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Abstract The application of CRISPR technology has greatly facilitated the creation of transgenic Caenorhabditis elegans lines. However, methods to insert multi-kilobase DNA constructs remain laborious even with these advances. Here, I describe a new approach for introducing large DNA constructs into the C. elegans genome at specific sites using a combination of Flp and Cre recombinases. The system utilizes specialized integrated landing sites that express GFP ubiquitously flanked by single loxP, FRT, and FRT3 sites. DNA sequences of interest are inserted into an integration vector that contains a sqt-1 self-excising cassette and FRT and FRT3 sites. Plasmid DNA is injected into the germline of landing site animals. Transgenic animals are identified as Rol progeny, and the sqt-1 marker is subsequently excised with heat shock Cre expression. Integration events were obtained at a rate of approximately one integration per three injected F0 animals—a rate substantially higher than any current approach. To demonstrate the robustness of the approach, I compared the efficiency of the Gal4/UAS, QF (and QF2)/QUAS, tetR(and rtetR)/tetO, and LexA/lexO bipartite expression systems by assessing expression levels in combinations of driver and reporter GFP constructs and a direct promoter GFP fusion each integrated at multiple sites in the genome. My data demonstrate that all four bipartite systems are functional in C. elegans. Although the new integration system has several limitations, it greatly reduces the effort required to create single-copy insertions at defined sites in the C. elegans genome.

Glaesserella parasuis is an important bacterial pathogen that affects the swine industry worldwide. Research on the pathogenic mechanism and genetically engineered vaccine remains undeveloped because an effective markerless and multiple-gene knockout system is unavailable for G. parasuis yet. To establish a markerless knockout, deleted allelic genes with kanamycin resistance (Kan R ) cassettes were introduced into the genome of G. parasuis by using natural transformation with suicide plasmids. Then, the Kan R cassette was excised with a thermosensitive plasmid pGF conferring a constitutive Flp expression. To realize the markerless and multiple-gene knockout, plasmid pGAF was constructed by placing the Flp gene under the control of an arabinose-inducible promoter. Firstly, pGAF was introduced into G. parasuis by electroporation, and the marked mutants were produced following natural transformation. Finally, the Kan R cassette was excised from the genome by the inducible expression of Flp upon arabinose action. Based on the natural transformation and the inducible expression of Flp, the markerless single-gene knockout mutants of Δ hsdR , Δ neuA2 , Δ espP2 , Δ apd , and Δ nanH were constructed. In addition, a five-gene knockout mutant of Δ hsdR Δ neuA2 Δ espP2 Δ apd Δ nanH was generated by successive natural transformation with five suicide plasmids. Taken together, a markerless and multiple-gene deletion system was established for G. parasuis in the present study for the first time. This system is simple, efficient, and easy to manipulate for G. parasuis ; thus, our technique will substantially aid the understanding of the etiology, pathogenesis, and genetic engineering of G. parasuis and other bacteria that can be naturally transformed in laboratory conditions. Key points • Flp recombinase excised the Kan R  gene flanked by FRT sites in  Glaesserella parasuis. • The regulatory expression of Flp enabled a multiple-gene knockout for G. parasuis. • The technique will promote the understanding of Glässer’s disease pathogens. Graphical abstract

Background The functional understanding of genetic interaction networks and cellular mechanisms governing health and disease requires the dissection, and multifaceted study, of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic effector alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical studies. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, its experimental implementation has been broadly restricted to manipulations of a limited set of common alleles that are often commercially produced at great expense, with costs and technical challenges associated with production of intersectional mouse lines hindering customized approaches to many researchers. Here, we present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production. Results Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study the way functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle. Conclusions The lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp–dependent, Cre-mediated Ca v 3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research. Most approaches to control gene expression in vivo require generation of knock-in mouse lines and often lack spatiotemporal control. Here the authors develop a photo-activatable Flp recombinase system and demonstrate its use by controlling object-exploration behavior in mice through Cav3.1 silencing.

Genetic mutations disrupting the structure and function of primary cilia cause various inherited retinal diseases in humans. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic ciliopathy characterized by retinal degeneration, obesity, postaxial polydactyly, intellectual disability, and genital and renal abnormalities. To gain insight into the mechanisms of retinal degeneration in BBS, we developed a congenital knockout mouse of Bbs8, as well as conditional mouse models in which function of the BBSome (a protein complex that mediates ciliary trafficking) can be temporally inactivated or restored. We demonstrate that BBS mutant mice have defects in retinal outer segment morphogenesis. We further demonstrate that removal of Bbs8 in adult mice affects photoreceptor function and disrupts the structural integrity of the outer segment. Notably, using a mouse model in which a gene trap inhibiting Bbs8 gene expression can be removed by an inducible FLP recombinase, we show that when BBS8 is restored in immature retinas with malformed outer segments, outer segment extension can resume normally and malformed outer segment discs are displaced distally by normal outer segment structures. Over time, the retinas of the rescued mice become morphologically and functionally normal, indicating that there is a window of plasticity when initial retinal outer segment morphogenesis defects can be ameliorated.

Summary The growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant‐based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large‐scale production due to high operational costs and batch‐to‐batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)‐derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harbouring the gene of interest (GOI) flanked by cis‐acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper‐inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast‐derived CUP2‐Gal4 copper‐responsive transcriptional activator for Rep/RepA expression, and (iv) a copper‐inducible Flp recombinase to minimize basal Rep/RepA expression. CuSO4 application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti‐SARS‐CoV‐2 antibody upon copper treatment. Notably, the system is functional in post‐harvest applications, a strategy with high potential impact for large‐scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost‐effective and scalable production of recombinant proteins in plants.

Summary The CRISPR‐Cas9 system has emerged as a potent molecular scalpel for precise genome editing, and profoundly revolutionized plant genetics and breeding, facilitating the development of innovative and improved plant varieties. Typically, the CRISPR‐Cas9 gene‐editing construct is delivered into target organisms via Agrobacterium tumefaciens‐mediated transformation or biolistic methods. However, the incorporation of the CRISPR‐Cas9 machinery increases the risk of off‐target effects, causing unintended genomic alterations. Additionally, the introduction of exogenous DNA sequences, such as antibiotic resistance marker, raises public concerns regarding the biosafety and regulatory oversight of genetically modified organisms (GMOs), potentially hindering regulatory approval and commercialization. Here, we have engineered an integrated system comprising RUBY, LoxP::FRT/FLP and CRISPR/Cas9‐sgRNA cassettes within a single construct, allowing visible color monitoring throughout process including genetic transform, positive transgenic and edited events screening, as well as exogenous DNA excision events, we refer to it as ‘a visual monitoring DNA‐free multi‐gene editing system (VMDFGE)’. This system was introduced into poplar through Agrobacterium tumefaciens‐mediated transformation, yielding transgenic poplars with a 75.0% visual screening rate, a 45.8% targeted mutation rate and a 54.5% excision rate for the entire integration system. This approach eliminates the concerns associated with off‐target effects and GMO regulatory challenges. It offers significant potential for improvement of poplar, other woody plants and crop species while removing the foreign DNA.

Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and λ-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriTRK2for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriTRK2to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.

Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.

We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based docking vector harbouring a selection gene, an eGFP reporter gene, and an Flp recombinase site for locus-directed gene insertion. PFV cells have insertion of a single docking vector with stable eGFP expression and generated phenotypic normal blastocysts with transgene expression after somatic cell nuclear transfer. PFV cells supported Flp mediated cassette exchange for transgene substitution of eGFP with dsRED, and the dsRED transgenic PFV cells generated blastocysts with transgene expression. Hence, the PFV cell line constitutes a valuable pig equivalent to transformed cell lines from other mammalian species suitable for locus-directed transgene expression in cell cultures and, in addition, for transgene analyses in the very early embryonic stages.