Functional Analysis of Seed Dormancy Genes by Biolistic Transient Gene Expression in Immature Embryos of Wheat.

Seed dormancy genes typically suppress germination and cell division. Therefore, overexpressing these genes can negatively affect tissue culture, interfering with the generation of transgenic plants and thus hampering the analysis of gene function. Transient expression in target cells is a useful approach for studying the function of seed dormancy genes. Here, we describe a protocol for transiently expressing genes related to seed dormancy in the scutellum of immature wheat (Triticum aestivum) embryos to analyze their effects on germination.

Mitochondrial Transformation in Baker's Yeast to Study Translation and Respiratory Complex Assembly.

Baker´s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

Biolistics-mediated transformation of hornworts and its application to study pyrenoid protein localization.

Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48-72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species.

Biolistic Transformation of Cryptococcus neoformans.

Biolistic transformation of Cryptococcus neoformans is used as a molecular tool to genetically alter or delete targeted genes. The DNA is introduced into the yeast on DNA-coated gold beads by a helium shock wave produced using a biolistic particle system. The procedure often involves insertion of a dominant selectable marker into the desired site by homologous recombination. To increase the likelihood of homologous recombination, large fragments of overlapping DNA are used. The two most used dominant selectable markers are nourseothricin and Geneticin. With the need to generate multiple gene deletions in the same strain, there are recyclable marker systems, such as the bacteriophage P1 Cre-loxP system or CRISPR that provide additional useful molecular tools. While newer strategies exist to generate deletions and introduce markers and other gene modifications, biolistic transformation has remained a viable tool to facilitate the construction of genetically modified yeast strains. This chapter provides a working protocol on how to delete and restore a gene in C. neoformans.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

Combined biolistic and cell penetrating peptide delivery for the development of scalable intradermal DNA vaccines.

Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET­gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery.

Biolistic transformation of the yeast Saccharomyces cerevisiae mitochondrial DNA.

The insertion of genes into mitochondria by biolistic transformation is currently only possible in the yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii. The fact that S. cerevisiae mitochondria can exist with partial (ρ- mutants) or complete deletions (ρ0 mutants) of mitochondrial DNA (mtDNA), without requiring a specific origin of replication, enables the propagation of exogenous sequences. Additionally, mtDNA in this organism undergoes efficient homologous recombination, making it well-suited for genetic manipulation. In this review, we present a summarized historical overview of the development of biolistic transformation and discuss iconic applications of the technique. We also provide a detailed example on how to obtain transformants with recombined foreign DNA in their mitochondrial genome.

Biolistic delivery of liposomes protected in metal-organic frameworks.

Needle-and-syringe-based delivery has been the commercial standard for vaccine administration to date. With worsening medical personnel availability, increasing biohazard waste production, and the possibility of cross-contamination, we explore the possibility of biolistic delivery as an alternate skin-based delivery route. Delicate formulations like liposomes are inherently unsuitable for this delivery model as they are fragile biomaterials incapable of withstanding shear stress and are exceedingly difficult to formulate as a lyophilized powder for room temperature storage. Here we have developed a approach to deliver liposomes into the skin biolistically-by encapsulating them in a nano-sized shell made of Zeolitic Imidazolate Framework-8 (ZIF-8). When encapsulated within a crystalline and rigid coating, the liposomes are not only protected from thermal stress, but also shear stress. This protection from stressors is crucial, especially for formulations with cargo encapsulated inside the lumen of the liposomes. Moreover, the coating provides the liposomes with a solid exterior that allows the particles to penetrate the skin effectively. In this work, we explored the mechanical protection ZIF-8 provides to liposomes as a preliminary investigation for using biolistic delivery as an alternative to syringe-and-needle-based delivery of vaccines. We demonstrated that liposomes with a variety of surface charges could be coated with ZIF-8 using the right conditions, and this coating can be just as easily removed-without causing any damage to the protected material. The protective coating prevented the liposomes from leaking cargo and helped in their effective penetration when delivered into the agarose tissue model and porcine skin tissue.

Biolistic Transformation of Chlamydomonas reinhardtii and Saccharomyces cerevisiae Mitochondria.

Chlamydomonas reinhardtii and Saccharomyces cerevisiae are currently the two micro-organisms in which genetic transformation of mitochondria is routinely performed. The generation of a large variety of defined alterations as well as the insertion of ectopic genes in the mitochondrial genome (mtDNA) are possible, especially in yeast. Biolistic transformation of mitochondria is achieved through the bombardment of microprojectiles coated with DNA, which can be incorporated into mtDNA thanks to the highly efficient homologous recombination machinery present in S. cerevisiae and C. reinhardtii organelles. Despite a low frequency of transformation, the isolation of transformants in yeast is relatively quick and easy, since several natural or artificial selectable markers are available, while the selection in C. reinhardtii remains long and awaits new markers. Here, we describe the materials and techniques used to perform biolistic transformation, in order to mutagenize endogenous mitochondrial genes or insert novel markers into mtDNA. Although alternative strategies to edit mtDNA are being set up, so far, insertion of ectopic genes relies on the biolistic transformation techniques.

Improvement of Gene Delivery and Mutation Efficiency in the CRISPR-Cas9 Wheat (Triticum aestivum L.) Genomics System via Biolistics.

Discovery of the CRISPR-Cas9 gene editing system revolutionized the field of plant genomics. Despite advantages in the ease of designing gRNA and the low cost of the CRISPR-Cas9 system, there are still hurdles to overcome in low mutation efficiencies, specifically in hexaploid wheat. In conjunction with gene delivery and transformation frequency, the mutation efficiency bottleneck has the potential to slow down advancements in genomic editing of wheat. In this study, nine bombardment parameter combinations using three gold particle sizes and three rupture disk pressures were tested to establish optimal stable transformation frequencies in wheat. Utilizing the best transformation protocol and a knockout cassette of the phytoene desaturase gene, we subjected transformed embryos to four temperature treatments and compared mutation efficiencies. The use of 0.6 µm gold particles for bombardment increased transformation frequencies across all delivery pressures. A heat treatment of 34 °C for 24 h resulted in the highest mutation efficiency with no or minimal reduction in transformation frequency. The 34 °C treatment produced two M0 mutant events with albino phenotypes, requiring biallelic mutations in all three genomes of hexaploid wheat. Utilizing optimal transformation and heat treatment parameters greatly increases mutation efficiency and can help advance research efforts in wheat genomics.

Optimized Proteolistic Protocol for the Delivery of the Cas9 Protein in Phaeodactylum tricornutum.

The CRISPR/Cas9 system coupled with proteolistics is a DNA-free nuclear transformation method based on the introduction of ribonucleoprotein (RNP) complexes into cells. The method has been set up for diatoms as an alternative to genetic transformation via biolistics and has the advantages of reducing off-target mutations, limiting the working time of the Cas9 endonuclease, and overcoming the occurrence of random insertions of the transgene in the genome. We present a point-by-point description of the protocol with modifications that make it more cost-effective, by reducing the amount of the enzyme while maintaining a comparable efficiency to the original protocol, and with an increased concentration of the selective drug which allows to reduce false positives.

Production of Recombinant Glycoproteins in Nicotiana tabacum BY-2 Suspension Cells.

This protocol describes a robust method to obtain transgenic Nicotiana tabacum BY-2 cells that produce glycoproteins of interest via Agrobacterium tumefaciens transformation. Compared to biolistics-based transformation, this procedure requires only standard laboratory equipment.

Light triggered nanoscale biolistics for efficient intracellular delivery of functional macromolecules in mammalian cells.

Biolistic intracellular delivery of functional macromolecules makes use of dense microparticles which are ballistically fired onto cells with a pressurized gun. While it has been used to transfect plant cells, its application to mammalian cells has met with limited success mainly due to high toxicity. Here we present a more refined nanotechnological approach to biolistic delivery with light-triggered self-assembled nanobombs (NBs) that consist of a photothermal core particle surrounded by smaller nanoprojectiles. Upon irradiation with pulsed laser light, fast heating of the core particle results in vapor bubble formation, which propels the nanoprojectiles through the cell membrane of nearby cells. We show successful transfection of both adherent and non-adherent cells with mRNA and pDNA, outperforming electroporation as the most used physical transfection technology by a factor of 5.5-7.6 in transfection yield. With a throughput of 104-105 cells per second, biolistic delivery with NBs offers scalable and highly efficient transfections of mammalian cells.

Using a Hand-Held Gene Gun for Genetic Transformation of Tetrahymena thermophila.

Biolistic bombardment is widely used as a means of delivering vector-coated microparticles into microorganisms, cultured cells, and tissues. The first particle delivery system contained a helium propulsion unit (the gun) mounted in a vacuum-controlled chamber. In contrast, the hand-held gene gun does not operate within a chamber. It is completely hand-held, easy, and efficient to use, and it requires minimal space on the laboratory bench top. This chapter describes protocols for using a hand-held gene gun to deliver transformation vectors for overexpression of genes or gene replacement into the macronucleus of Tetrahymena thermophila. The protocols provide helpful information for preparing Tetrahymena for biolistic bombardment, preparation of vector-coated microcarriers, and basic gene gun operating procedures.

Transplastomic Tomato Plants Expressing Insect-Specific Double-Stranded RNAs: A Protocol Based on Biolistic Transformation.

Expressing insecticidal double-stranded RNA (dsRNA) molecules in plant plastids is a novel approach for in planta production of dsRNA that has enormous potential for developing improved plant-mediated RNA interference (RNAi) strategies for insect pest control. In this chapter, we describe the design of a transformation vector containing an expression cassette which can be used to stably transform plastids of tomato plants for production and accumulation of dsRNA . Such dsRNA can trigger the mechanisms of RNAi in pest insects and selectively suppress the expression of target genes, resulting in lethality. We also describe a protocol for detection of full-length dsRNA molecules in plastids using an RT-PCR-based method.

Biolistic Inoculation of Fruit Trees with Full-Length Infectious cDNA Clones of RNA Viruses.

Long life cycle and lack of efficient and robust virus inoculation technique are the major technical challenges for studying virus infection in perennial woody plants such as fruit trees. Biolistic technology also called particle bombardment is a physical approach that can directly introduce virions or viral full-length cDNA infectious clones into target cells and tissues by high velocity microcarrier particles. The flexibility and high efficiency of the biolistic inoculation method facilitate research on fruit tree virology and the screening and identification of fruit tree germplasms resistant to viruses. Here, we describe a detailed protocol for the biolistic inoculation of peach with of a cDNA infectious clone of Plum pox virus (PPV) using the Helios gene gun, a biolistic particle delivery system.

A Review of Brain-Targeted Nonviral Gene-Based Therapies for the Treatment of Alzheimer's Disease.

Diseases of the central nervous system (CNS) are difficult to treat owing to the complexity of the brain and the presence of a natural blood-brain-barrier (BBB). Alzheimer's disease (AD) is one of the major progressive and currently incurable neurodegenerative disorders of the CNS, which accounts for 60-80% of cases of dementia. The pathophysiology of AD involves the accumulation of amyloid beta (Aß) plaques and neurofibrillary tangles (NFTs) in the brain. Additionally, synaptic loss and imbalance of neuronal signaling molecules are characterized as important markers of AD. Existing treatments of AD help in the management of its symptoms and aim toward the maintenance of cognitive functions, behavior, and attenuation of gradual memory loss. Over the past decade, nonviral gene therapy has attracted increasing interest due to its various advantages over its viral counterparts. Moreover, advancements in nonviral gene technology have led to their increasing contributions in clinical trials. However, brain-targeted nonviral gene delivery vectors come across various extracellular and intracellular barriers, limiting their ability to transfer the therapeutic gene into the target cells. Chief barriers to nonviral gene therapy have been discussed briefly in this review. We have also highlighted the rapid advancement of several nonviral gene therapies for AD, which are broadly categorized into physical and chemical methods. These methods aim to modulate Aß, beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), apolipoprotein E, or neurotrophic factors' expression in the CNS. Overall, this review discusses challenges and recent advancements of nonviral gene therapy for AD.

Optimizing Agrobacterium-Mediated Transformation and CRISPR-Cas9 Gene Editing in the tropical japonica Rice Variety Presidio.

Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties.

Methods to improve the immunogenicity of plasmid DNA vaccines.

DNA vaccines have emerged as innovative approaches that have great potential to overcome the limitations of current conventional vaccines. Plasmid DNA vaccines are often safer than other vaccines because they carry only antigen genetic information, are more stable and easier to produce, and can stimulate both humoral and cellular immune responses. Although the results of ongoing clinical trials are very promising, some limitations compromise the immunogenicity of these vaccines. Thus, this review describes different strategies that can be explored to improve the immunogenicity of plasmid DNA vaccines, including the optimization of the plasmid vector backbone, the use of different methods for vaccine delivery, the use of alternative administration routes and the inclusion of adjuvants. In combination, these improvements could lead to the successful clinical use of plasmid DNA vaccines.

Rapid Ultrastructural Changes in the PSD and Surrounding Membrane after Induction of Structural LTP in Single Dendritic Spines.

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.SIGNIFICANCE STATEMENT To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon-spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP.