Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease

In this study, we report a highly efficient transgenesis technique for Xenopus tropicalis based on a method described first for Medaka. This simple procedure entails co-injection of meganuclease I-SceI and a transgene construct flanked by two I-SceI sites into fertilized eggs. Approximately 30% of injected embryos express transgenes in a promoter-dependent manner. About 1/3 of such embryos show incorporation of the transgene at the one-cell stage and the remainder are 'half-transgenics' suggesting incorporation at the two-cell stage. Transgenes from both classes of embryos are shown to be transmitted and expressed in offspring. The procedure also works efficiently in Xenopus laevis. Because the needle injection procedure does not significantly damage embryos, a high fraction develop normally and can, as well, be injected with a second reagent, for example an mRNA or antisense morpholino oligonucleotide, thus allowing one to perform several genetic manipulations on embryos at one time. This simple and efficient technique will be a powerful tool for high-throughput transgenesis assays in founder animals, and for facilitating genetic studies in the fast-breeding diploid frog, X. tropicalis.     

High-throughput transgenesis in Xenopus using I-SceI meganuclease

In this report we describe an easy, highly efficient transgenesis method for Xenopus. The method is very simple; a commercially available meganuclease, I-SceI, is incubated with a transgene construct carrying its recognition sites, and is subsequently microinjected into fertilized eggs. Approximately 30% (in Xenopus tropicalis) or 20% (in Xenopus laevis) of injected embryos exhibit non-mosaic, promoter-dependent transgene expression, and transgenes from the founder animals are transmitted to offspring. The method is compatible with mRNA or antisense morpholino oligonucleotide injection, and these secondary reagents can be introduced simultaneously or sequentially with a transgene to test their interaction. This high-throughput transgenic technique will be a powerful tool for studying the complex wiring of regulatory networks at the genome-wide level, as well as for facilitating genetic studies in the rapidly breeding diploid frog, X. tropicalis.     

I-SceI Meganuclease-mediated transgenesis in the acorn worm, Saccoglossus kowalevskii

Hemichordates are a phylum of marine invertebrate deuterostomes that are closely related to chordates, and represent one of the most promising models to provide insights into early deuterostome evolution. The genome of the hemichordate, Saccoglossus kowalevskii, reveals an extensive set of non-coding elements conserved across all three deuterostome phyla. Functional characterization and cross-phyla comparisons of these putative regulatory elements will enable a better understanding of enhancer evolution, and subsequently how changes in gene regulation give rise to morphological innovation. Here, we describe an efficient method of transgenesis for the characterization of non-coding elements in S. kowalevskii. We first test the capacity of an I-SceI transgenesis system to drive ubiquitous or regionalized gene expression, and to label specific cell types. Finally, we identified a minimal promoter that can be used to test the capacity of putative enhancers in S. kowalevskii. This work demonstrates that this I-SceI transgenesis technique, when coupled with an understanding of chromatin accessibility, can be a powerful tool for studying how evolutionary changes in gene regulatory mechanisms contributed to the diversification of body plans in deuterostomes.     

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo 'gain-of-function' and 'loss-of-function' studies.     

Progress and biotechnological prospects in fish transgenesis

The history of transgenesis is marked by milestones such as the development of cellular transdifferentiation, recombinant DNA, genetic modification of target cells, and finally, the generation of simpler genetically modified organisms (e.g. bacteria and mice). The first transgenic fish was developed in 1984, and since then, continuing technological advancements to improve gene transfer have led to more rapid, accurate, and efficient generation of transgenic animals. Among the established methods are microinjection, electroporation, lipofection, viral vectors, and gene targeting. Here, we review the history of animal transgenesis, with an emphasis on fish, in conjunction with major developments in genetic engineering over the past few decades. Importantly, spermatogonial stem cell modification and transplantation are two common techniques capable of revolutionizing the generation of transgenic fish. Furthermore, we discuss recent progress and future biotechnological prospects of fish transgenesis, which has strong applications for the aquaculture industry. Indeed, some transgenic fish are already available in the current market, validating continued efforts to improve economically important species with biotechnological advancements.     

New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements

We have developed a new method for the transgenesis of the silkworm, Bombyx mori. This method couples the use of recombinant baculoviruses with the use of the piggyBac transposable element. One recombinant AcNPV, designated the helper virus, is designed to express the piggyBac transposase under the control of the Drosophila hsp70 promoter. Another recombinant AcNPV encoded the gene to be incorporated into the silkworm genome, in this case a green fluorescent protein (GFP) gene, under the control of B. mori actin A3 promoter and franked by the piggyBac inverted terminal repeats. Preblastoderm eggs were inoculated with a fine needle coated with a mixture of these two recombinant baculoviruses. Most of the inoculated larvae hatched and a high proportion of the newly hatched G0 larvae expressed the GFP marker. Transgenesis was confirmed by Southern blot analysis of G1 insects, sequencing the insertion site junctions isolated by inverse PCR, and the marker segregated in Mendelian fashion, as evidenced by the appearance of green fluorescence in G2 insects. Thus, transgenic silkworms were easily and efficiently obtained using this new method.     

A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection

Creating transgenic mice is an important technology for genetic studies and is currently performed by pronuclear microinjection of plasmid DNA into fertilized eggs. Since survival of injected embryos and integration of plasmid DNA are not efficient, total efficiency is only around 3% with a standard protocol. To circumvent this problem, here we describe a novel transgenesis method, the Tol2-mediated cytoplasmic injection method (Tol2:CI). We injected a foreign DNA cloned in a Tol2-transposon vector together with the transposase mRNA into the cytoplasm of fertilized eggs. As expected, the survival rate of the injected embryos was increased drastically. Also, the foreign DNA was transposed from the plasmid to the genome and transmitted to the next generation very efficiently. Together, the overall transgenic efficiency became more than 20%. Considering its simplicity and perfect compatibility with existing pronuclear microinjection facilities, we propose that the Tol2:CI method is applicable to high throughput functional genomics studies.     

An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi

RNA interference (RNAi) mediates gene silencing in many eukaryotes and has been widely used to investigate gene functions. A common method to induce sustained RNAi is introducing plasmids that synthesize short hairpin RNAs (shRNAs) using Pol III promoters. While these promoters synthesize shRNAs and elicit RNAi efficiently, they lack cell specificity. Monitoring shRNA expression levels in individual cells by Pol III promoters is also difficult. An alternative way to deliver RNAi is to use Pol II-directed synthesis of shRNA. Previous efforts in developing a Pol II system have been sparse and the results were conflicting, and the usefulness of those Pol II vectors has been limited due to low efficacy. Here we demonstrate a new Pol II system that directs efficient shRNA synthesis and mediates strong RNAi at levels that are comparable with the commonly used Pol III systems. In addition, this system synthesizes a marker protein under control of the same promoter as the shRNA, thus providing an unequivocal indicator, not only to the cells that express the shRNA, but also to the levels of the shRNA expression. This system may be adapted for in vivo shRNA expression and gene silencing.     

Microalgae--novel highly efficient starch producers

The freshwater alga Chlorella, a highly productive source of starch, might substitute for starch‐rich terrestrial plants in bioethanol production. The cultivation conditions necessary for maximizing starch content in Chlorella biomass, generated in outdoor scale‐up solar photobioreactors, are described. The most important factor that can affect the rate of starch synthesis, and its accumulation, is mean illumination resulting from a combination of biomass concentration and incident light intensity. While 8.5% DW of starch was attained at a mean light intensity of 215 µmol/(m² s¹), 40% of DW was synthesized at a mean light intensity 330 µmol/(m² s¹). Another important factor is the phase of the cell cycle. The content of starch was highest (45% of DW) prior to cell division, but during the course of division, its cellular level rapidly decreased to about 13% of DW in cells grown in light, or to about 4% in those kept in the dark during the division phase. To produce biomass with high starch content, it is necessary to suppress cell division events, but not to disturb synthesis of starch in the chloroplast. The addition of cycloheximide (1 mg/L), a specific inhibitor of cytoplasmic protein synthesis, and the effect of element limitation (nitrogen, sulfur, phosphorus) were tested. The majority of the experiments were carried out in laboratory‐scale photobioreactors, where culture treatments increased starch content to up to about 60% of DW in the case of cycloheximide inhibition or sulfur limitation. When the cells were limited by phosphorus or nitrogen supply, the cellular starch content increased to 55% or 38% of DW, respectively, however, after about 20 h, growth of the cultures stopped producing starch, and the content of starch again decreased. Sulfur limited and cycloheximide‐treated cells maintained a high content of starch (60% of DW) for up to 2 days. Sulfur limitation, the most appropriate treatment for scaled‐up culture of starch‐enriched biomass, was carried out in an outdoor pilot‐scale experiment. After 120 h of growth in complete mineral medium, during which time the starch content reached around 18% of DW, sulfur limitation increased the starch content to 50% of DW. Biotechnol. Bioeng. 2011; 108:766–776. © 2010 Wiley Periodicals, Inc.     

Endothelial lipase mediates efficient lipolysis of triglyceride-rich lipoproteins

Triglyceride-rich lipoproteins (TRLs) are circulating reservoirs of fatty acids used as vital energy sources for peripheral tissues. Lipoprotein lipase (LPL) is a predominant enzyme mediating triglyceride (TG) lipolysis and TRL clearance to provide fatty acids to tissues in animals. Physiological and human genetic evidence support a primary role for LPL in hydrolyzing TRL TGs. We hypothesized that endothelial lipase (EL), another extracellular lipase that primarily hydrolyzes lipoprotein phospholipids may also contribute to TRL metabolism. To explore this, we studied the impact of genetic EL loss-of-function on TRL metabolism in humans and mice. Humans carrying a loss-of-function missense variant in LIPG, p.Asn396Ser (rs77960347), demonstrated elevated plasma TGs and elevated phospholipids in TRLs, among other lipoprotein classes. Mice with germline EL deficiency challenged with excess dietary TG through refeeding or a high-fat diet exhibited elevated TGs, delayed dietary TRL clearance, and impaired TRL TG lipolysis in vivo that was rescued by EL reconstitution in the liver. Lipidomic analyses of postprandial plasma from high-fat fed Lipg^-/- mice demonstrated accumulation of phospholipids and TGs harboring long-chain polyunsaturated fatty acids (PUFAs), known substrates for EL lipolysis. In vitro and in vivo, EL and LPL together promoted greater TG lipolysis than either extracellular lipase alone. Our data positions EL as a key collaborator of LPL to mediate efficient lipolysis of TRLs in humans and mice.     

Intermolecular ligation mediates efficient cotransformation in Phytophthora infestans

The processing of DNA molecules during transformation was characterized in the oomycete Phytophthora infestans. Linear and circular forms of nonreplicating transformation vectors supported similar rates of stable transformation. Remarkably, digestion of plasmids within the selectable marker genes neomycin phosphotransferase (npt) or hygromycin phosphotransferase (hpt) had little effect on the recovery of drug-resistant transformants, and the cleaved sites were shown to be reconstituted in the transformants. An assay for the transient expression of -glucuronidase (GUS) in protoplasts treated with partial or disrupted GUS genes demonstrated that active genes could be reconstituted through intramolecular and/or intermolecular ligation between compatible ends, while incompatible ends were inefficiently joined. Stable transformation studies also demonstrated that complementing portions of incomplete npt or hpt genes joined through homologous recombination. Based on the indication of efficient ligation between DNA molecules during transformation, an efficient procedure for cotransformation was developed. The frequency of cotransformation between vectors expressing selected genes (npt or hpt) and nonselected sequences (GUS, -galactosidase, or streptomycin phosphotransferase) approached unity when the plasmids were linearized with the same restriction enzyme before transformation. In contrast, cotransformation between circular plasmids or those cut with different enzymes occurred infrequently (10%). Hybridization analysis of DNA from cotransformants demonstrated that linearized plasmids became colocalized within genomic DNA, while circular plasmids typically inserted at unliked sites.     

Transposon-mediated BAC transgenesis in zebrafish

Bacterial artificial chromosomes (BACs) are widely used in studies of vertebrate gene regulation and function because they often closely recapitulate the expression patterns of endogenous genes. Here we report a step-by-step protocol for efficient BAC transgenesis in zebrafish using the medaka Tol2 transposon. Using recombineering in Escherichia coli, we introduce the iTol2 cassette in the BAC plasmid backbone, which contains the inverted minimal cis-sequences required for Tol2 transposition, and a reporter gene to replace a target locus in the BAC. Microinjection of the Tol2-BAC and a codon-optimized transposase mRNA into fertilized eggs results in clean integrations in the genome and transmission to the germline at a rate of ～15%. A single person can prepare a dozen constructs within 3 weeks, and obtain transgenic fish within approximately 3-4 months. Our protocol drastically reduces the labor involved in BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates.     

Stella-Cre mice are highly efficient Cre deleters

Cre-loxP recombination is widely used for genetic manipulation of the mouse genome. Here, we report generation and characterization of a new Cre line, Stella-Cre, where Cre expression cassette was targeted to the 3' UTR of the Stella locus. Stella is specifically expressed in preimplantation embryos and in the germline. Cre-loxP recombination efficiency in Stella-Cre mice was investigated at several genomic loci including Rosa26, Jak2, and Npm1. At all the loci examined, we observed 100% Cre-loxP recombination efficiency in the embryos and in the germline. Thus, Stella-Cre mice serve as a very efficient deleter line.     

Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes

Autophagy is a membrane trafficking pathway that carries cytosolic components to the lysosome for degradation. During this process, the autophagosome, a double-membraned organelle, is generated de novo, sequesters cytoplasmic proteins and organelles, and delivers them to lysosomes. However, the mechanism by which autophagosomes are targeted to lysosomes has not been determined. Here, we observed the real-time behavior of microtubule-associated protein light chain 3 (LC3), which localizes to autophagosomes, and showed that autophagosomes move in a microtubule- and dynein-dynactin motor complex-dependent manner. After formation, autophagosomes show a rapid vectorial movement in the direction of the centrosome, where lysosomes are usually concentrated. Microinjection of antibodies against LC3 inhibited this movement; furthermore, using FRAP, we showed that anti-LC3 antibody injection caused a defect in targeting of autophagosomes to lysosomes. Collectively, our data demonstrate the functional significance of autophagosome movement that enables effective delivery from the cytosol to lysosomes.     

A rapid RIG-I signaling relay mediates efficient antiviral response

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Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.