated with pSP72-pFOG::Venus-B1-GATAa-B2 and pSP72-pFOG::B1-GATAa-B2-HA. C, D, E projections of confocal stacks of images from embryos either electroporated (C, E) or microinjected with mRNA (D) and expressing ensconsin-3GFP (C, D), or ci-aurora kinase (E). Due to the mosaic expression of the transgene, some cells express the marker at a lower level (C, F, G).

Figure 4. Donor vectors of different recombination specificities.

A) list of generated vectors with the identity of the recombination sites and type of element cloned. B) Sequence of the recombination sites used in the different donor vectors.

Figure 5. Cloning of cis-regulatory sequences in the R3-R5 cassette.

A) scheme of the recombination of an L3-cis-reg-L5 entry clone into the R3-R5 cassette of a destination vector. B-F) Activity of the indicated constructs introduced in embryos by electroporation. B, C) The reporter was GATAa-venus. D-F) The reporter was NLS-LacZ.

Figure 6. Simultaneous introduction of a cis-regulatory region in the R3-R5 cassette and an ORF in the RfA cassette.

A) Experimental scheme. B) Comparison of the activity by electroporation of the indicated constructs. The weaker fluorescence observed in C is not representative. Due to mosaic expression of the transgene, only some cells express the fluorescent marker.

Figure 7. Strategy for the replacement of a cis-regulatory (A) or ORF ((B) segment by the corresponding Gateway cassette.

Figure 8. Simultaneous introduction of an enhancer and basal promoter into a destination vector.

A) Experimental scheme. B-F) Results of the electroporation of the indicated constructs generated by conventional cloning (B, D) or by Gateway cloning (C, E, F). The numbers indicate the proportion of embryos with the shown lacZ staining.

Figure 9. Chordate expression constructs are suitable for mammalian cells and zebrafish embryos.

A) Transfection in mammalian cells of the indicated murine enhancers. The Y-axis shows the percentage of activity (% of LacZ positive cells) of tested Gateway-cloned enhancers relative to a control CMV-lacZ construct. The left most two columns were obtained in independent experiments from the other columns, restricted to Hela cells. B-D) Transient transgenesis by microinjection into zebrafish eggs of the indicated constructs. Left panel, lateral view, right panel, dorsal view, of 30 hour post-fertilization stage zebrafish embryos. No change in the intensity or localisation is observed when Gateway expression constructs are used (B, D) instead of pCS2+-based constructs (C). The numbers indicate the proportion of embryos with the shown lacZ or GFP staining.