Introduction Of Foreign DNA Research Articles

Microalgal Phycoremediation: A Glimpse into a Sustainable Environment.

Microalgae are continually exposed to heavy metals and metalloids (HMMs), which stifles their development and reproduction due to the resulting physiological and metabolic abnormalities, leading to lower crop productivity. They must thus change their way of adapting to survive in such a hostile environment without sacrificing their healthy growth, development, reproductive capacity, or survival. The mode of adaptation involves a complex relationship of signalling cascades that govern gene expression at the transcriptional and post-transcriptional levels, which consequently produces altered but adapted biochemical and physiochemical parameters. Algae have been reported to have altered their physicochemical and molecular perspectives as a result of exposure to a variety of HMMs. Hence, in this review, we focused on how microalgae alter their physicochemical and molecular characteristics as a tolerance mechanism in response to HMM-induced stress. Furthermore, physiological and biotechnological methods can be used to enhance extracellular absorption and clean up. The introduction of foreign DNA into microalgae cells and the genetic alteration of genes can boost the bio-accumulation and remediation capabilities of microalgae. In this regard, microalgae represent an excellent model organism and could be used for HMM removal in the near future.

Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing.

Epichloë festucae var. lolii and Epichloë sp. LpTG-3 are filamentous fungal endophytes of perennial ryegrass (Lolium perenne) that have a substantial impact on New Zealand's agricultural economy by conferring biotic advantages to the host grass. Overall, Epichloë endophytes contribute NZ$200 million to the economy annually, with strain AR37 estimated to contribute NZ$3.6 billion to the New Zealand economy over a 20-year period. This strain produces secondary metabolites, including epoxyjanthitrems, which are a class of indole diterpenes, associated with the observed effects of AR37 on livestock and insect pests. Until very recently, AR37 was intractable to genetic modification but this has changed with the application of CRISPR-Cas9 based gene editing techniques. In this paper, gene inactivation by CRISPR-Cas9 was used to deconvolute the genetic basis for epoxyjanthitrem biosynthesis, including creating an AR37 strain that has been edited to remove the biosynthesis of all indole diterpenes. We show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) through an AMA1-based plasmid that simultaneously expresses the CRISPR-Cas9 system and selectable marker. Genetic modification events in these transformants were investigated through genome sequencing and in planta chemistry.

Towards the discovery of virulence and survival factors in Fusobacterium using host‐mimicking DNA methylation to transcend genetic barriers

Fusobacterium nucleatum (Fn) is an anaerobic, Gram‐negative, oral bacterium that disseminates from the mouth, and contributes to preterm birth, tissue infections, and acceleration of multiple cancers including colorectal and pancreatic. It is well‐established that most Fusobacteriumexhibit genetic recalcitrance, mainly owing to robust restriction modification systems. These systems allow bacteria to distinguish self from non‐self DNA via two enzymatic activities: a restriction endonuclease and a modification methyltransferase. Though the association of Fnin diseases is well‐established, the majority of our experimental work stems from the strain F. nucleatum 23726 because it is genetically tractable. To enable the study of Fn clinical isolates that are currently genetically intractable, here we show that we are able to fool host restriction modification systems that protect the bacteria against the introduction of foreign DNA by using FnDNA methyltransferases to protect our gene knockout plasmids against nuclease digestion. This genetic breakthrough has enabled the first mutants in F. nucleatum 25586, a feat that was never accomplished in decades of trying. Furthermore, we created a deletion library of genes predicted to be involved in host cell invasion and bacterial intracellular survival, which is enabling detailed mechanistic studies of these virulence proteins in Fn‐driven disease. Our results demonstrate the simplicity and convenience of this host‐mimicking approach, which enables circumvention of innate restriction modification barriers ‐ the most common underlying cause of bacterial genetic intractability. We anticipate in future applications our system could be expanded to study novel genetic elements of which are fundamental to the pathophysiology of a wide range of relevant Fusobacterium species.

A Comprehensive Toolkit for Quick and Easy Visualization of Marker Proteins, Protein-Protein Interactions and Cell Morphology in Marchantia polymorpha.

Even though stable genomic transformation of sporelings and thalli of Marchantia polymorpha is straightforward and efficient, numerous problems can arise during critical phases of the process such as efficient spore production, poor selection capacity of antibiotics or low transformation efficiency. It is therefore also desirable to establish quick methods not relying on stable transgenics to analyze the localization, interactions and functions of proteins of interest. The introduction of foreign DNA into living cells via biolistic mechanisms has been first reported roughly 30 years ago and has been commonly exploited in established plant model species such as Arabidopsis thaliana or Nicotiana benthamiana. Here, we report the fast and reliable transient biolistic transformation of Marchantia thallus epidermal cells using fluorescent protein fusions. We present a catalog of fluorescent markers which can be readily used for tagging of a variety of subcellular compartments. Moreover, we report the functionality of the bimolecular fluorescence complementation (BiFC) in M. polymorpha with the example of the p-body markers MpDCP1/2. Finally, we provide standard staining procedures for live cell imaging in M. polymorpha, applicable to visualize cell boundaries or cellular structures, to complement or support protein localizations and to understand how results gained by transient transformations can be embedded in cell architecture and dynamics. Taken together, we offer a set of easy and quick tools for experiments that aim at understanding subcellular localization, protein–protein interactions and thus functions of proteins of interest in the emerging early diverging land plant model M. polymorpha.

Plastid Transformation: How Does it Work? Can it Be Applied to Crops? What Can it Offer?

In recent years, plant genetic engineering has advanced agriculture in terms of crop improvement, stress and disease resistance, and pharmaceutical biosynthesis. Cells from land plants and algae contain three organelles that harbor DNA: the nucleus, plastid, and mitochondria. Although the most common approach for many plant species is the introduction of foreign DNA into the nucleus (nuclear transformation) via Agrobacterium- or biolistics-mediated delivery of transgenes, plastid transformation offers an alternative means for plant transformation. Since there are many copies of the chloroplast genome in each cell, higher levels of protein accumulation can often be achieved from transgenes inserted in the chloroplast genome compared to the nuclear genome. Chloroplasts are therefore becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes. This review provides a comprehensive historical and biological perspective on plastid transformation, with a focus on current and emerging approaches such as the use of single-walled carbon nanotubes (SWNTs) as DNA delivery vehicles, overexpressing morphogenic regulators to enhance regeneration ability, applying genome editing techniques to accelerate double-stranded break formation, and reconsidering protoplasts as a viable material for plastid genome engineering, even in transformation-recalcitrant species.

Locating and Characterizing a Transgene Integration Site by Nanopore Sequencing.

The introduction of foreign DNA into cells and organisms has facilitated much of modern biological research, and it promises to become equally important in clinical practice. Locating sites of foreign DNA incorporation in mammalian genomes has proven burdensome, so the genomic location of most transgenes remains unknown. To address this challenge, we applied nanopore sequencing in search of the site of integration of Tg(Pou5f1-EGFP)2Mnn (also known as Oct4:EGFP), a widely used fluorescent reporter in mouse germ line research. Using this nanopore-based approach, we identified the site of Oct4:EGFP transgene integration near the telomere of Chromosome 9. This methodology simultaneously yielded an estimate of transgene copy number, provided direct evidence of transgene inversions, revealed contaminating E. coli genomic DNA within the transgene array, validated the integrity of neighboring genes, and enabled definitive genotyping. We suggest that such an approach provides a rapid, cost-effective method for identifying and analyzing transgene integration sites.

Comparison of different kinds of nonviral vectors for gene delivery to human periodontal ligament stem cells

Background/purposeInducing human-periodontal-ligament-stem-cell (hPDLSC) differentiation by DNA transfection is a promising way for periodontal tissue engineering. However, there are only a few studies that focus on the introduction of foreign DNA into hPDLSCs by nonviral methods that are relatively safe. Hence, the major purposes of this study were to compare the transfection efficiency and toxicity of nonviral-gene-transfer methods on hPDLSCs, and to search for the best approach and optimal protocol for transferring genes into hPDLSCs using nonviral vectors. Materials and methodsThe hPDLSCs were transfected by (1) Lipofectamine 2000, (2) polyethylenimine, (3) GBfectene-Elite transfection reagent, (4) X-tremeGENE HP DNA Transfection Reagent, and (5) Magnet-Assisted Transfection (MATra), compared to (6) lentiviral vectors harboring a green-fluorescent-protein gene. The transfection efficiency was measured by a fluorescence microscope and flow cytometry. Meanwhile, the cell morphology and growth status were observed to estimate the cytotoxicity. ResultsAmong these methods, the transfection efficiency of the former four methods was not very satisfactory (< 6%) compared to that of lentiviral vectors (positive control, 95%). However, MATra was the most effective nonviral method (11%). Moreover, the cellular toxicity was lower than that of the former four methods. ConclusionThe transfection efficiency of hPDLSCs with MATra was higher than the other nonviral transfection reagents in this study, but it was far less than the viral vectors. Saving from the interaction between the positive and negative charges, and increasing the opportunity of contact between the plasmid and the cytomembrane might be the key to enhancing the transfection efficiency for hPDLSCs in application of periodontal tissue engineering.

Current Technologies and Related Issues for Mushroom Transformation

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

Improvement of adventitious organogenesis for regeneration of transgenic plants in blackberry

The introduction of foreign DNA into the plant genome by Agrobacterium tumefaciens is a promising technique of targeted gene transfer which depends on good working regeneration system. The aim of the work was to elaborate the system for efficient adventitious organogenesis and transgenic plant regeneration in Rubus fruticosus L. using explants from mature plants. Regeneration of putative transgenic shoots took place from flag explants cultivated vertically on MS medium with 1 mg l-1 TDZ and 0.02 mg l-1 IBA followed by transfer on MS medium with 1 mg l-1 BAP, 0.02 mg l-1 IBA and 0.1 mg l-1 GA3 supplemented with 10-15 mg l-1 hygromycin after transformation by A. tumefaciens strain LBA 4404 carrying plasmid pCambia 1304. Four putative transgenic plants of cv. 'Cacanska Bestrna' were rooted and acclimatized.

Manipulation of epigenetic factors and the DNA repair machinery for improving the frequency of plant transformation

Plant genetic engineering involves the introduction of foreign DNA into the plant genome in order to enhance/modify plant traits. In transgenic plants, it is difficult to achieve stable and predictable transgene expression over subsequent generations. Largely, this is due to the lack of critical understanding of plant perception and response to the artificially introduced foreign DNA. Recent reports have revealed components of the epigenetic module that may affect transgene stability at both pre- and post-integration steps. Furthermore, the integration of the transgene has been shown to be strictly dependent on the DNA repair machinery. In this review, we briefly summarize genetic and epigenetic factors whose manipulation can enhance the efficiency of plant transformation and the quality of genetically engineered transgenic plants.

CRISPR Regulation of Intraspecies Diversification by Limiting IS Transposition and Intercellular Recombination

Mobile genetic elements (MGEs) and genetic rearrangement are considered as major driving forces of bacterial diversification. Previous comparative genome analysis of Porphyromonas gingivalis, a pathogen related to periodontitis, implied such an important relationship. As a counterpart system to MGEs, clustered regularly interspaced short palindromic repeats (CRISPRs) in bacteria may be useful for genetic typing. We found that CRISPR typing could be a reasonable alternative to conventional methods for characterizing phylogenetic relationships among 60 highly diverse P. gingivalis isolates. Examination of genetic recombination along with multilocus sequence typing suggests the importance of such events between different isolates. MGEs appear to be strategically located at the breakpoint gaps of complicated genome rearrangements. Of these MGEs, insertion sequences (ISs) were found most frequently. CRISPR analysis identified 2,150 spacers that were clustered into 1,187 unique ones. Most of these spacers exhibited no significant nucleotide similarity to known sequences (97.6%: 1,158/1,187). Surprisingly, CRISPR spacers exhibiting high nucleotide similarity to regions of P. gingivalis genomes including ISs were predominant. The proportion of such spacers to all the unique spacers (1.6%: 19/1,187) was the highest among previous studies, suggesting novel functions for these CRISPRs. These results indicate that P. gingivalis is a bacterium with high intraspecies diversity caused by frequent insertion sequence (IS) transposition, whereas both the introduction of foreign DNA, primarily from other P. gingivalis cells, and IS transposition are limited by CRISPR interference. It is suggested that P. gingivalis CRISPRs could be an important source for understanding the role of CRISPRs in the development of bacterial diversity.

Functional genomic signatures of sponge bacteria reveal unique and shared features of symbiosis

Sponges form close relationships with bacteria, and a remarkable phylogenetic diversity of yet-uncultured bacteria has been identified from sponges using molecular methods. In this study, we use a comparative metagenomic analysis of the bacterial community in the model sponge Cymbastela concentrica and in the surrounding seawater to identify previously unrecognized genomic signatures and functions for sponge bacteria. We observed a surprisingly large number of transposable insertion elements, a feature also observed in other symbiotic bacteria, as well as a set of predicted mechanisms that may defend the sponge community against the introduction of foreign DNA and hence contribute to its genetic resilience. Moreover, several shared metabolic interactions between bacteria and host include vitamin production, nutrient transport and utilization, and redox sensing and response. Finally, an abundance of protein-protein interactions mediated through ankyrin and tetratricopeptide repeat proteins could represent a mechanism for the sponge to discriminate between food and resident bacteria. These data provide new insight into the evolution of symbiotic diversity, microbial metabolism and host-microbe interactions in sponges.

Introduction of foreign DNA into the water flea, Daphnia magna, by electroporation

Daphnids inhabit a diverse array of aquatic environments and they are a good model for understanding response and adaptation to environmental changes and they have been used one of standard organisms in ecotoxicology. Recent progress of genomics changed the tools for analyzing responses of daphnids, because gene expression changes can be observed before the emergence of prominent adverse effect such as immobility of the organism. Thus understanding of biological changes from gene expression level can be one of the sensitive tools for the evaluation of environmental response of organisms. However, there was no technique for genetic manipulation in daphnids. Hence, we have developed a gene introduction technique based on electroporation. There are two critical points for the successful introduction of foreign DNA into D. magna. (1) Injection of DNA into blood stream. (2) Usage of very low voltage for the electroporation. The injected DNA containing green fluorescent protein (GFP) could be introduced daphnids and the expression of GFP could be detected in living daphnids. This is the first report of gene introduction to daphnids and, together with the emerging genome sequences, will be useful for the expanding our use of daphnid in ecotoxicology.

In vitro male germ cell cultures of zebrafish

Transgenic modification of sperm before fertilization has the advantages of a much shorter timeline for the production of transgenic animals. A culture system using primary cultures of zebrafish male germ cells, in which the differentiation of spermatogonia to functional sperm can occur in vitro, allows us to introduce foreign DNA into the cultured sperm and to produce transgenics from the sperm. This chapter describes methods for the co-culture of male germ cells and a Sertoli cell feeder layer and the introduction of foreign DNA with retroviruses. This male germ cell culture system should prove useful not only in producing genetically modified sperm, but also in analyzing the regulatory function of Sertoli cells for spermatogenesis in vertebrates.

Crop improvement through modification of the plant's own genome.

Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes. Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA. Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA. Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene. By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA. Further refinements are accomplished by employing Omega-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively. The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. The modified plants represent the first example of genetically engineered plants that only contain native DNA.

New non-viral method for gene transfer into primary cells

The availability of genetically altered cells is an essential prerequisite for many scientific and therapeutic applications including functional genomics, drug development, and gene therapy. Unfortunately, the efficient gene transfer into primary cells is still problematic. In contrast to transfections of most cell lines, which can be successfully performed using a variety of methods, the introduction of foreign DNA into primary cells requires a careful selection of gene transfer techniques. Whereas viral strategies are time consuming and involve safety risks, non-viral methods proved to be inefficient for most primary cell types. The Nucleofector technology is a novel gene transfer technique designed for primary cells and hard-to-transfect cell lines. This non-viral gene transfer method is based on a cell type specific combination of electrical parameters and solutions. In this report, we show efficient transfer of DNA expression vectors and siRNA oligonucleotides into a variety of primary cell types from different species utilizing the Nucleofector technology, including human B-CLL cells, human CD34+ cells, human lymphocytes, rat cardiomyocytes, human, porcine, and bovine chondrocytes, and rat neurons.

High-efficiency nonviral transfection of primary chondrocytes.

The introduction of foreign DNA into mammalian cells is an essential investigative tool in molecular biology. Nonviral approaches to transfection offer the advantage of relatively simple vector design, production, and purification and, for tissue engineering applications, avoid many of the potential risks associated with virus-mediated transfection methods. Unfortunately, primary cells, and in particular chondrocytes, are notoriously refractory to conventional transfection approaches, and optimized transfection efficiencies in these cells are extremely low (1-1.5%). In this chapter, we present three protocols that have proved useful in transfecting primary chondrocytes at high efficiency (~70%). The first uses radiofrequency electroporation, a transfection method that frequently works extremely well in cell types that are difficult to transfect. It should be noted that electroporation is not limited to DNA but that essentially any molecule can be introduced into the cell using this approach. In addition to the primary protocol, we present two additional reliable, albeit less efficient backup protocols, the first using exponential decay electroporation and the second FuGENE 6 transfection.

Optimization of Transfection

When embarking upon any transfection procedure, a critical first step is to optimize conditions. Every mammalian cell type has a characteristic set of requirements for optimal introduction of foreign DNA; there is a tremendous degree of variability in the transfection conditions that work, even among cell types that are very similar to one another. Often, an experimenter must screen a wide variety of cell types for a desired regulatory trait, such as an appropriate response to a particular effector molecule. It is thus helpful to have a straightforward, systematic approach to optimizing transfection efficiency. This unit provides guidelines to optimizing transfection experiments.

Foreign DNA: integration and expression in transgenic plants.

Gene transfer to plants is now a routine procedure, and the number of species amenable to transformation is increasing rapidly. The introduction of foreign DNA into plants allows the creation of engineered plant lines with improved production traits, resistance to pests, pathogens or herbicides, or the ability to synthesize speciality recombinant proteins such as human hormones, enzymes and antibodies. Many of these plants are now reaching commercial status (1). Although the applications of plant genetic engineering are diverse, there are only two basic strategies for stable plant transformation. The first depends on the natural ability of soil bacteria, such as Agrobacterium tumefaciens, to transfer a small segment of DNA from a resident plasmid into the plant cell (2). The second is ‘direct DNA transfer’ and encompasses a range of techniques where DNA is either physically forced into the plant cell, or the cell is persuaded to take up DNA from the surrounding medium. Examples of these techniques include particle bombardment (3), electroporation (4), the transformation of protoplasts mediated by polyethylene glycol (PEG) (5), and transformation using silicon carbide whiskers (6).KeywordsTransgenic PlantParticle BombardmentTrans GeneIllegitimate RecombinationTransgenic LocusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Transgenic technology and applications in swine

The introduction of foreign DNA into the genome of livestock and its stable integration into the germ line has been a major technical advance in agriculture. Production of transgenic livestock provides a method to rapidly introduce “new” genes into cattle, swine, sheep and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Several recent developments will profoundly impact the use of transgenic technology in livestock production. These developments are: 1) the ability to isolate and maintain in vitro embryonic stem (ES) cells from preimplantation embryos, embryonic germ (EG) and somatic cells from fetuses; and somatic cells from adults, and 2) the ability to use these embryonic and somatic cells as nuclei donors in nuclear transfer or “cloning” strategies. Cell based (ES, EG, and somatic cells) strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. There are many potential applications of transgenic methodology to develop new and improved strains of livestock. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition and increased disease resistance. Development of transgenic farm animals will allow more flexibility in direct genetic manipulation of livestock.