BMC Biotechnology

The suitability of rice yellow mottle virus RYMV as a gene expression vector in plant was assessed using a construct carrying promastigote surface antigen (PSA) C-terminal coding sequence of the parasite protozoan Leishmania. RYMV ORF1 encoding P1 protein has been deleted from the RYMV native genome. The C-terminal PSA gene was substituted for the viral coat protein. PSA is present at the surface of the parasite and displays vaccine properties against canine and human leishmaniosis. RYMV-based vector allowed PSA expression in Nicotiana benthamiana. Q-pcr analysis showed that chimeric RYMV carrying PSA gene is able to replicate in N. benthamiana leaves. P19 silencing suppressor in combination with the lacked viral vector ORF encoding RYMV Coat Protein (CP) enhanced significantly RYMV tool replication in N. benthamiana. RYMV CP played a key role on viral RNA stabilization and acts as a weak silencing suppressor. The original RYMV-based expression vector allowed PSA protein expression enhancement in N. benthamiana without any symptoms. RYMV-based vector could be suitable for functional genomic studies in monocots by VIGS (Viral Induced Gene Silencing) technology.

BackgroundA basic requirement for synthetic biology is the availability of efficient DNA assembly methods. Numerous methods have been previously reported to accomplish this task. One such method has been reported, which allows parallel assembly of multiple DNA fragments in a one-tube reaction, called Golden Gate Assembly. This study aims to further simplify that method and make it more suitable for small labs and students. MethodsPrior to amplification of the parental plasmids used in building the modules were domesticated using a variation of SDM (Site Directed Mutagenesis) called SPRIP. After careful design and amplification of the desired modules, using a high-fidelity polymerase, amplified PCR fragments that enter the one-step-one-pot reaction were stored in Zymo DNA/RNA Shield at -20 degrees C and thawed whenever needed to be used as fragments or modules in the assembly. The fragments were designed to posses unique overhangs using NEB Golden Gate assembly tool and Snapgene, amplification of modules was performed using a Q5 high fidelity polymerase from preexisting plasmids or gene fragments, clean-up of the PCR products (fragments) was performed in one tube per assembly using Zymo DNA Clean and Concentrator-5, assembled using BsaI and T4 ligase, DpnI digestion performed for eliminating the background plasmids that remain after the PCR reaction and the resulting assembled product was transformed into competent E.coli cells. Transformants were screened using diagnostic digest, transfected into HEK293T cells and the fluorescence was evaluated using fluorescent microscopy and flow cytometry. ResultsHerein presented is a simple and inexpensive alternate protocol to build modular plasmids using the Golden Gate Assembly method. A total of p37 S/MARs mammalian expression vectors were designed and constructed using 6 modules previously amplified by PCR and stored in the appropriate buffer to eliminate exo- and endonuclease activity and to protect the DNA from freeze thaw cycles. The existing modules were interchangeable and new modules were easily amplified and stored for use when needed. The mammalian expression vectors constructed showed the desired restriction pattern and GFP expression in bacteria and in mammalian cells. A comparison of 7 pNoname variants was conducted using flow cytometry. Interestingly, no pNoname variant harbouring the SV40 promoter showed expression in tested HEK293T cells. It appears that using the Ef1a promoter in combination with the BGH polyA signal provides the best expression in S/MARS vectors harboring the DTS40 region, as measured by flow cytometry. ConclusionsProvided the design steps are respected and the fragments are stored and labeled appropriately, multiple plasmid variants and combinations of the pre-designed modules can be assembled in one day, easier and using less resources than the established protocols, with good efficiency. The simplicity of the design and the affordability of the method could make modular cloning of plasmid constructs more accessible to small labs and students.

A limitation to the non-vertebrate 3Rs model Galleria mellonella has been the lack of genetic toolkit. A common requirement for genetic tractability is a method to introduce exogenous material to the unicellular embryo, the most common of which is microinjection. This short article describes a detailed method for rearing Galleria mellonella to collect large amounts of staged embryos and to dechorionate and microinject embryos with limited mortality. Research HighlightsO_ST_ABSScientific BenefitsC_ST_ABSMicroinjection allows the introduction of a wide variety of substances, such as DNA, RNA or drugs into Galleria embryos, providing the technology needed for genetic engineering, gene editing and functional studies in this important model organism. 3Rs BenefitsGalleria is being increasingly used as a partial animal replacement model, especially in the field of infection biology. However, uptake has been limited by the lack of genetic and molecular tools. This protocol takes a step towards removing these barriers by providing a means to introduce substances that can create transgenic or genetically engineered Galleria. Practical BenefitsProtocol for injecting substances into Galleria, using for the most part easily accessible equipment. Current ApplicationsGenerating stable transgenic and gene-edited Galleria lines. Potential ApplicationsAny technique requiring the introduction of substances to Galleria embryos. This includes applying existing techniques such as pBac-mediated transgenesis or CRISPR/Cas-based gene-editing to this organism,in order to generate engineered strains of Galleria. It could also include injection of synthetic mRNAs encoding proteins fused to fluorescent genes (such as GFP) in order to visualise their dynamics in living embryos; and the injection of drugs that perturb particular cell or developmental processes in order to learn more about early Galleria development.

Regenerating fertile Arabidopsis thaliana plants from tissue culture cells with transformed plastid genomes is difficult, because of somaclonal variation in tissue culture cells. For nuclear gene transformation, tissue culture limitations were overcome in Arabidopsis by direct transformation of the female gametes using the floral dip protocol and identification of transgenic events in the seed progeny. During Agrobacterium transformation the VirD2 protein guides the T-complex, consisting of single stranded transferred-DNA (T-DNA) coated with VirE2 proteins, to the plant nucleus. To enable floral dip transformation of the plastid DNA, we retargeted VirD2 to chloroplasts. We show plastid targeting of VirD2 in a split GFP assay, where VirD2-GFP11 complements GFP1-10 in chloroplasts. Floral dip transformation of plastids will avoid tissue culture altogether, making plastid transformation readily available for the research community.

Fluorescent RNA aptamers are an increasingly used tool for quantifying transcription and for visualising RNA interactions, both in vitro and in vivo. However when tested in the commercially available, E. coli extract based Expressway cell-free expression system, no fluorescence is detected. The same experimental setup is shown to successfully produce fluorescent RNA aptamers when tested in another buffer designed for in vitro transcription, and RNA purification of the Expressway reaction products show that transcription does occur, but does not result in a fluorescent product. In this paper we demonstrate the incompatibility of a narrow selection of RNA aptamers in one particular cell-free expression system, and consider that similar issues may arise with other cell-free expression systems, RNA aptamers, and their corresponding fluorophores.

Adeno-associated viruses (AAVs) have emerged as powerful tools for delivery of genes to a variety of cell types including pancreatic endocrine cells. Currently, AAV serotype 8 (AAV8) is the main AAV vector employed for infecting pancreatic cells for transgene transfer. We aimed to address whether alternative serotypes (AAV2, AAV6, and AAV9) commonly used for gene transfer can be effective in transducing pancreatic cells efficiently. We also screened the additives heparin and neuraminidase to further understand the interaction between the individual AAV types included in this work and the cells for optimal infection. Murine pancreatic {beta}-cells and -cells as well as fibroblasts were infected with AAV serotypes 2, 6, and 9 carrying the transgene for enhanced green fluorescent protein (eGFP). AAV2 outperformed AAV9 in transducing pancreatic cells, while AAV6 induced cytotoxicity. Both AAV2 and AAV9 displayed slightly higher tropism for -cells than for {beta}-cells. Compared to the pancreatic cells, the fraction of GFP-expressing cells at various multiplicities of infection was consistently lower for fibroblasts. Incubation of AAV2 with heparin prior to transduction failed to induce any GFP expression in {beta}-cells, indicating that the primary site used for initial interaction with pancreatic cells are heparan sulfate proteoglycans. Treatment of {beta}-cells with neuraminidase prior to AAV9 infection appeared to improve the number of GFP-positive cells, but the increase was not statistically significant. These findings expand the repertoire of available serotypes for AAV-mediated delivery of transgenes to pancreatic endocrine cells and may contribute to gene therapy strategies for pancreas pathologies.

The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production. Main conclusionWe generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb.

Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard.

Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus) comprises the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompass most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I (CHLI). Second, we developed a tobacco etch virus (genus Potyvirus)-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed to obtain plants exhibiting a white phenotype with their four alleles edited through in vitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus, these findings must be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.

Culex quinquefasciatus Say is a brown, medium sized mosquito distributed widely in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and is responsible for vectoring many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical / chemical) are increasingly ineffective; use of insecticides also imposes some hazards to both human and ecosystem health. Recent advances in genome editing have allowed the development of genetic methods of insect control, which is species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that has been shown to be functional in a range of species. We demonstrate here, the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated exogenous sequence comprising a sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8% similar to rates achieved in other mosquito species. Precise knock-in at the intended locus was confirmed by sequencing. Insertion homozygotes displayed a white eye phenotype in early-mid stage larvae and a recessive lethal phenotype by pupation. This work shows an alternative and efficient method for genetic engineering of C. quinquefasciatus, providing a new tool for researchers interested in developing genetic control tools for this vector.

Rod photoreceptor formation in the postnatal mouse is a widely used model system for studying mammalian photoreceptor development. This experimental paradigm provides opportunities for both gain and loss-of-function studies which can be accomplished through in vivo plasmid delivery and electroporation. However, the cis-regulatory elements used to implement this approach have not been fully evaluated or optimized for the unique transcriptional environment of photoreceptors. Here we report that the use of a photoreceptor cis-regulatory element from the Crx gene in combination with broadly active promoter elements can increase the targeting of developing rod photoreceptors in the mouse. This can lead to greater reporter expression, as well as enhanced misexpression and loss-of-function phenotypes in these cells. This study also highlights the importance of identifying and testing relevant cis-regulatory elements when planning cell subtype specific experiments. The use of the specific hybrid elements in this study will provide a more efficacious gene delivery system to study mammalian photoreceptor formation.

It is commonplace in East Africa for 100% of cassava fields to be infected with Cassava mosaic disease (CMD) and/or Cassava brown streak disease (CBSD), resulting in annual losses of more than US$1.25 billion and reduced food and economic security for farming households. The vector of both diseases is the African cassava species of the whitefly Bemisia tabaci. Since the late 1990s, there has been an unprecedented increase in whitefly populations, to the extent that they are referred to as "super-abundant". Research efforts since the late 1990s has focused mainly on developing plant resistance to the viral pathogens and paid scant attention to understanding the root causes of disease epidemics or the control of whitefly infestation. Here, we aimed at developing long-term whitefly-control solutions using an in-planta RNA interference (RNAi) approach. First, transcriptome analysis identified candidate genes that play key roles in whitefly biology: osmoregulation, sugar metabolism and transport, symbiosis with endosymbiotic bacteria and detoxification of phytotoxins. Then, fifteen RNAi inverted repeat constructs were produced, designed to target the candidate genes and 140 independent transgenic lines were generated in cassava variety NASE 13. Whole plant bioassays showed insecticidal activity of transgenic plants, reaching 58% lethality for adults within 7 days and 75-90% lethality of nymphs after 25 days, compared to control plants. Target genes were confirmed to be downregulated by up to 2.5-fold in adult whiteflies and nymphs. We used population dynamics modelling to predict the potential of the RNAi technology to control whiteflies under field conditions in East Africa.

Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga, Chlamydomonas reinhardtii and used to control disease in shrimp. Here we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high level transcription of both strands of the WSSV gene element in the chloroplast. New vectors were designed that allow rapid Golden Gate-mediated assembly of a transformation plasmid in which the dual promoter-WSSV DNA cassette is targeted into the chloroplast genome, with selection based on the restoration of photosynthesis. PCR analysis of transformant lines confirmed the integration of the cassette and homoplasmy of the polyploid genome. Transcribed sense and antisense VP28-RNA were hypothesised to form an RNA duplex in the chloroplast stroma, and quantitative RT-PCR indicated that [~]100 g dsRNA is produced per litre of transgenic microalgae culture. This represents an [~]10,000-fold increase in dsRNA relative to previous reports using convergent psaA promoters. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. Survival of shrimp fed with dsRNA-expressing C. reinhardtii was significantly enhanced (68.3%) relative to the negative control. The study suggests that this new dsRNA production platform is significantly more efficient than that reported previously, and merits further scale-up and downstream processing studies.

Apoptosis is an essential physiological process involved in embryonic development, immune responses, and tissue homeostasis. Despite many studies on pro-apoptotic genes, few reports have directly compared the lethality-inducing potential between them under identical conditions. In this study, we evaluated the lethality-inducing potential of three representative pro-apoptotic genes, Bax, Casp3, and Casp9, in mouse early embryos under defined conditions using the doxycycline (Dox)-inducible tetracycline-regulated gene expression system (Tet-On system) in combination with the PiggyBac transposon system. All genes were transcriptionally induced by Dox, and Bax showed the strongest lethal effect, followed by Casp9, while Casp3 did not show any effect. Notably, Bax expression severely impaired blastocyst formation and led to the intense accumulation of the DNA damage marker {gamma}H2AX. These findings suggest that introducing upstream apoptotic regulators leads to the more efficient and widespread activation of the apoptotic cascade. Additionally, an unexpected Dox-dependent increase in the expression of reverse tetracycline-controlled transactivator, which is typically driven by a constitutive promoter, was observed, raising the possibility of unanticipated regulatory mechanisms within the Tet-On system. Overall, this study is expected to contribute to a deeper understanding of apoptotic mechanisms and future advancements in regenerative medicine, reproductive engineering, and cancer research.

In 2020 we suffered from a major global pandemic caused by the SARS-CoV-2 coronavirus. Efforts to contain the virus include the development of rapid tests and vaccines, which require a ready supply of viral proteins. Here we report the production of two SARS-CoV-2 proteins by transient transformation of tobacco, leading to high expression within three days, and subsequent purification of the intact proteins. Such efforts may help to develop testing resources to alleviate the major impacts of this global pandemic.

Abstract/SummaryIntegrase-mediated Programmable Genomic Integration (I-PGI) uses a Cas9 nickase (nCas9) with a reverse transcriptase (RT), to write a large serine integrase (LSI) target site (attB/P, here called "beacon") in a programmed location. Co-delivery of the LSI and a DNA template containing the cognate recognition site results in precise integration of the template in a specific genomic location. While we were able to achieve high-fidelity beacon placement in a range of primate cycling and non-dividing cells, when translating our technology into an in vivo rodent model (liver) we surprisingly observed very low beacon fidelity, with the vast majority of beacons being unsuitable for integration. This phenomenon was independent of mouse strain, but was specific to non-dividing cells, as a cycling mouse hepatocyte cell line (Hepa1-6) demonstrated very high levels of fidelity. To address this issue we utilized neonatal mice, which have a much higher proportion of proliferating hepatocytes than adult mice. This resulted in a significant increase in the placement of high-fidelity beacons, and achieved functional gene expression after I-PGI in a therapeutically relevant target site. In an alternate approach, we engineered transgenic mice with intact beacons placed in specific genomic locations, allowing us to optimize integrase and DNA template dosing and kinetics. In summary, we have identified a previously undescribed challenge when using RT-based editing to write long sequences (~40 bp) in non-dividing rodent hepatocytes. This phenomenon was specific to rodents and was not observed in primate dividing or non-dividing cells. This previously unidentified challenge using RTs will limit the use of I-PGI in mouse models, however here we describe two methods that address this issue.

This work demonstrates the reconstitution of active methylxanthine synthesis enzymes in human cells and their potential use as inducible reporter enzymes. A variety of plant enzymes involved in caffeine synthesis have been characterized in vitro and several of these methylxanthine synthesis enzymes have been heterologously-expressed in yeast or bacteria. In this work, enzymes from Coffea arabica, Camellia sinensis, and Paullinia cupana have been heterologously-expressed in human cells. We demonstrate that the enzymes tested exhibit similar patterns of activity with a set of xanthine substrates in human cells when compared to previous reports of in vitro activity. We demonstrate that the activity of these enzymes can be used as a reporter for juxtacrine signaling using synNotch-induced expression in the presence of an appropriate substrate. When used in combination with synthetic caffeine receptors, this work has potential for use as an in vivo reporter (e.g. enabling non-invasive monitoring of cell-cell interactions after a cellular transplant) or in synthetic intercellular signaling a methylxanthine, such as caffeine, acting as a synthetic paracrine hormone.

Application of plasmid encoding synthetic dsRNA targeted IMNV genome (Infectious Myonecrosis Virus) can reduce viral replication in the shrimp industry by activating RNA interference (RNAi) response. Application of dsRNA plasmid as antiviral for IMNV in shrimp-farm need a huge quantity of plasmid. Bioreactor can be used for large-scale plasmid production to achieve high plasmid yield. Plasmid production in the bioreactor can be improved by selection of the host organism, the recombinant plasmid vector, the fermentation media, and the fermentation strategy. This research aim is to determine the fermentation media and fermentation strategy to produce recombinant dsRNA plasmid with high plasmid yield. Selection of fermentation media was conducted in a baffled flask with three different media. Then, the optimum media was used for optimization in bioreactor production with the addition of feeding media. As a result, plasmid production in TB media has a higher biomass growth rate and plasmid production rate than production in M9+Mod and LB+ media. Plasmid production in TB media in baffled-flask resulted in plasmid yield in 2.318 mg/L, 14-fold higher than M9+Mod (0.165 mg/L), and 34-fold higher than LB (0.068 mg/L). In bioreactor production, plasmid production in fed-batch fermentation in bioreactor resulted plasmid yield in 1.018 mg/L, 5-fold higher than batch fermentation (1.882 mg/L). Plasmid was confirmed in agarose gel electrophoresis at [~]5750 bp and insert gene at 700 bp. The cultivation technique developed should be workable for the pilot scale. Downstream processing in plasmid production should be able to achieve plasmid with high concentration and purity.

PurposeTargeted regulation of transfected extra-chromosomal plasmid DNA typically requires the integration of 9 - 20 bp docking sites into the plasmid. Here, we report an elegant approach, The Dpn Adaptor Linked Effector (DAL-E) system, to target fusion proteins to 6-methyladenosine in GATC, which appears frequently in popular eukaryotic expression vectors and is absent from endogenous genomic DNA. Methods: The DNA-binding region from the DpnI endonuclease binds 6-methyladenosine within the GATC motif. We used a Dpn-transcriptional activator (DPN7-TA) fusion to induce gene expression from transiently transfected pDNAs. ResultsWe validated methylation-dependent activity of DPN7-TA with a panel of target pDNAs. We observed stronger transactivation when GATC targets were located upstream of the transcriptional start site in the target pDNA. Conclusion: DAL-E, consisting of a 108 aa, 12 kD DNA-binding adaptor and a 4 bp recognition site, offers a genetically-tractable, tunable system that can potentially be redesigned to recruit a variety of regulators (e.g. activators, silencers, epigenome editors) to transfected plasmid DNA. LAY SUMMARYTransfection of plasmid DNA (pDNA) is a commonly used method for introducing exogenous genetic material into mammalian cells. Once introduced into cells not all pDNAs express this genetic material at sufficient levels. Current techniques to improve transgene expression are limited and are not always feasible for all plasmids. This report presents a new method to improve gene expression from pDNA. The Dpn Adaptor Linked Effector (DAL-E) binds to methylated adenines in the pDNA resulting in increased expression. This technique has exciting implications for improved genetic engineering of mammalian cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=124 HEIGHT=200 SRC="FIGDIR/small/466616v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1116db0org.highwire.dtl.DTLVardef@1386f99org.highwire.dtl.DTLVardef@26c63corg.highwire.dtl.DTLVardef@1a09673_HPS_FORMAT_FIGEXP M_FIG C_FIG

Baculovirus technology has been the most commonly used expression system for insect cells both due to its potential to generate a large amount of recombinant protein as well as the benefit of post-translational modifications. The most commonly used system to generate recombinant baculoviruses is the Tn7 transposition-based technology known as Bac-to-Bac. Although improvements have been made to this system to further improve quality and reproducibility of baculovirus production, recent data suggests that improved strains still have potential issues with contamination of non-recombinant baculovirus caused by improper transposition into a Tn7 site in the E. coli chromosome. Here we describe a new option for alteration of the E. coli genome to completely block the native Tn7 attachment site, leading to far fewer false positive bacmid colonies being selected and eliminating all risk of non-recombinant baculovirus production.