Stable Nuclear Transformation Research Articles

Stable nuclear transformation methods for Euglena gracilis and its application to a related Euglenida

Euglenida is a taxonomic group of single-celled flagellates that have a variety of nutrient uptake strategies, making this an excellent model for studying the evolutionary acquisition of secondary chloroplasts. Among Euglenida, Euglena gracilis is the most extensively studied at the biochemical and molecular levels. However, the lack of a widely adopted method of nuclear genome transformation has hindered genetic studies in this organism. Herein, we present a novel nuclear transformation electroporation method that utilizes the 5′ adjacent region sequences of endogenous genes in E. gracilis. We used a NanoLuc reporter to evaluate promoter activity to identify four endogenous promoter candidates with superior luciferase transcriptional activity compared with the activity of the commonly used CaMV 35S promoter in E. gracilis nuclear transformation methods. We used G418 selection to obtain stable E. gracilis transformants harboring the transgene in their genomic DNA. Furthermore, we extended the applicability of our method to Rapaza viridis, a kleptoplastic species in Euglenida. Introduction of the DNA constructs developed for E. gracilis into R. viridis via electroporation generated stable G418-resistant strains exhibiting robust luciferase activity. The introduced promoter sequences functioned effectively, and accurate 5′ end modifications of mRNA were observed in both E. gracilis and R. viridis transformants. These findings demonstrate the broad applicability of our nuclear transformation method across multiple Euglenida species, facilitating molecular biology and genetic diversity studies in this taxonomic group.

Production of Bovine Rotavirus VP6 Subunit Vaccine in a Transgenic Fodder Crop, Egyptian Clover (Berseem, Trifolium alexandrinum) that Elicits Immune Responses in Rabbit.

Group A rotavirus causes acute gastroenteritis in young ones of animals worldwide and is responsible for a high rate of their morbidity and mortality leading to huge economic losses. Developing affordable and safer vaccine on large scale is imperative to reach cattle population worldwide for the long-term control of diarrhea. Rotavirus middle capsid protein layer, VP6, is the most immunogenic and highly conserved protein that induces immune responses against rotavirus. In the present study, bovine group A rotavirus VP6 protein has been expressed for the first time in a highly nutritious and palatable forage crop, Trifolium alexandrinum, using Agrobacterium tumefaciens-mediated stable nuclear transformation. Transgenic nature of the shoots regenerated from cotyledon explants and rooted on hygromycin-containing medium was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qPCR), and protein expression and quantification by Western blot and enzyme-linked immune-sorbent assay (ELISA), respectively. The transformation efficiency of 2.10% was obtained. The highest amount of VP6 protein produced in a transgenic line was 402ng/g fresh weights (0.03% of total soluble protein). Oral feeding of transgenic leafy shoots expressing VP6 protein stimulated systemic immunity by inducing significantly higher titers of anti-VP6 serum IgG antibodies in rabbit to reduce rotavirus infection. These transgenic fodder plants offer safer vaccine produced on large scale at low cost with reduced regulatory issues to improve livestock's health and wealth. These plants would be used as alternative to the current live attenuated vaccines to protect young calves against rotavirus infection.

Production of a viral surface protein in Nannochloropsis oceanica for fish vaccination against infectious pancreatic necrosis virus

Nannochloropsis oceanica is a unicellular oleaginous microalga of emerging biotechnological interest with a sequenced, annotated genome, available transcriptomic and proteomic data, and well-established basic molecular tools for genetic engineering. To establish N. oceanica as a eukaryotic host for recombinant protein synthesis and develop molecular technology for vaccine production, we chose the viral surface protein 2 (VP2) of a pathogenic fish virus that causes infectious pancreatic necrosis as a model vaccine. Upon stable nuclear transformation of N. oceanica strain CCMP1779 with the codon-optimized VP2 gene, a Venus reporter fusion served to evaluate the strength of different endogenous promoters in transformant populations by qPCR and flow cytometry. The highest VP2 yields were achieved for the elongation factor promoter, with enhancer effects by its N-terminal leader sequence. Individual transformants differed in their production capability of reporter-free VP2 by orders of magnitude. When subjecting the best candidates to kinetic analyses of growth and VP2 production in photobioreactors, recombinant protein integrity was maintained until the early stationary growth phase, and a high yield of 4.4% VP2 of total soluble protein was achieved. The maximum yield correlated with multiple integrations of the expression vector into the nuclear genome. The results demonstrate that N. oceanica was successfully engineered to constitute a robust platform for high-level production of a model subunit vaccine. The molecular methodology established here can likely be adapted in a straightforward manner to the production of further vaccines in the same host, allowing their distribution to fish, vertebrates, or humans via a microalgae-containing diet.Key points• We engineered N. oceanica for recombinant protein production.• The antigenic surface protein 2 of IPN virus could indeed be expressed in the host.• A high yield of 4.4% VP2 of total soluble protein was achieved in N. oceanica.

Recombinant Expression of Thrombolytic Agent Reteplase in Marine Microalga Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta).

Tetraselmis subcordiformis, a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis, and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis. This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.

A marine Chlamydomonas sp. emerging as an algal model.

The freshwater microalga Chlamydomonas reinhardtii, which lives in wet soil, has served for decades as a model for numerous biological processes, and many tools have been introduced for this organism. Here, we have established a stable nuclear transformation for its marine counterpart, Chlamydomonas sp. SAG25.89, by fusing specific cis-acting elements from its Actin gene with the gene providing hygromycin resistance and using an elaborated electroporation protocol. Like C. reinhardtii, Chlamydomonas sp. has a high GC content, allowing reporter genes and selection markers to be applicable in both organisms. Chlamydomonas sp. grows purely photoautotrophically and requires ammonia as a nitrogen source because its nuclear genome lacks some of the genes required for nitrogen metabolism. Interestingly, it can grow well under both low and very high salinities (up to 50g · L-1 ) rendering it as a model for osmotolerance. We further show that Chlamydomonas sp. grows well from 15 to 28°C, but halts its growth at 32°C. The genome of Chlamydomonas sp. contains some gene homologs the expression of which is regulated according to the ambient temperatures and/or confer thermal acclimation in C. reinhardtii. Thus, knowledge of temperature acclimation can now be compared to the marine species. Furthermore, Chlamydomonas sp. can serve as a model for studying marine microbial interactions and for comparing mechanisms in freshwater and marine environments. Chlamydomonas sp. was previously shown to be immobilized rapidly by a cyclic lipopeptide secreted from the antagonistic bacterium Pseudomonas protegens PF-5, which deflagellates C. reinhardtii.

Stable transformation of unicellular green alga Coccomyxa subellipsoidea C-169 via electroporation.

In this study, we have shown the applicability of electroporation and hygromycin B as a convenient selectable marker for stable nuclear transformation of Coccomyxa subellipsoidea C-169. Since it is the first sequenced eukaryotic microorganism from polar environment, this offers unique opportunities to study adaptation mechanisms to cold.

Construction of a novel vector for the nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii and its stable expression

Plasmid pBI221aadAGUS which carried both of GUS (β-glucuronidase) and aadA (aminoglyoside transferase) genes besides of the 35S cauliflower mosaic virus promoter was constructed and used for stable nuclear transformation of Chlamydomonas reinhardtii. The vector was transformed into the alga by particle gun bombardment and two positive colonies were selected on spectinomycin–containing medium. The restriction analysis of the DNA of the positive colonies showed that aadA was inserted in two orientations. The presence of introduced genes in the transformed colonies was confirmed by (PCR) using primers specific to GUS and aadA genes. The expression of aadA and GUS genes was revealed in all colonies that were grown on spectinomycin in liquid culture for 3–4 generations. The usefulness of this vector, differing in the orientation of the aadA cassette, was manisfested by transforming C. reinhardtii to spectinomycin resistance in the stable expression. This constructed plasmid-based expression vector system would help to unravel the functions of various genes in the green alga.

PEG-mediated, Stable, Nuclear and Chloroplast Transformation of Cyanidioschizon merolae.

The ability to achieve nuclear or chloroplast transformation in plants has been a long standing goal, especially in microalgae research. Over past years there has been only little success, but transient and stable nuclear transformation has been achieved in multiple species. Our newly developed method allows for relatively simple transformation of Cyanidioschizon merolae in both nuclear and chloroplast genome by means of homologous recombination between the genome and a transformation vector. The use of chloramphenicol resistance gene as the selectable marker allows for plate-based efficient selection of mutant colonies. Overall, the method allows the generation of mutant strains within 6 months.

Stable Expression of Adalimumab in Nicotiana tabacum.

Production of monoclonal antibodies and pharmaceutical proteins in transgenic plants has been the focus of many research efforts for close to 30years. Use of plants as bioreactors reduces large-scale production costs and minimizes risk for human pathogens contamination. Stable nuclear transformation of the plant genome offers a clear advantage in agricultural protein production platforms, limited only by the number of hectares that can be cultivated. We report here, for the first time, successful and stable expression of adalimumab in transgenic Nicotiana tabacum plants. The plant-derived adalimumab proved fully active and was shown to rescue L929 cells from the in vitro lethal effect of rhTNFα just as effectively as commercially available CHO-derived adalimumab (Humira). These results indicate that agricultural biopharming is an efficient alternative to mammalian cell-based expression platforms for the large-scale production of recombinant antibodies.

Nuclear transformation and functional gene expression in the oleaginous microalga Monoraphidium neglectum

Photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of a range of bio-products and genetic engineering is an increasingly important strategy to improve the natural cellular features. For the vast majority of microalgal strains, however, genetic engineering is not yet possible and the establishment of efficient genetic tools for a broad range of microalgal species is an important task to reach biotechnological success. Stable DNA transformation is one of the crucial steps for most genetic engineering approaches. In this context, we report the first successful and stable nuclear genetic transformation of the biotechnologically promising oleaginous microalga M. neglectum. Transformation was achieved by electroporation and the efficiency could progressively be improved by implementation of an additional cell pretreatment step, aiming at weakening the rigid natural cell wall. Furthermore, a first reporter transgene was established for M. neglectum. As a result of this work, genetic engineering strategies now become accessible which are required to successfully establish M. neglectum as a novel host for biotechnological applications.

Chloramphenicol acetyltransferase-a new selectable marker in stable nuclear transformation of the red alga Cyanidioschyzon merolae.

In this study, we have shown the applicability of chloramphenicol acetyltransferase as a new and convenient selectable marker for stable nuclear transformation as well as potential chloroplast transformation of Cyanidioschyzon merolae-a new model organism, which offers unique opportunities for studding the mitochondrial and plastid physiology as well as various evolutionary, structural, and functional features of the photosynthetic apparatus.

Heterologous Expression of Moss Light-harvesting Complex Stress-related 1 (LHCSR1), the Chlorophyll a-Xanthophyll Pigment-protein Complex Catalyzing Non-photochemical Quenching, in Nicotiana sp.

Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called non-photochemical quenching, occurs within the photosystem II antenna system by the action of two essential light-harvesting complex (LHC)-like proteins, photosystem II subunit S (PSBS) in plants and light-harvesting complex stress-related (LHCSR) in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, which are present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems Nicotiana benthamiana and Nicotiana tabacum using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes, and is correctly folded with chlorophyll a and xanthophylls but without chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo, implying that the recombinant protein obtained is a good material for future structural and functional studies.

Transformation of the Diatom Phaeodactylum tricornutum with its Endogenous (E)-4-Hydroxy-3-methylbut-2-enyl Diphosphate Reductase Gene

Phaeodactylum tricornutum is a model diatom that its genomic information and biological tools are well established. In this study, a gene encoding (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (PtHDR), a terminal enzyme of the methylerythritol phosphatepathway regulating chlorophyll and carotenoid biosynthesis, was isolated from P. tricornutum. The isolated gene was cloned into pPha-T1 vector containing fcpA promoter to prepare pPha-T1-HDR plasmid. As a positive control, pPha-T1-eGFP plasmid was constructed with egfp gene. Stable nuclear transformation was carried out with these plasmids by particle bombardment method and zeocin resistant colonies of P. tricornutum were selected on f/2 agar plate. In result, transformation efficiency was evaluated according to the amount of plasmid DNA coated with gold particles. Integration of introduced plasmids was confirmed with genomic DNA of each transformant by polymerase chain reaction. The eGFP fluorescence was visible in the cytoplasm, indicating that eGFP was successively expressed in P. tricornutum system. The transcript level of exogenous Pthdr gene was evaluated with the obtained transformants. The results presented here demonstrated that introduction of Pthdr gene into P. tricornutum chromosome succeeded and expression of PtHDR was enhanced under the fcpA promoter.

English

Although microalgae have valuable resources with great necessity in many biotechnological applications, no tools have been developed yet for a stable genetic transformation without antibiotic marker genes in these organisms. Chlorella is one of the most useful genus for biotechnological applications. The transfer of foreign DNA (vector or linear DNA cassette) into Chlorella by electroporation has very low stability and it is hard to screen the transformants without antibiotic marker genes. However, the marker genes have some disadvantages to host cells. To avoid the negative effects caused by the marker genes, we tried to develop a stable marker-free nuclear transformation system in Chlorella. For this, linear gene expression cassettes (LGEC) were constructed with functional domains, which are responsible for transformation, of SV40 large T antigen. The LGECs were transferred into Chlorella via electroporation and durability of the LGECs were confirmed in transgenic Chlorella. Transcription levels of the LGECs were also determined at different cell cycle sates. The LGECs integrated into the chromosomal DNA of Chlorella were stably replicated and were expressed successfully at G0-, G1-, and G2-phases. This study presents a stable marker-free nuclear transformation system with potential for biotechnological applications. Key words: Chlorella vulgaris, marker-free nuclear transformation, SV40 large T antigen, microalga.

Stable nuclear transformation of the oleaginous microalga Neochloris oleoabundans by electroporation

Biodiesel from microalgae is technically feasible, but not yet economically viable. A potential approach to improve microalgae as an economically viable biodiesel feedstock is to increase microalgal lipid content via genetic engineering. Genetic manipulation of microalgae requires the accessibility to stable nuclear transformation. In this study, we describe a strategy for developing a stable nuclear transformation system of the oleaginous microalga Neochloris oleoabundans using electroporation. The hygromycin B-resistant gene Hyg3, which was used as a positively selectable marker, consisted of aph7” gene encoding aminoglycoside phosphotransferase of Streptomyces hygroscopicus and intron1 of Chlamydomonas reinhardtii rbcS2 gene, under the control of C. reinhardtii HSP70A-RBCS2 hybrid promoter. The transformation frequency was 5.2 × 10−4 transformants mg−1 DNA. The transformants showed stable hygromycin B-resistant phenotype for at least 6 months in the absence of the antibiotic selection. Co-transformation frequency of unselectable green fluorescent protein gene (Gfp) adapted to C. reinhardtii codon usage (ChGfp) and selectable Hyg3 gene was 2.6 × 10−4 transformants mg−1 DNA; up to 90 % of the transformants exhibited green fluorescent protein (GFP) activity. The ChGfp and Hyg3 gene were integrated into the nuclear genome of N. oleoabundans. The GFP fluorescence signal of the transformants under confocal laser scanning microscope was visible. The successful stable nuclear transformation system not only provides a basis for molecular genetics study, but also enables subsequent genetic engineering in the microalga to increase lipid content for biodiesel production. The strategy for developing the stable nuclear transformation system presented in this study may be applicable to other microalgal species without sequenced genome.

The inducible nitA promoter provides a powerful molecular switch for transgene expression in Volvox carteri.

BackgroundThe multicellular green alga Volvox carteri represents an attractive model system to study various aspects of multicellularity like cellular differentiation, morphogenesis, epithelial folding and ECM biogenesis. However, functional and molecular analyses of such processes require a wide array of molecular tools for genetic engineering. So far there are only a limited number of molecular tools available in Volvox.ResultsHere, we show that the promoter of the V. carteri nitrate reductase gene (nitA) is a powerful molecular switch for induction of transgene expression. Strong expression is triggered by simply changing the nitrogen source from ammonium to nitrate. We also show that the luciferase (g-luc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of the unicellular alga Chlamydomonas reinhardtii, is a suitable reporter gene in V. carteri. Emitted light of the chemiluminescent reaction can be easily detected and quantified with a luminometer. Long-term stability of inducible expression of the chimeric nitA/g-luc transgenes after stable nuclear transformation was demonstrated by transcription analysis and bioluminescence assays.ConclusionTwo novel molecular tools for genetic engineering of Volvox are now available: the nitrate-inducible nitA promoter of V. carteri and the codon-adapted luciferase reporter gene of G. princeps. These novel tools will be useful for future molecular research in V. carteri.

Engineering, expression in transgenic plants and characterisation of E559, a rabies virus-neutralising monoclonal antibody.

Rabies post-exposure prophylaxis (PEP) currently comprises administration of rabies vaccine together with rabies immunoglobulin (RIG) of either equine or human origin. In the developing world, RIG preparations are expensive, often in short supply, and of variable efficacy. Therefore, we are seeking to develop a monoclonal antibody cocktail to replace RIG. Here, we describe the cloning, engineering and production in plants of a candidate monoclonal antibody (E559) for inclusion in such a cocktail. The murine constant domains of E559 were replaced with human IgG1κ constant domains and the resulting chimeric mouse-human genes were cloned into plant expression vectors for stable nuclear transformation of Nicotiana tabacum. The plant-expressed, chimeric antibody was purified and biochemically characterized, was demonstrated to neutralize rabies virus in a fluorescent antibody virus neutralization assay, and conferred protection in a hamster challenge model.

Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens.

Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has already been widely recognized as a viable alternative for industrial-scale production of biopharmaceuticals. For expression of terpenoid biosynthetic genes, and reconstruction of heterologous pathways, Physcomitrella has unique attributes that makes it a very promising biotechnological host. These features include a high native tolerance to terpenoids, a simple endogenous terpenoid profile, convenient genome editing using homologous recombination, and cultivation techniques that allow up-scaling from single cells in microtiter plates to industrial photo-bioreactors. Beyond its use for functional characterization of terpenoid biosynthetic genes, engineered Physcomitrella can be a green biotechnological platform for production of terpenoids. Here, we describe two complementary and simple procedures for stable nuclear transformation of Physcomitrella with terpenoid biosynthetic genes, selection and cultivation of transgenic lines, and metabolite analysis of terpenoids produced in transgenic moss lines. We also provide tools for metabolic engineering through genome editing using homologous recombination.

A Codon-Optimized Bacterial Antibiotic Gene Used as Selection Marker for Stable Nuclear Transformation in the Marine Red Alga Pyropia yezoensis

Marine macroalgae play an important role in marine coastal ecosystems and are widely used as sea vegetation foodstuffs and for industrial purposes. Therefore, there have been increased demands for useful species and varieties of these macroalgae. However, genetic transformation in macroalgae has not yet been established. We have developed a dominant selection marker for stable nuclear transformation in the red macroalga Pyropia yezoensis. We engineered the coding region of the aminoglycoside phosphotransferase gene aph7″ from Streptomyces hygroscopicus to adapt codon usage of the nuclear genes of P. yezoensis. We designated this codon-optimized aph7″ gene as PyAph7. After bombarding P. yezoensis cells with plasmids containing PyAph7 under the control of their endogenous promoter, 1.9 thalli (or individuals) of hygromycin-resistant strains were isolated from a 10-mm square piece of the bombarded thallus. These transformants were stably maintained throughout the asexual life cycle. Stable expression of PyAph7was verified using Southern blot analysis and genomic PCR and RT-PCR analyses. PyAph7 proved to be a new versatile tool for stable nuclear transformation in P. yezoensis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10126-013-9549-5) contains supplementary material, which is available to authorized users.

Stable nuclear transformation of Eudorina elegans

BackgroundA fundamental step in evolution was the transition from unicellular to differentiated, multicellular organisms. Volvocine algae have been used for several decades as a model lineage to investigate the evolutionary aspects of multicellularity and cellular differentiation. There are two well-studied volvocine species, a unicellular alga (Chlamydomonas reinhardtii) and a multicellular alga with differentiated cell types (Volvox carteri). Species with intermediate characteristics also exist, which blur the boundaries between unicellularity and differentiated multicellularity. These species include the globular alga Eudorina elegans, which is composed of 16–32 cells. However, detailed molecular analyses of E. elegans require genetic manipulation. Unfortunately, genetic engineering has not yet been established for Eudorina, and only limited DNA and/or protein sequence information is available.ResultsHere, we describe the stable nuclear transformation of E. elegans by particle bombardment using both a chimeric selectable marker and reporter genes from different heterologous sources. Transgenic algae resistant to paromomycin were achieved using the aminoglycoside 3′-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus, an actinobacterium, under the control of an artificial promoter consisting of two V. carteri promoters in tandem. Transformants exhibited an increase in resistance to paromomycin by up to 333-fold. Co-transformation with non-selectable plasmids was achieved with a rate of 50 - 100%. The luciferase (gluc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of C. reinhardtii, was used as a reporter gene. The expression of gluc was mediated by promoters from C. reinhardtii and V. carteri. Heterologous heat shock promoters induced an increase in luciferase activity (up to 600-fold) at elevated temperatures. Long-term stability and both constitutive and inducible expression of the co-bombarded gluc gene was demonstrated by transcription analysis and bioluminescence assays.ConclusionsHeterologous flanking sequences, including promoters, work in E. elegans and permit both constitutive and inducible expression of heterologous genes. Stable nuclear transformation of E. elegans is now routine. Thus, we show that genetic engineering of a species is possible even without the resources of endogenous genes and promoters.