Bacterial artificial chromosomes as recombinant reporter constructs to investigate gene expression and regulation in echinoderms

Abstract

Genome sequences contain all the necessary information-both coding and regulatory sequences-to construct an organism. The developmental process translates this genomic information into a three-dimensional form. One interpretation of this translation process can be described using gene regulatory network (GRN) models, which are maps of interactions among regulatory gene products in time and space. As high throughput investigations reveal increasing complexity within these GRNs, it becomes apparent that efficient methods are required to test the necessity and sufficiency of regulatory interactions. One of the most complete GRNs for early development has been described in the purple sea urchin, Strongylocentrotus purpuratus. This work has been facilitated by two resources: a well-annotated genome sequence and transgenes generated in bacterial artificial chromosome (BAC) constructs. BAC libraries played a central role in assembling the S. purpuratus genome sequence and continue to serve as platforms for generating reporter constructs for use in expression and regulatory analyses. Optically transparent echinoderm larvae are highly amenable to transgenic approaches and are therefore particularly well suited for experiments that rely on BAC-based reporter transgenes. Here, we discuss the experimental utility of BAC constructs in the context of understanding developmental processes in echinoderm embryos and larvae.

Figure 1:

Homologous recombination can be used to generate BAC-based fluorescent reporter constructs. Two strategies for modifying BAC sequences through homologous recombination are shown; specific protocols are detailed in Sharan et al. [61]. A BAC is identified that contains the gene or region of interest (indicated by pink rectangles) using either bioinformatics or hybridization-based library screens. BAC stocks are typically maintained in DH10B Escherichia coli strains. A recombination cassette is designed in which homology arms (narrow, light blue) flank the exon or sequence to be replaced. The BAC and recombination cassette are introduced via electroporation into a modified bacterial strain (either EL250 or SW105; shown as green ovals) that contains the defective prophage λ in which the recombination machinery is repressed. Raising the temperature to 42 °C de-represses these genes to initiate recombination. To identify recombinant clones, the recombination cassette may include an antibiotic resistance gene (e.g. kanamycin resistance; kan^R) flanked by FRT sites (orange ovals). The antibiotic resistance is removed upon arabinose-dependent activation of the flp recombinase. Alternatively, a two-step process in which galK expression is first used to positively select recombinant clones grown on minimal media and then used as a means of negative selection to seamlessly construct BAC recombinants. (A colour version of this figure is available online at: https://academic.oup.com/bfg.)

Figure 2:

Recombinant BACs can be generated using multiple fluorescent proteins to trace expression of alternatively spliced genes. An example of this technique is shown based on the strategy in Schrankel et al. [31]. Exons that are specifically expressed in distinct isoforms serve as targets for recombination using fluorescent proteins. Through this strategy, ‘double-fluorescent protein’ BAC constructs can be generated such that GFP indicates expression of isoform 1, whereas RFP specifically reflects expression of isoform 2. (A colour version of this figure is available online at: https://academic.oup.com/bfg.)

Figure 3:

Recombinant BACs can be used to rescue protein function in the context of endogenous perturbation. (A) Constructing reporter and rescue BACs. The GeneX structure is shown within the original BAC (coding sequence, red; noncoding sequence, white). The most common technique for functional perturbation in echinoderm embryos is the use of MASO reagents [70], which often anneal to the transcript near the start of translation (indicated by blue line). To generate a MASO-resistant BAC, the annealing site can be modified by introducing a complementary oligonucleotide containing point mutations into the EL250 or SW105 cells and activating the recombination machinery as shown in Figure 1. The recombination frequency is much higher than introducing a larger DNA fragment and recombinants can be isolated using a pooled polymerase chain reaction strategy. Based on the location of the fluorescent protein, these reporter BACs may or may not be resistant to the MASO. If not, they can be modified as described above (dashed green arrows). (B) An experimental strategy for perturbation and functional rescue of GeneX. The endogenous expression pattern of hypothetical GeneX is shown in red [in mesenchyme blastula sea urchin embryos, this territory corresponds to aboral non-skeletal mesoderm (NSM)]. Microinjection of the GeneX::gfp BAC recapitulates the GeneX expression pattern but is mosaic so is only expressed in a portion of the endogenous pattern. In this context, striped cells express both endogenous GeneX and the GFP reporter. In contrast, MASO perturbation affects embryos globally (indicated by light pink cells). Protein function can be rescued in a mosaic subset by co-injecting the GeneX::MASO^R BAC (blue), which is resistant to MASO perturbation, as well as a GeneX::gfp BAC to trace integration. Here, the endogenous GeneX translation is blocked by the MASO, but GFP and GeneX are expressed from the BAC clones in a portion of the endogenous territory (striped cells). (A colour version of this figure is available online at: https://academic.oup.com/bfg.)