FRAGLER: A Fragment Recycler Application Enabling Rapid and Scalable Modular DNA Assembly.

Rapid and flexible plasmid construct generation at scale is one of the most limiting first steps in drug discovery projects. These hurdles can partly be overcome by adopting modular DNA design principles, automated sequence fragmentation, and plasmid assembly. To this end we have designed a robust, multimodule golden gate based cloning platform for construct generation with a wide range of applications. The assembly efficiency of the system was validated by splitting sfGFP and sfCherry3C cassettes and expressing them in E. coli followed by fluorometric assessment. To minimize timelines and cost for complex constructs, we developed a software tool named FRAGLER (FRAGment recycLER) that performs codon optimization, multiple sequence alignment, and automated generation of fragments for recycling. To highlight the flexibility and robustness of the platform, we (i) generated plasmids for SarsCoV2 protein reagents, (ii) automated and parallelized assemblies, and (iii) built modular libraries of chimeric antigen receptors (CARs) variants. Applying the new assembly framework, we have greatly streamlined plasmid construction and increased our capacity for rapid generation of complex plasmids.

Gonadotropins in European sea bass: Endocrine roles and biotechnological applications.

Follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of the gonadal function in vertebrates. They act through specific receptors located in certain cell types found in the gonads. In fish, the differential roles of these hormones are being progressively elucidated due to the development of suitable tools for their study. In European sea bass (Dicentrarchus labrax), isolation of the genes coding for the gonadotropin subunits and receptors allowed in first instance to conduct expression studies. Later, to overcome the limitation of using native hormones, recombinant dimeric gonadotropins, which show different functional characteristics depending on the cell system and DNA construct, were generated. In addition, single gonadotropin beta-subunits have been produced and used as antigens for antibody production. This approach has allowed the development of detection methods for native gonadotropins, with European sea bass being one of the few species where both gonadotropins can be detected in their native form. By administering recombinant gonadotropins to gonad tissues in vitro, we were able to study their effects on steroidogenesis and intracellular pathways. Their administration in vivo has also been tested for use in basic studies and as a biotechnological approach for hormone therapy and assisted reproduction strategies. In addition to the production of recombinant hormones, gene-based therapies using somatic gene transfer have been offered as an alternative. This approach has been tested in sea bass for gonadotropin delivery in vivo. The hormones produced by the genes injected were functional and have allowed studies on the action of gonadotropins in spermatogenesis.

Distinct expression profiles of three melatonin receptors during early development and metamorphosis in the flatfish Solea senegalensis.

Melatonin actions are mediated through G protein-coupled transmembrane receptors. Recently, mt1, mt2, and mel1c melatonin receptors were cloned in the Senegalese sole. Here, their day-night and developmental expressions were analyzed by quantitative PCR. These results revealed distinct expression patterns of each receptor through development. mel1c transcripts were more abundant in unfertilized ovulated oocytes and declined during embryonic development. mt1 and mt2 expression was higher at the earliest stages (2-6 days post-fertilization), decreasing before (mt2) or during (mt1) metamorphosis. Only mt1 and mel1c expression exhibited day-night variations, with higher nocturnal mRNA levels. These results suggest different roles and transcriptional regulation of these melatonin receptors during flatfish development and metamorphosis.

foxl2 and foxl3 are two ancient paralogs that remain fully functional in teleosts.

FOXL2 is a forkhead transcription factor involved in mammalian development and regulation of reproduction. Two foxl2 paralogs, foxl2a and foxl2b, have been described in various teleost species and were considered as fish-specific duplicates. Here, we report the isolation and characterization of foxl2a (foxl2) and foxl2b (foxl3) in European sea bass (Dicentrarchus labrax), together with the identification of these two genes in non-teleost genomes. Phylogenetic and synteny analyses indicate that these paralogs originated from an ancient genome duplication event that happened long before the teleost specific duplication. While foxl2/foxl2a has been maintained in most vertebrate lineages, foxl2b, which we propose to rename as foxl3, was repeatedly lost in tetrapods. Gonadal expression patterns of the sea bass genes point to a strong sexual dimorphism, and the mRNA levels of foxl2 in ovary and foxl3 in testis vary significantly during the reproductive cycle. When overexpressed in cultured ovarian follicular cells, foxl2 and foxl3 produced functional transcription factors able to control the expression of reproduction-related genes. Taken together, these data suggest that Foxl2 may play a conserved role in ovarian maturation, while Foxl3 could be involved in testis physiology.