Transgenic black soldier flies for production of carotenoids.

The black soldier fly (BSF), Hermetia illucens, has gained traction recently as a means to achieve closed-loop production cycles. BSF can subsist off mammalian waste products and their consumption of such waste in turn generates compost that can be used in agricultural operations. Their environmental impact is minimal and BSF larvae are edible, with a nutritional profile high in protein and other essential vitamins. Therefore, it is conceivable to use BSF as a mechanism for both reducing organic waste and maintaining a low-impact food source for animal livestock or humans. The main drawback to BSF as a potential human food source is they are deficient in fat-soluble vitamins such as Vitamins A, D, and E. While loading BSF with essential vitamins may be achieved via diet-based interventions, this undercuts the goal of a closed-loop as specialized diets would require additional supply chains. An alternative is to genetically engineer BSF that can synthesize these essential vitamins. Here we describe a BSF line that has been engineered with the two main carotenoid biosynthetic genes, CarRA and CarB for production of provitamin carotenoids within the Vitamin A family. Our data describe the manipulation of the BSF genome to insert transgenes for expression of functional protein products.

Intracellular Calcium Mobilization Is Required for Sonic Hedgehog Signaling.

Graded Shh signaling across fields of precursor cells coordinates patterns of gene expression, differentiation, and morphogenetic behavior as precursors form complex structures, such as the nervous system, the limbs, and craniofacial skeleton. Here we discover that intracellular calcium mobilization, a process tightly controlled and readily modulated, regulates the level of Shh-dependent gene expression in responding cells and affects the development of all Shh-dependent cell types in the zebrafish embryo. Reduced expression or modified activity of ryanodine receptor (RyR) intracellular calcium release channels shifted the allocation of Shh-dependent cell fates in the somitic muscle and neural tube. Mosaic analysis revealed that RyR-mediated calcium mobilization is required specifically in Shh ligand-receiving cells. This work reveals that RyR channels participate in intercellular signal transduction events. As modulation of RyR activity modifies tissue patterning, we hypothesize that alterations in intracellular calcium mobilization contribute to both birth defects and evolutionary modifications of morphology.

Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.

Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle.

Mutations affecting the seemingly unrelated gene products, SepN1, a selenoprotein of unknown function, and RyR1, the major component of the ryanodine receptor intracellular calcium release channel, result in an overlapping spectrum of congenital myopathies. To identify the immediate developmental and molecular roles of SepN and RyR in vivo, loss-of-function effects were analyzed in the zebrafish embryo. These studies demonstrate the two proteins are required for the same cellular differentiation events and are needed for normal calcium fluxes in the embryo. SepN is physically associated with RyRs and functions as a modifier of the RyR channel. In the absence of SepN, ryanodine receptors from zebrafish embryos or human diseased muscle have altered biochemical properties and have lost their normal sensitivity to redox conditions, which likely accounts for why mutations affecting either factor lead to similar diseases.