Biolistic Research Articles

Expression of Heat-labile Enterotoxin of Escherichia coli in Biolistic Transformed Hairy Roots of Daucus carota L.

Expression and secretion of recombination proteins in transgenic hairy roots have opened door to simple, feasible and economic option compared to animal and bacterial systems. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heatlabile enterotoxin (LTB) in Daucus carota L hairy root where transformation is achieved by biolistic gene gun method. Agrobacterium rhizogenes strain (R-1000) cells containing synthetic LTB gene in pMYO51T plant expression vector under the CaMV 35S promoter introduced to Daucus carota L roots by biolistic gene gun method. PCR amplification confirmed the integration of synthetic LTB gene in carrot hairy roots. Western blot analysis confirmed production of LTB pentamer in similar banding pattern to the native bacterial derived LTB. 0.50-0.70% of LTB protein estimated in carrot hairy roots. GM1-ganglioside assay indicated formation of biological active pentamers. LTB protein with biochemical properties identical to native LTB protein in the hairy roots of edible carrot roots opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

Assessment of biolistic and Agrobacterium-mediated genetic transformation methods in Cenchrus ciliaris

Cenchrus ciliaris, commonly known as buffelgrass, is an apomictic perennial range grass usually grown in arid/semi-arid regions. Because of the difficulties faced in conventional breeding of this polymorphic polyploid grass, the development of an efficient protocol for genetic transformation is warranted. Such a protocol would enable functional genomic studies needed to elucidate the mechanism of apomixis in this species. The embryogenic calli for genetic transformation experiments were obtained by in vitro culture of the immature inflorescences of buffelgrass cv. IGFRI-3108. Four developmental stages of immature inflorescences were compared for callus induction, and the most mature stage produced most callus. Embryogenic calli were bombarded at three distances, 6 cm (L1), 9 cm (L2), or 12 cm (L3) with a marker gene uid A present in pCAMBIA1301, under the same vacuum (85 kPa) and at constant pressure (900 psi). The Agrobacterium-mediated genetic transformation was also performed using the same construct. Transient and stable expression as well as PCR amplification of the GUS gene was used for comparative analysis as well as for validation of the transformants. Transient GUS expression was present in a significantly higher percentage of bombarded calli (56.33%) than of Agrobacterium treated calli (11.17%), but the number of GUS positive cells per callus was similar. Among the three different bombardment distances, transient GUS expression was highest at L2, but stable GUS expression was highest at L1. Shoot development from the bombarded calli could be accomplished, which failed from the Agrobacterium-mediated transformed calli. Thus, the results indicate that C. ciliaris cv. IGFRI-3108 can be successfully transformed through Biolistic particle bombardment, while Agrobacterium-mediated transformation requires further optimization of transformation protocols.

An AMA1/MSP119 Adjuvanted Malaria Transplastomic Plant-Based Vaccine Induces Immune Responses in Test Animals

Malaria is a tropical human disease, caused by protozoan parasites, wherein a significant number of the world's population is at risk. Annually, more than 219 million new cases are reported. Although there are prevention treatments, there are no highly and widely effective licensed anti-malarial vaccines available for use. Opportunities for utilization of plant-based vaccines as novel platforms for developing safe, reliable, and affordable treatments offer promise for developing such a vaccine against malaria. In this study, a Malchloroplast candidate vaccine was designed, composed of segments of AMA1 and MSP1 proteins, two epitopes of Plasmodium falciparum, along with a GK1 peptide from Taenia solium as adjuvant, and this was expressed in tobacco chloroplasts. Transplastomic tobacco lines were generated using biolistic transformation, and these were confirmed to carry the synthetic gene construct. Expression of the synthetic GK1 peptide was confirmed using RT-PCR and Western blots. Furthermore, the GK1 peptide was detected by HPLC at levels of up to 6µgg-1 dry weight of tobacco leaf tissue. The plant-derived Malchloroplast candidate vaccine was subsequently tested in BALB/c female mice following subcutaneous administration, and was found to elicit specific humoral responses. Furthermore, components of this candidate vaccine were recognized by antibodies in Plasmodium falciparum malaria patients and were immunogenic in test mice. Thus, this study provided a 'proof of concept' for a promising plant-based candidate subunit vaccine against malaria.

Clinical Pathology, Immunopathology and Advanced Vaccine Technology in Bovine Theileriosis: A Review.

Theileriosis is a blood piroplasmic disease that adversely affects the livestock industry, especially in tropical and sub-tropical countries. It is caused by haemoprotozoan of the Theileria genus, transmitted by hard ticks and which possesses a complex life cycle. The clinical course of the disease ranges from benign to lethal, but subclinical infections can occur depending on the infecting Theileria species. The main clinical and clinicopathological manifestations of acute disease include fever, lymphadenopathy, anorexia and severe loss of condition, conjunctivitis, and pale mucous membranes that are associated with Theileria-induced immune-mediated haemolytic anaemia and/or non-regenerative anaemia. Additionally, jaundice, increases in hepatic enzymes, and variable leukocyte count changes are seen. Theileria annulata and Theileria parva induce an incomplete transformation of lymphoid and myeloid cell lineages, and these cells possess certain phenotypes of cancer cells. Pathogenic genotypes of Theileria orientalis have been recently associated with severe production losses in Southeast Asia and some parts of Europe. The infection and treatment method (ITM) is currently used in the control and prevention of T. parva infection, and recombinant vaccines are still under evaluation. The use of gene gun immunization against T. parva infection has been recently evaluated. This review, therefore, provides an overview of the clinicopathological and immunopathological profiles of Theileria-infected cattle and focus on DNA vaccines consisting of plasmid DNA with genes of interest, molecular adjuvants, and chitosan as the most promising next-generation vaccine against bovine theileriosis.

Creating a tobacco line with a weaker antifeedant property against colorado potato beetle

Background . Genetic modification of plants is one of the promising strategies to increase their resistance to insect pests. The development of metabolic or RNA interference systems for plant protection requires appropriate models of host-insect interactions. Nicotiana tabacum L. is a classical model plant used in molecular and metabolic engineering. We consider tobacco as a model for developing protective strategies against Colorado potato beetle ( Leptinotarsa decemlineata Say, CPB). Normally, tobacco is toxic for CPB due to high content of nicotine and related alkaloids in leaves. Modification of the tobacco genome could provide tobacco genotypes with altered metabolism suitable for CPB feeding. It is known that different mutations in Berberine Bridge-Like (BBL) genes cause different alterations in tobacco leaf alkaloid levels. In the current study, the Cas9/gRNA system targeting members of the BBL gene family of tobacco was used to create a line which can serve as a diet for CPB. Results . In order to obtain tobacco with modified alkaloid content, two gRNAs matching target sequences in six BBL genes were selected. Each gRNA was cloned into a gRNA/Cas9 generic vector. The created constructs were mixed and used for biolistic transformation of tobacco leaf explants together with the pBI121 plasmid harboring the kanamycin resistance gene nptII and the reporter E.coli betaglucuronidase (GUS) gene. Regenerants were selected on 100 mg/l of kanamycin and checked for transgene presence by histochemical GUS-assay. Unexpectedly, the regenerated plants displayed a variety of adverse phenotypic effects including different degree of growth and rooting inhibition, early flowering, increased number of internodes, changes in leaf shape, fusion of flowers, longostyly, and partial sterility. Only one from seven obtained calli produced a population of regenerated plants without severe phenotypic abnormalities. The NtaBBL5-14 line of clonally propagated plants was selected from this population and used for a CPB feeding experiment. It was shown that CPB larvae consume the leaves of NtaBBL5-14 line ten times more efficiently than the leaves of control plants (97±0.5% vs. 9±3% in 24 h respectively). Conclusion . The NtaBBL5-14 tobacco line is suitable for CPB feeding and can be further used as a model for studies in plant-pest interaction. The modification of other genes regulating nicotine metabolism can be a promising strategy to obtain tobacco plants edible for CPB with less pleiotropic effects.

Electroporation-mediated genetic transformation of oil palm (Elaeis guineensis)

Abstract. Darmawan C, Wiendi NMA, Utomo C, Liwang T. 2020. Electroporation-mediated genetic transformation of oil palm (Elaeis guineensis). Biodiversitas 21: 3720-3726. Novel traits introduction to existing varieties may shorten the duration of oil palm genetic improvement. This approach heavily relies on efficient plant transformation and regeneration methods. Commonly used methods such as biolistic and Agrobacterium-mediated transformation still generated low efficiency for oil palm. Therefore, it is important to find alternative transformation methods that have the potency to give better results. Electroporation is usually used for delivering genes into bacteria or plant protoplast where cell wall does not exist, however, in this research, it was used to deliver plasmid into plant tissues. The aim of this research is to establish an optimum electroporation protocol for delivering genes into oil palm calli. Electric field strength of 250, 500, 750, 1,000, and 1,250 V/cm were applied for oil palm calli electroporation. Transient GUS assay was not applicable for initial detection presumably due to oil palm endogenous GUS-like protein activity after electroporation. Delivered gene could be detected in survived calli from all tested electric field strength treatments and the highest calli growth rates were observed from 250 V/cm electroporation treatment. Overall results showed that electroporation could be used to deliver specific genes into oil palm calli and might be developed further to increase its efficiency.

Low-temperature pretreatment of explants and high maltose concentration during callus culture improves particle-bombardment-mediated stable transgene expression in common wheat.

Biolistic transformation systems are widely used to introduce foreign genes into common wheat (Triticum aestivum L.); however, these techniques often generate high transgene copy numbers and complex transgene integration patterns that hinder the stable expression of the transgenes. To improve the efficiency of stable transgene expression, we examined the effect of low-temperature pretreatment of wheat flower spikes and of high maltose concentration (HMC) in the medium during the subsequent callus culture. Tillers of the spring wheat cultivar Bobwhite were stored at 5°C without water for one week before the isolation of their immature scutellar tissues, and the resulting particle-bombarded explants were cultured on 15% maltose for a month. Together, these treatments significantly increased the number of recovered transgenic lines expressing the reporter gene. The low-temperature pretreatment eliminated the negative effects of HMC, and HMC improved the efficiency of stable transgene expression. Southern blot analysis revealed that transgenic lines recovered after HMC treatment integrated a lower copy number of transgenes than those cultured at normal (4%) maltose concentration. These findings suggest that the HMC-mediated reduction of the transgene copy number results from the suppression of plasmid DNA rearrangement before or during transgene integration into the wheat genome.

Pretreatment and posttreatment in the biolistic transformation of tea plant (Camellia sinensis) somatic embryos.

The tea plant (Camellia sinensis) contains various metabolic substances, including catechins and caffeine, for which genetic transformation techniques are essential for investigating the associated metabolic pathways. In this study, we sought to optimize the conditions and culture process for particle bombardment-mediated transformation of tea plant somatic embryos. We describe somatic embryo pretreatment for effective transient transformation in biolistic bombardment and the posttreatment conditions of somatic embryos for accelerating differentiation after bombardment. For the purpose of transformation, we used the somatic embryos of C. sinensis var. assamica 'Tingamira normal,' which were cultured in Murashige and Skoog (MS) medium containing 2 mg l-1 indole-3-butyric acid (IBA) and 4 mg l-1 6-benzyladenine (BA) at 25°C ±2°C under a 16-h photoperiod. With respect to the optimization of particle bombardment conditions for tea somatic embryos, we examined the effects of different Au colloid particle diameters and bombardment pressures, and accordingly established bombardment with 1.0-µm-diameter DNA-coated Au colloid at 1,100 psi as optimal conditions for introducing DNA for the transient expression of GUS. Additionally, we found that transplantation of tea somatic embryos from IBA/BA medium to a hormone-free medium prior to bombardment and incubation in the dark post-bombardment increased the frequency of secondary embryo production. Furthermore, osmotic treatment by culturing the somatic embryos in medium supplemented with 0.4 M mannitol improved transient transformation efficiency. After transformation, the culture of somatic embryos on filter papers or Kimwipes soaked in MS medium facilitated rapid and effective development of the somatic embryos.

An attempt to establish an Agrobacterium-mediated transient expression system in medicinal plants.

Genetic transformation has always been an important method for studying medical plant secondary metabolic regulation, among which stable transformation has a good reproducibility. However, it was time-consuming to obtain a stable transformed hairy root or transgenic plants, which was difficult to satisfy the great demand of researches on medical plant secondary metabolism-related genes. Moreover, Agrobacterium tumefaciens-mediated transient transformation has been extensively applied in studies of functional genes because of its simpleness, low cost, and short period. However, presently, researches on medical plant functional genes commonly used stable genetic transformation and some high-cost and high-difficulty transient transformation methods, such as gene gun and protoplast transformation. Thus, in this study, we selected the seedlings of Nicotiana benthamiana, Salvia miltiorrhiza, and Prunella vulgaris as the experimental material, with the methods of Agrobacterium tumefaciens injection, fast Agrobacterium-mediated seedling transformation (FAST), and FAST and mechanical damage. The results demonstrated that the injection transient transformation system of pCAMBIA1301 vector mediated by A. tumefaciens and the transient transformation of seedling system were not established in S. miltiorrhiza. In addition, the instantaneous transformation system of N. benthamiana and P. vulgaris seedlings was basically set up by FAST method. Besides, using the method of FAST and mechanical damage, the transient genetic transformation system of P. vulgaris seedlings was established for the first time. A. tumefaciens-mediated transient transformation of seedlings with pEAQ vectors provided an effective way and reference for the further study of functional genes of the medicinal plants N. benthamiana and P. vulgaris.

Breakthrough concepts in immune-oncology: Cancer vaccines at the bedside.

Clinical approval of the immune checkpoint blockade (ICB) agents for multiple cancer types has reinvigorated the long-standing work on cancer vaccines. In the pre-ICB era, clinical efforts focused on the Ag, the adjuvants, the formulation, and the mode of delivery. These translational efforts on therapeutic vaccines range from cell-based (e.g., dendritic cells vaccine Sipuleucel-T) to DNA/RNA-based platforms with various formulations (liposome), vectors (Listeria monocytogenes), or modes of delivery (intratumoral, gene gun, etc.). Despite promising preclinical results, cancer vaccine trials without ICB have historically shown little clinical activity. With the anticipation and expansion of combinatorial immunotherapeutic trials with ICB, the cancer vaccine field has entered the personalized medicine arena with recent advances in immunogenic neoantigen-based vaccines. In this article, we review the literature to organize the different cancer vaccines in the clinical space, and we will discuss their advantages, limits, and recent progress to overcome their challenges. Furthermore, we will also discuss recent preclinical advances and clinical strategies to combine vaccines with checkpoint blockade to improve therapeutic outcome and present a translational perspective on future directions.

Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide.

The purpose of this study was to express an antimicrobial peptide in the chloroplast to further develop the plastid engineering of H. pluvialis. Homologous targeting of the 16S-trnI/trnA-23S region and four endogenous regulatory elements, including the psbA promoter, rbcL promoter, rbcL terminator, and psbA terminator in H. pluvialis, were performed to construct a chloroplast transformation vector for H. pluvialis. The expression of codon-optimized antimicrobial peptide piscidin-4 gene (ant1) and selection marker gene (bar, biolaphos resistance gene) in the chloroplast of H. pluvialis was controlled by the rbcL promoter and psbA promoter, respectively. Upon biolistic transformation and selection with phosphinothricin, integration and expression of ant1 in the chloroplast genome were detected using polymerase chain reaction (PCR), southern blotting, and western blotting. Using this method, we successfully expressed antimicrobial peptide piscidin-4 in H. pluvialis. Hence, our results showed H. pluvialis promises as a platform for expressing recombinant proteins for biotechnological applications, which will further contribute to promoting genetic engineering improvement of this strain.

Genomic variation between PRSV resistant transgenic SunUp and its progenitor cultivar Sunset

BackgroundThe safety of genetically transformed plants remains a subject of scrutiny. Genomic variants in PRSV resistant transgenic papaya will provide evidence to rationally address such concerns.ResultsIn this study, a total of more than 74 million Illumina reads for progenitor ‘Sunset’ were mapped onto transgenic papaya ‘SunUp’ reference genome. 310,364 single nucleotide polymorphisms (SNPs) and 34,071 small Inserts/deletions (InDels) were detected between ‘Sunset’ and ‘SunUp’. Those variations have an uneven distribution across nine chromosomes in papaya. Only 0.27% of mutations were predicted to be high-impact mutations. ATP-related categories were highly enriched among these high-impact genes. The SNP mutation rate was about 8.4 × 10− 4 per site, comparable with the rate induced by spontaneous mutation over numerous generations. The transition-to-transversion ratio was 1.439 and the predominant mutations were C/G to T/A transitions. A total of 3430 nuclear plastid DNA (NUPT) and 2764 nuclear mitochondrial DNA (NUMT) junction sites have been found in ‘SunUp’, which is proportionally higher than the predicted total NUPT and NUMT junction sites in ‘Sunset’ (3346 and 2745, respectively). Among all nuclear organelle DNA (norgDNA) junction sites, 96% of junction sites were shared by ‘SunUp’ and ‘Sunset’. The average identity between ‘SunUp’ specific norgDNA and corresponding organelle genomes was higher than that of norgDNA shared by ‘SunUp’ and ‘Sunset’. Six ‘SunUp’ organelle-like borders of transgenic insertions were nearly identical to corresponding sequences in organelle genomes (98.18 ~ 100%). None of the paired-end spans of mapped ‘Sunset’ reads were elongated by any ‘SunUp’ transformation plasmid derived inserts. Significant amounts of DNA were transferred from organelles to the nuclear genome during bombardment, including the six flanking sequences of the three transgenic insertions.ConclusionsComparative whole-genome analyses between ‘SunUp’ and ‘Sunset’ provide a reliable estimate of genome-wide variations and evidence of organelle-to-nucleus transfer of DNA associated with biolistic transformation.

Autophagy Regulates Fungal Virulence and Sexual Reproduction in Cryptococcus neoformans.

Autophagy (macroautophagy) is an evolutionarily conserved degradation pathway involved in bulk degradation of cytoplasmic organelles, old protein, and other macromolecules and nutrient recycling during starvation. Extensive studies on functions of autophagy-related genes have revealed that autophagy plays a role in cell differentiation and pathogenesis of pathogenic fungi. In this study, we identified and characterized 14 core autophagy machinery genes (ATGs) in C. neoformans. To understand the function of autophagy in virulence and fungal development in C. neoformans, we knocked out the 14 ATGs in both α and a mating type strain backgrounds in C. neoformans, respectively, by using biolistic transformation and in vivo homologous recombination. Fungal virulence assay showed that virulence of each atgΔ mutants was attenuated in a murine inhalation systemic-infection model, although virulence factor production was not dramatically impaired in vitro. Fungal mating assays showed that all the 14 ATGs are essential for fungal sexual reproduction as basidiospore production was blocked in bilateral mating between each atgΔ mutants. Fungal nuclei development assay showed that nuclei in the bilateral mating of each atgΔ mutants failed to undergo meiosis after fusion, indicating autophagy is essential for regulating meiosis during mating. Overall, our study showed that autophagy is essential for fungal virulence and sexual reproduction in C. neoformans, which likely represents a conserved novel virulence and sexual reproduction control mechanism that involves the autophagy-mediated proteolysis pathway.

Independent overexpression of OsRab16A and AtDREB1A exhibit enhanced drought tolerance in transgenic aromatic rice variety Pusa Sugandhi 2

Rice (Oryza sativa L.), the staple food in South-East Asian countries, is a water-intensive crop so that its productivity is highly affected by drought, the most severe environmental stress factor. We report here the development of transgenic rice via overexpression of full length OsRab16A (driven by endogenous promoter) or AtDREB1A driven by rd29A promoter in the background of the aromatic indica rice cultivar, Pusa Sugandhi 2 (PS2) through biolistic transformation. Both the transgenes were stably introgretted in T2 generations as indicated by the results of polymerase chain reaction (PCR). Reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses showed that the expression of both the transgenes was induced by drought stress in all the T2 transgenic plants examined, which were highly tolerant to water deficit stress during both the vegetative and reproductive stages without their morphological or agronomic traits being affected. Protein immunoblot analysis with Rab16A (= anti dehydrin) antiserum showed detectable accumulation of Rab16A protein in the transgenics in response to drought stress and exogenous abscisic acid (ABA) application. The physiological studies revealed that the expression of the transgene under drought stress in T2 transgenics was associated with lesser shoot or root length inhibition, improved leaf relative water content, stable maintenance of chlorophyll, lesser hydrogen peroxide accumulation with higher catalase activity and an increased accumulation of the osmoprotectant proline, as compared with the wild type (WT) plants. The transgenic plants also showed significantly higher filled grain, spikelet fertility and grain yield under stressed conditions. All these findings highlighted the tremendous potentiality of both OsRab16A and AtDREB1A in conferring drought tolerance without affecting the normal phenotype and physiology of the plants. To our knowledge, this is the first report on the development of a drought-tolerant transgenic aromatic rice variety through overexpression of genes, one involved in ABA-dependent pathway (OsRab16A), while the other in ABA-independent pathway (AtDREB1A) of stress tolerance, thereby highlighting the significance of both the pathways in drought tolerance of the aromatic rice varieties.

Highly Efficient Biolistic Transformation of Embryogenic Cell Suspensions of Banana Via a Liquid Medium Selection System

An efficient biolistic transformation system of banana combined with a liquid medium selection system was developed during this study. An embryogenic cell suspension (ECS) of Musa acuminata cv. Baxi (AAA) was bombarded with a particle delivery system. After 7 days of restoring culture in liquid M2 medium, embryogenic cells were transferred to a liquid selection M2 medium supplemented with 10 μg/mL hygromycin for resistance screening. The untransformed cell clusters were inhibited or killed, and a small number of transformants proliferated in the liquid selection medium. After the 0th, first, second, and third generation of antibiotic screening, there were 0, 65, 212, and 320, respectively, vitality-resistant buds obtained from a 0.5-mL packed cell volume (PCV) of embryogenic cell suspension. The β-glucuronidase (GUS) staining, polymerase chain reaction (PCR) analysis, and Southern blot hybridization results all demonstrated a 100% positive rate of regenerated resistant seedlings. Interestingly, the number of buds obtained through third-generation screening was almost equal to that obtained from the original ECS in M2 medium without antibiotics. These results suggested that the liquid medium selection system facilitated the proliferation of a positive transgenic ECS, which significantly improved the regeneration rate of transformants. This protocol is suitable for the genetic transformation of all banana genotypes and is highly advantageous to varieties with low callusing potential.

Transforming Candida hispaniensis, a promising oleaginous and flavogenic yeast.

Candida hispaniensis is an oleaginous yeast with a great potential for production of single cell oil according to its naturally high lipid accumulation capacity. Its unusual small genome size trait is also attractive for fundamental research on genome evolution. Our physiological study suggests a great potential for lipid production, reaching 224 mg/g of cell dry weight in glucose minimum medium. C. hispaniensis is also able to secrete up to 34.6 mg/L of riboflavin promising further riboflavin production improvements by cultivation optimization and genetic engineering. However, while its genome sequence has been released very recently, no genetic tools have been described up to now for this yeast limiting its use for fundamental research and for exploitation in an industrial biotechnology. We report here the first genetic modification of C. hispaniensis by introducing a heterologous invertase allowing the growth on sucrose using a biolistic transformation approach using a dedicated vector. The first genetic tool and transformation method developed here appear as a proof of concept, and while it would benefit from further optimization, heterogeneous expression of invertase allows for metabolism of an additional sugar and shows heterologous enzyme production capacity.

DNA vaccines: prime time is now.

Recently newer synthetic DNA vaccines have been rapidly advanced to clinical study and have demonstrated an impressive degree of immune potency and tolerability. Improvements in DNA delivery over prior needle and syringe approaches include jet delivery, gene gun delivery, among others. Among the most effective of these new delivery methods, advanced electroporation (EP), combined with other advances, induces robust humoral and cellular immunity in both preventative as well as therapeutic studies. Advancements in the design of the DNA inserts include leader sequence changes, RNA and codon optimizations, improved insert designs, increased concentrations of DNA, and skin delivery, appear to complement newer delivery strategies. These advances also provide a framework for the in vivo production of synthetic DNA biologics. In this review, we focus on recent studies of synthetic DNA vaccines in the clinic for the prevention or treatment of infectious diseases with a focus on adaptive electroporation for delivery, and briefly summarize novel preclinical data advancing the in vivo delivery of DNA-encoded antibody-like biologics.

Gene transfer to plants by electroporation: methods and applications.

Developing gene transfer technologies enables the genetic manipulation of the living organisms more efficiently. The methods used for gene transfer fall into two main categories; natural and artificial transformation. The natural methods include the conjugation, transposition, bacterial transformation as well as phage and retroviral transductions, contain the physical methods whereas the artificial methods can physically alter and transfer genes from one to another organisms' cell using, for instance, biolistic transformation, micro- and macroinjection, and protoplast fusion etc. The artificial gene transformation can also be conducted through chemical methods which include calcium phosphate-mediated, polyethylene glycol-mediated, DEAE-Dextran, and liposome-mediated transfers. Electrical methods are also artificial ways to transfer genes that can be done by electroporation and electrofusion. Comparatively, among all the above-mentioned methods, electroporation is being widely used owing to its high efficiency and broader applicability. Electroporation is an electrical transformation method by which transient electropores are produced in the cell membranes. Based on the applications, process can be either reversible where electropores in membrane are resealable and cells preserve the vitality or irreversible where membrane is not able to reseal, and cell eventually dies. This problem can be minimized by developing numerical models to iteratively optimize the field homogeneity considering the cell size, shape, number, and electrode positions supplemented by real-time measurements. In modern biotechnology, numerical methods have been used in electrotransformation, electroporation-based inactivation, electroextraction, and electroporative biomass drying. Moreover, current applications of electroporation also point to some other uncovered potentials for various exploitations in future.

Ex Vivo Imaging of Mitochondrial Dynamics and Trafficking in Astrocytes.

Mitochondria are essential organelles involved in energy supply and calcium homeostasis. The regulated distribution of mitochondria in polarized cells, particularly neurons, is thought to be essential to these roles. Altered mitochondrial function and impairment of mitochondrial distribution and dynamics is implicated in a number of neurologic disorders. Several recent reports have described mechanisms regulating the activity-dependent distribution of mitochondria within astrocyte processes and the functional consequences of altered mitochondrial transport. Here we provide an ex vivo method for monitoring the transport of mitochondria within the processes of astrocytes using organotypic "slice" cultures. These methods can be easily adapted to investigate a wide range of mitochondrial behaviors, including fission and fusion dynamics, mitophagy, and calcium signaling in astrocytes and other cell types of the central nervous system. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of brain slices Basic Protocol 2: Preparation of gene gun bullets Basic Protocol 3: Gene gun transfection of slices Basic Protocol 4: Visualization and tracking of mitochondrial movement Alternate Protocol: Transduction of EGFP-mito via viral injection of the neonatal mouse brain.

Transformation of Cryptococcus neoformans by electroporation using a transient CRISPR-Cas9 expression (TRACE) system

The basidiomycete Cryptococcus neoformans is not only a clinically important pathogen, but also a model organism for studying microbial pathogenesis and eukaryotic biology. One key factor behind its rise as a model organism is its genetic amenability. The widely used methods for transforming the C. neoformans species complex are Agrobacterium-mediated transformation (AMT) for random insertional mutagenesis and biolistic transformation for targeted mutagenesis. Electroporation was introduced to C. neoformans in early 1990s. Although electroporation is economic and yields a large number of transformants, introduced DNA rarely integrates into cryptococcal genome, which limits its use. Biolistic transformation, although costly and inefficient, has been the only method used in targeted mutagenesis in the past two decades. Several modifications, including the use of a donor DNA with split markers, a drug-resistant selection marker, and a recipient strain deficient in non-homologous end joining (NHEJ), have since modestly increased the frequency of genome integration and the rate of homologous replacement of the DNA introduced by electroporation. However, electroporation was not the method of choice for transformation until the recent adoption of CRISPR-Cas9 systems. We have developed a Transient CRISPR-Cas9 coupled with Electroporation System (TRACE), which dramatically facilitates targeted mutagenesis in the Cryptococcus species complex. TRACE combines the high transformation efficiency of electroporation with the high rates of DNA integration due to the transiently expressed CRISPR-Cas9. Here, we briefly discussed the history of electroporation for Cryptococcus transformation and provided detailed procedures for electroporation and the cassettes construction of the TRACE system for various genetic manipulations.