DNA Delivery Methods Research Articles

Efficient delivery of DNA into bovine preimplantation embryos by multiwall carbon nanotubes.

The pellucid zone (PZ) is a protective embryonic cells barrier against chemical, physical or biological substances. This put, usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells. Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection, however its ability to cross the PZ and mediated gene transfer is unknown. Our data confirm that multiwall carbon nanotubes (MWNTs) can cross the PZ and delivery of pDNA into in vitro-fertilized bovine embryos. The degeneration rate and the expression of genes associated to cell viability were not affected in embryos exposed to MWNTs. Those embryos, however, had lower cell number and higher apoptotic cell index, but this did not impair the embryonic development. This study shows the potential utility of the MWNT for the development of new method for delivery of DNA into bovine embryos.

An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

Summary CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T0 transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.

Refinement of the Diatom Episome Maintenance Sequence and Improvement of Conjugation-Based DNA Delivery Methods.

Conjugation of episomal plasmids from bacteria to diatoms advances diatom genetic manipulation by simplifying transgene delivery and providing a stable and consistent gene expression platform. To reach its full potential, this nascent technology requires new optimized expression vectors and a deeper understanding of episome maintenance. Here, we present the development of an additional diatom vector (pPtPBR1), based on the parent plasmid pBR322, to add a plasmid maintained at medium copy number in Escherichia coli to the diatom genetic toolkit. Using this new vector, we evaluated the contribution of individual yeast DNA elements comprising the 1.4-kb tripartite CEN6-ARSH4-HIS3 sequence that enables episome maintenance in Phaeodactylum tricornutum. While various combinations of these individual elements enable efficient conjugation and high exconjugant yield in P. tricornutum, individual elements alone do not. Conjugation of episomes containing CEN6-ARSH4 and a small sequence from the low GC content 3′ end of HIS3 produced the highest number of diatom exconjugant colonies, resulting in a smaller and more efficient vector design. Our findings suggest that the CEN6 and ARSH4 sequences function differently in yeast and diatoms, and that low GC content regions of greater than ~500 bp are a potential indicator of a functional diatom episome maintenance sequence. Additionally, we have developed improvements to the conjugation protocol including a high-throughput option utilizing 12-well plates and plating methods that improve exconjugant yield and reduce time and materials required for the conjugation protocol. The data presented offer additional information regarding the mechanism by which the yeast-derived sequence enables diatom episome maintenance and demonstrate options for flexible vector design.

Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Mammalian Cell Engineering

The CRISPR/Cas9 genome‐editing platform is a versatile and powerful technology to efficiently create genetically engineered living cells and organisms. This system requires a complex of Cas9 endonuclease protein with a gene‐targeting guide RNA (gRNA) to introduce double‐strand DNA breaks (DSBs) at specific locations in the genome. DSBs are then repaired by the error‐prone Non‐Homologous End Joining (NHEJ) pathway, resulting in insertions and/or deletions (indels) which disrupt the targeted locus.The success of CRISPR genome editing experiments is limited by the intracellular delivery and expression of Cas9 protein and gRNA. Many methods for achieving CRISPR mediated cleavage have been identified and the choice of DNA, RNA or ribonucleoprotein (RNP) format is dictated by experimental goal and cell type. Transfection of each type of molecule requires specific considerations for efficient functional delivery. We performed transfections using different combinations of molecules including: plasmid DNA, messenger RNA, Cas9 protein and gRNA to maximize targeting of the Cyclophilin B (PPIB) gene in HEK 293T/17, U2OS, and other mammalian cell types. Our results extend the utility of the CRISPR/Cas9 system by identifying optimal transfection conditions for intracellular delivery of Cas9 and gRNA in different formats.

Gene Electrotransfer: A Mechanistic Perspective.

Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.

High-frequency, precise modification of the tomato genome.

BackgroundThe use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.ResultsHere, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences.ConclusionsHigh-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0796-9) contains supplementary material, which is available to authorized users.

DNA vaccines, electroporation and their applications in cancer treatment

Numerous animal studies and recent clinical studies have shown that electroporation-delivered DNA vaccines can elicit robust Ag-specific CTL responses and reduce disease severity. However, cancer antigens are generally poorly immunogenic, requiring special conditions for immune response induction. To date, many different approaches have been used to elicit Ag-specific CTL and anti-neoplastic responses to DNA vaccines against cancer. In vivo electroporation is one example, whereas others include DNA manipulation, xenogeneic antigen use, immune stimulatory molecule and immune response regulator application, DNA prime-boost immunization strategy use and different DNA delivery methods. These strategies likely increase the immunogenicity of cancer DNA vaccines, thereby contributing to cancer eradication. However, cancer cells are heterogeneous and might become CTL-resistant. Thus, understanding the CTL resistance mechanism(s) employed by cancer cells is critical to develop counter-measures for this immune escape. In this review, the use of electroporation as a DNA delivery method, the strategies used to enhance the immune responses, the cancer antigens that have been tested, and the escape mechanism(s) used by tumor cells are discussed, with a focus on the progress of clinical trials using cancer DNA vaccines.

Optimization of a plasma facilitated DNA delivery method

Plasma-based methods have recently emerged as a technique for augmenting plasmid DNA delivery to skin. This delivery modality relies on the deposition of ionized gas molecules on to targeted cells or tissue to establish an electric field. It is hypothesized that this electric field results in the dielectric breakdown of cell membranes, making cells permeable to exogenous molecules. This in vivo investigation sought to optimize the intradermal delivery of a luciferase expressing plasmid DNA by modulating the total exposure to the plasma source and the plasmid DNA dose. Varying the plasma exposure time from 2, 5, 10, and 20min allowed the conditions resulting in the highest expression of luciferase to be found. These conditions correlated to the 10minute exposure time for a plasma derived from either +8kV or −8kV, when the generator was operated 3cm from the epidermal tissue surface with a helium flow rate of 15L/min. Exposing the injected flank skin for 10min resulted in a rise of 37.3-fold for a plasma created with +8kV and 27.1-fold for a plasma created with −8kV. When using this treatment time with 50, 100, or 200μg of a luciferase expressing plasmid, it was found that 100μg resulted in the highest peak luminescence.

HIV DNA Vaccine: Stepwise Improvements Make a Difference.

Inefficient DNA delivery methods and low expression of plasmid DNA have been major obstacles for the use of plasmid DNA as vaccine for HIV/AIDS. This review describes successful efforts to improve DNA vaccine methodology over the past ~30 years. DNA vaccination, either alone or in combination with other methods, has the potential to be a rapid, safe, and effective vaccine platform against AIDS. Recent clinical trials suggest the feasibility of its translation to the clinic.

Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle.

The recent single-cell manipulation technology using atomic force microscopy (AFM) not only allows high-resolution visualization and probing of biomolecules and cells but also provides spatial and temporal access to the interior of living cells via the nanoneedle technology. Here we review the development and application of single-cell manipulations and the DNA delivery technology using a nanoneedle. We briefly describe various DNA delivery methods and discuss their advantages and disadvantages. Fabrication of the nanoneedle, visualization of nanoneedle insertion into living cells, DNA modification on the nanoneedle surface, and the invasiveness of nanoneedle insertion into living cells are described. Different methods of DNA delivery into a living cell, such as lipofection, microinjection, and nanoneedles, are then compared. Finally, single-cell diagnostics using the nanoneedle and the perspectives of the nanoneedle technology are outlined. The nanoneedle-based DNA delivery technology provides new opportunities for efficient and specific introduction of DNA and other biomolecules into precious living cells with a high spatial resolution within a desired time frame. This technology has the potential to be applied for many basic cellular studies and for clinical studies such as single-cell diagnostics.

Genetic Transformation of Rice: Problems, Progress and Prospects

Plant genetic engineering has become one of the most important molecular tools in the modern molecular breeding of crops. Now a days, production of transgenic plants is a routine process in some crop species. Transgenes are delivered into plants to confer novel traits such as improving nutritional qualities, resistance to pests. It is possible to insert genes from plants at evolutionary distant from the host plant, as well as from fungi, viruses, bacteria and even animals. Genetic transformation requires penetration of the transgene through the plant cell wall, facilitated by biological or physical methods. Over the last few decades, a significant progress has been made in the development of new and efficient transformation methods. Despite a variety of available DNA delivery methods, Agrobacterium and Biolistic mediated transformation remain two predominantly applied approaches. The objective of this article is to review the currently used methods for genetic plant transformation, their biological requirements and critical parameters

Comparison of intradermal and intramuscular delivery followed by in vivo electroporation of SIV Env DNA in macaques

A panel of SIVmac251 transmitted Env sequences were tested for expression, function and immunogenicity in mice and macaques. The immunogenicity of a DNA vaccine cocktail expressing SIVmac239 and three transmitted SIVmac251 Env sequences was evaluated upon intradermal or intramuscular injection followed by in vivo electroporation in macaques using sequential vaccination of gp160, gp120 and gp140 expressing DNAs. Both intradermal and intramuscular vaccination regimens using the gp160 expression plasmids induced robust humoral immune responses, which further improved using the gp120 expressing DNAs. The responses showed durability of binding and neutralizing antibody titers and high avidity for > 1 y. The intradermal DNA delivery regimen induced higher cross-reactive responses able to neutralize the heterologous tier 1B-like SIVsmE660_CG7V. Analysis of cellular immune responses showed induction of Env-specific memory responses and cytotoxic granzyme B+ T cells in both vaccine groups, although the magnitude of the responses were ~10x higher in the intramuscular/electroporation group. The cellular responses induced by both regimens were long lasting and could be detected ~1 y after the last vaccination. These data show that both DNA delivery methods are able to induce robust and durable immune responses in macaques.

Development of Poly(β-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA

Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy.

A Novel Method for Genetic Transformation of Yeast Cells Using Oligoelectrolyte Polymeric Nanoscale Carriers

The genetic transformation of target cells is a key tool in modern biological research, as well as in many gene therapy and biotechnology applications. Here we describe a new method for delivery of DNA into several industrially important species of yeast, including Saccharomyces cerevisiae. Our method is based on the use of a novel nanoscale oligoelectrolyte polymer possessing a comb-like structure as a carrier molecule. Direct comparisons to standard transformation methods clearly show that our approach: (i) yields two times more transformants of Hansenula polymorpha NCYC 495 compared to electroporation approaches and 15 times more transformants compared to lithium acetate protocols, as well as (ii) 5 times more Pichia pastoris GS115 transformants compared to electroporation and 79 times more transformants compared to lithium acetate. Taken together, these results clearly indicate genetic transformation of yeasts using oligoelectrolyte polymer carriers is a highly effective means of gene delivery.

PLANT TISSUE CULTURE IN BIOTECHNOLOGY: RECENT ADVANCES IN TRANSFORMATION THROUGH SOMATIC EMBRYOGENESIS

118 The development of cell, tissue and organ culture methods was rapidly accelerated in the second half of the previous century after establishing robust cell culture techniques and media formulation for in vitro growth of plant material [1–8]. A stunning number of articles have been published on in vitro induction and maintenance of non-differentiated cells and the regeneration the plants from them whether through organogenesis or somatic embryogenesis. Fine protocols were established for the culture of enzymatically isolated single cells and protoplasts [9], which were able to regenerate into plants [10–12]. In vitro culture methods became an essential part of many micropropagation protocols. Culture of plant cells and organs in bioreactors were used for the production of different secondary metabolites and pharmaceuticals. In vitro techniques were used for production of mutants, haploids, virus-free material, and also for maintenance and preservation of rare genotypes and specific cell cultures [13–15]. The first positive results on Agro bac te rium-mediated transformation in plants were reported in 1983 [16–18]. A simple method for transferring genes into plants through the inoculation of leaf discs with Agrobacterium tumefaciens followed by in vitro culture and regeneration of whole plants was reported by Horsch et al. [19]. Different methods for DNA delivery into plant cell, including electroporation [20], PEG treatment [21, 22], microinjection [23], sonication [24], biolistics or particle bombardment [25], silicon carbide Whiskers treatment [26, 27], were used for specific transformation purposes and different types of cells and genotypes. However, Agrobac terium-mediated transformation became a preferred method. Agrobacterium tumefaciens is a natural vector system for transgenes delivery into a wide range of plants species, providing an efficient and «clean» insertion of DNA into the plant genome and deserved to be called the «tzar of genetic engineering» [28]. In the past 30 years the discovery and application of new transformation technologies essentially sped up the improvement of major cultivated crops. The first, really commercially grown plants hit the market in the mid — 1990s. Practically all transformation systems were based on in vitro culture methods. It was impossible to transform whole plant organism at once. All techniques were based on transforming single cells of callus, leaves, pollen, roots or other organs and than regeneration of plants through somatic embryogenesis or organogenesis. Routine and highly efficient transformation methods for many important crop and particular genotypes were implemented in many biotechnology companies. UDK 575327/275.854

DNA Tattooing - Pain Or Gain?

Tattooing is one of a number of DNA delivery methods which results in an efficient expression of an introduced gene in the epidermal and dermal layers of the skin. The tattoo procedure causes minor mechanical injuries followed by hemorrhage, necrosis, inflammation and regeneration of the skin and thus non-specifically stimulates the immune system. DNA vaccines delivered by tattooing have been shown to induce higher specific humoral and cellular immune responses than intramuscularly injected DNA.

A simple plant gene delivery system using mesoporous silica nanoparticles as carriers.

A facile DNA delivery method would greatly facilitate studies of plant functional genomics. However, plant cell walls limit the utilization of nanoparticles on plant research. Here, we employed functionalized mesoporous silica nanoparticles (MSNs) to develop a MSN-mediated plant transient gene expression system. In this system, MSNs served as carriers to deliver foreign DNA into intact Arabidopsis thaliana roots without the aid of mechanical force. Gene expression was detected in the epidermal layer and in the more inner cortical and endodermal root tissues by both fluorescence and antibody labeling. This is a novel alternative to the conventional gene-gun or ultrasonic methods. In addition, the parameters that affect the MSN uptake and the mechanism and subcellular distribution of particles were also analyzed. The present study may provide valuable information on the manipulation of functional nanoparticles in plants and have significant impact on plant biotechnology.

GENETIC TRANSFORMATION OF COMMON BEANS (Phaseolus vulgaris L.) CALLI BY BIOLISTIC GUN AND VIA Agrobacterium tumefaciens

Common bean is one of the most recalcitrant species for in vitro manipulation and transformation. In this study efficient genetic transformation methods were developed and performed in commercialized bean varieties calli by utilized microparticle bombardment (direct biolistic gun) and indirect A. tumefaciens-based technique. Hypocotyl segments cultured on Gamborgs medium supplemented with 1 mg l-l kinetin and 2 mg l-l 2,4-dichlorophenoxy acetic acid proved the best for callus induction and maintenance. Co-transformed calli were double selected on selective medium using 150 mg l-l kanamycin and 0.8 mol l-l mannitol. Transient genes expressions were obtained in calli cells and the results showed that the cells shot by biolistic DNA delivery method can be transformed to get kanamycin resistant and mannitol tolerant. The presence and integration of the neomycin phosphotransferase II (npt II) and osmosis protector mannitol-1-phosphate dehydrogenase (mtlD) genes into P. vulgaris L. genome were confirmed and verified by double selection tests. Transgenic calli were selected after co-cultivated with Agrobacterium method on selective medium containing 5 mg l-l phosphinotricin (PPT). Transient genes expressions were obtained in the calli and the results showed that the common beans calli co-cultivated with Agrobacterium can be transformed to get phosphinotricin resistant and β-glucuronidase reporter positive tissues. The presence and integration of the phosphinotricin acetyl transferase and β-glucuronidase genes into P. vulgaris L. genome were confirmed and verified by marker selection and histochemical assay tests. These results suggest an efficient biolistic gun, and Agrobacterium-mediated transformation methods for stable integration of economically important genes into common beans calli, and that these transformations systems could be useful for future studies on transferring economically important genes into common bean plants.

HER2/neu DNA vaccination by intradermal gene delivery in a mouse tumor model

DNA vaccines are potential tools for the induction of immune responses against both infectious disease and cancer. The dermal application of DNA vaccines is of particular interest since the epidermal and dermal layers of the skin are characterized by an abundance of antigen-presenting cells (APCs). The aim of our study was to compare tumor protection as obtained by two different methods of intradermal DNA delivery (gene gun and jet injector) in a well-established HER2/neu mouse tumor model. BALB/c mice were immunized twice with a HER2/neu-coding plasmid by gene gun or jet injector. Mice were then subcutaneously challenged with HER2/neu+ syngeneic D2F2/E2 tumor cells. Protection against subsequent challenges with tumor cells as well as humoral and T-cell immune responses induced by the vaccine were monitored. Gene gun immunization was far superior to jet injector both in terms of tumor protection and induction of HER2/neu-specific immune responses. After gene gun immunization, 60% of the mice remained tumor-free until day 140 as compared with 25% after jet injector immunization. Furthermore, gene gun vaccination was able to induce both a strong TH1-polarized T-cell response with detectable cytotoxic T-lymphocyte (CTL) activity and a humoral immune response against HER2/neu, whereas the jet injector was not. Although the disadvantages that were associated with the use of the jet injector in our model may be overcome with methodological modifications and/or in larger animals, which exhibit a thicker skin and/or subcutaneous muscle tissue, we conclude that gene gun delivery constitutes the method of choice for intradermal DNA delivery in preclinical mouse models and possibly also for the clinical development of DNA-based vaccines.

Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts

The red yeasts of the Pucciniomycotina have rarely been transformed with DNA molecules. Transformation methods were recently developed for a species of Sporobolomyces, based on selection using uracil auxotrophs and plasmids carrying the wild-type copies of the URA3 and URA5 genes. However, these plasmids were ineffective in the transformation of closely related species. Using the genome-sequenced strain of Rhodotorula graminis as a starting point, the URA3 and URA5 genes were cloned and tested for the transformation ability into different Pucciniomycotina species by biolistic and Agrobacterium-mediated transformations. Transformation success depended on the red yeast species and the origin of the URA3 or URA5 genes, which may be related to the high G + C DNA content found in several species. A new vector was generated to confer resistance to nourseothricin, using a native promoter from R. graminis and the naturally high G + C nourseothricin acetyltransferease gene. This provides a second selectable marker in these species. Targeted gene disruption was tested in Sporobolomyces sp. IAM 13481 using different lengths of homologous DNA with biolistic and Agrobacterium transformation methods. Both DNA delivery methods were effective for targeted replacement of a gene required for carotenoid pigment biosynthesis. The constructs also triggered transgene silencing. These developments open the way to identify and manipulate gene functions in a large group of basidiomycete fungi.