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
Radical Fluoroalkylation Reactions Barata-Vallejo, Sebastián; Cooke, Maria Victoria; Postigo, Al
ACS catalysis,
08/2018, Volume:
8, Issue:
8
Journal Article
Peer reviewed
Open access
Recent protocols and reactions for catalytic radical perfluoroalkylations will be described. The production of perfluoroalkyl radicals (RF = C n F2n+1, n ≥ 2), which effect both addition and ...substitution reactions on organic substrates, can be realized through a range of diverse methods such as the well-established visible-light transition-metal-mediated photocatalysis, organic-dye-photocatalyzed reactions, electron donor–acceptor complexes, and more recently frustrated Lewis pairs. Thus, perfluoroalkylation reactions of carbon–carbon multiple bonds, isocyanides, nitrones, hydrazones, β-keto esters, α-cyano arylacetates, sulfides, and (hetero)arenes will be described. Special emphasis will be placed on examples published after 2015, where higher fluorinated series of fluoroalkylating reagents are studied.
Free‐living protozoa (FLP) are ubiquitous in natural ecosystems where they play an important role in the reduction of bacterial biomass and the regeneration of nutrients. However, it has been shown ...that some species such as Acanthamoeba castellanii, Acanthamoeba polyphaga, and Tetrahymena pyriformis can act as hosts of pathogenic bacteria. There is a growing concern that FLP might contribute to the maintenance of bacterial pathogens in the environment. In addition to survival and/or replication of bacterial pathogens in FLP, resistance to antimicrobial agents and increased virulence of bacteria after passage through protozoa have been reported. This review presents an overview of FLP in food‐associated environments and on foods, and discusses bacterial interactions with FLP, with focus on the foodborne pathogens Campylobacter jejuni, Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. The consequences of these microbial interactions to food safety are evaluated.
Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter ...transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
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•23 new driver lines and 26 new reporter lines for a wide range of applications•TIGRE2.0 reporters have viral-like transgene expression level in diverse cell types•New calcium- or voltage-sensing reporters with the former functionally characterized•Comparative analysis of new optogenetic effectors with complementary properties
An expanded toolkit of transgenic mouse lines for exploring the organization, function, and development of mammalian neural circuits.
SUMMARY
Meristemoids, which are stomatal precursor cells, exhibit self‐renewal and differentiation abilities. However, the only known core factor associated with meristemoid division termination and ...fate transition is the heterodimer formed by the basic helix–loop–helix proteins MUTE and SCREAMs (SCRMs). FOUR LIPS (FLP), a well‐known transcription factor that restricts guard mother cell (GMC) division, is a direct target of MUTE. Whether FLP involves in meristemoid differentiation is unknown. Through sensitized genetic screening of flp‐1, we identified a mute‐like (mutl) mutant with arrested meristemoids. The mutant carried a novel allele of the MUTE locus, i.e., mute‐4. Intriguingly, mute‐4 is a hypomorphic allele that exhibits wild‐type appearance with slightly delayed meristemoid‐to‐GMC transition, whereas it renders an unexpected mutl epidermis with most meristemoids arrested and very few stomata when combined with flp (flp mute‐4), suggesting that FLP is a positive regulator during this transition process. Consistently, the expression of FLP increased during GMC commitment, and the number of cells at this stage was markedly increased in flp. flp scrm double mutants produced arrested meristemoids similar to mute, and FLP was able to interact physically with SCRM. Taken together, our results demonstrate that FLP functions together with MUTE and SCRMs to direct meristemoid‐to‐GMC fate transition.
Significance Statement
The MUTE•SCREAM heterodimer is a master core complex coordinating meristemoid‐to‐guard mother cell (GMC) transition during stomatal development. However, whether additional transcription factors contribute to this process is difficult to determine, as the lack of MUTE function precludes meristemoid progression to GMC. We isolated the weak mutant mute‐4 and surprisingly found that FOUR LIPS, previously reported to limit GMC symmetric division, could form a heterodimer with SCREAMs to promote meristemoid‐to‐GMC fate transition.
Growth differentiation factor 15 (GDF15) is a stress-induced secreted protein whose circulating levels increase in obesity and fibrotic liver disease, like NAFLD. GDF15 KO mice develop more severe ...fibrosis after carbon tetrachloride (CCL4) treatment, suggesting a protective effect of GDF15. Despite this, the tissue specific sites of GDF15 production during liver injury are unknown and the potential for local action is understudied. To determine the hepatocyte specific contribution of Gdf15 to changes in circulating GDF15 and effects on fibrosis, we utilized a Gdf15 KO-first mouse strain where endogenous expression of Gdf15 can be restored in a tissue specific fashion via Flp recombinase (FLP). Gdf15 KO-first mice received AAV8-TBG-Gfp (KO+GFP) or AAV8-TBG-Flp (liver specific Gdf15 expression; KO+FLP) and were treated with CCL4 3X per week for 3 weeks alongside wild type (WT) controls. CCL4 increased circulating GDF15 7X in WT and 3.5X in KO+FLP compared with WT vehicle (VEH), and levels were undetected in KO+GFP mice. CCL4 caused significant BW loss in WT and KO+FLP mice and had no effect on KO+GFP mice. CCL4 reduced feeding similarly in all groups despite marked differences in circulating GDF15. Liver gene expression markers of fibrosis were upregulated in KO+GFP mice compared with WT and KO+FLP mice. Interestingly, despite differences in fibrosis, circulating cytokines and liver expression of inflammatory markers were reduced in KO+GFP and KO+FLP compared with WT mice. These data demonstrate that hepatocyte Gdf15 protects against CCL4-induced fibrotic liver injury independent of feeding and inflammation. They also suggest that GDF15 may have local, GFRAL-independent effects within liver to remodel the extracellular matrix that require further study. Disclosure J. Kastroll: None. M. J. Jurczak: None. F. Bello: None. A. Vandevender: None. I. J. Sipula: None. J. Alder: None.
The well-characterized cell line Chinese hamster ovary (CHO) has been used to produce numerous biopharmaceuticals and is an important tool for basic research. However, introducing foreign DNA into ...specially modified CHO cells such as DG44 and Lec 3.2.8.1 can sometimes be an arduous process. Here we show that the Flp-intm plasmid can be modified to produce a fluorescent tracer protein tag (mCherrytm) as a fusion reporter, to allow for the rapid selection of single-cell sorted, isogenic Flp-intm-ready DG44 and Lec 3.2.8.1 cell lines. These two cell lines are stable and viable and may be useful for applications such as antibody production and crystallographic studies. Here we provide key details on how the modified pFRT/CherryZeo plasmid may be used to incorporate Flp-intm technology into virtually any desired target cell line in a fast, safe and reliable manner.
The well-characterized cell line Chinese hamster ovary (CHO) has been used to produce numerous biopharmaceuticals and is an important tool for basic research. However, introducing foreign DNA into ...specially modified CHO cells such as DG44 and Lec 3.2.8.1 can sometimes be an arduous process. Here we show that the Flp-in
plasmid can be modified to produce a fluorescent tracer protein tag (mCherry
) as a fusion reporter, to allow for the rapid selection of single-cell sorted, isogenic Flp-in
-ready DG44 and Lec 3.2.8.1 cell lines. These two cell lines are stable and viable and may be useful for applications such as antibody production and crystallographic studies. Here we provide key details on how the modified pFRT/CherryZeo plasmid may be used to incorporate Flp-in
technology into virtually any desired target cell line in a fast, safe and reliable manner.
To decipher neural circuits underlying brain functions, viral tracers are widely applied to map input and output connectivity of neuronal populations. Despite the successful application of retrograde ...transsynaptic viruses for identifying presynaptic neurons of transduced neurons, analogous anterograde transsynaptic tools for tagging postsynaptically targeted neurons remain under development. Here, we discovered that adeno-associated viruses (AAV1 and AAV9) exhibit anterograde transsynaptic spread properties. AAV1-Cre from transduced presynaptic neurons effectively and specifically drives Cre-dependent transgene expression in selected postsynaptic neuronal targets, thus allowing axonal tracing and functional manipulations of the latter input-defined neuronal population. Its application in superior colliculus (SC) reveals that SC neuron subpopulations receiving corticocollicular projections from auditory and visual cortex specifically drive flight and freezing, two different types of defense behavior, respectively. Together with an intersectional approach, AAV-mediated anterograde transsynaptic tagging can categorize neurons by their inputs and molecular identity, and allow forward screening of distinct functional neural pathways embedded in complex brain circuits.
•AAV1 and AAV9 exhibit anterograde transsynaptic spread properties•AAV1-Cre can transsynaptically tag input-defined postsynaptic neurons•Corticocollicular input-defined SC neurons mediate different defense behaviors•With an intersectional approach, the tagging can be postsynaptic cell-type specific
Zingg et al. revealed anterograde transsynaptic spread properties of adeno-associated viruses. This novel viral application is useful for tracing and manipulating neural circuits in a postsynaptic cell-type- and input-specific manner, thus facilitating dissection of neural circuits underlying different behaviors.
The resolution and dimensionality with which biologists can characterize cell types have expanded dramatically in recent years, and intersectional consideration of such features (e.g., multiple gene ...expression and anatomical parameters) is increasingly understood to be essential. At the same time, genetically targeted technology for writing in and reading out activity patterns for cells in living organisms has enabled causal investigation in physiology and behavior; however, cell-type-specific delivery of these tools (including microbial opsins for optogenetics and genetically encoded Ca2+ indicators) has thus far fallen short of versatile targeting to cells jointly defined by many individually selected features. Here, we develop a comprehensive intersectional targeting toolbox including 39 novel vectors for joint-feature-targeted delivery of 13 molecular payloads (including opsins, indicators, and fluorophores), systematic approaches for development and optimization of new intersectional tools, hardware for in vivo monitoring of expression dynamics, and the first versatile single-virus tools (Triplesect) that enable targeting of triply defined cell types.
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•Multiple recombinase-dependent expression of 15 new molecular payloads in single AAVs•Intersectional Ca2+ imaging, cell labeling, and optogenetic inhibition or excitation•Creation and in vivo validation of triple-feature-dependent viruses (Triplesect)•Design of a widely adaptable in vivo quantitative expression tracking system
Fenno et al. enable versatile functional access to cell types defined by the presence of multiple (2 or 3) features, creating diverse expression-control logic contained in single viruses. This result is a comprehensive toolset enabling multiple-feature-dependent optogenetic inhibition and excitation and structure- or activity-based fluorescence imaging with diverse new indicators.
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