Genome editing of rodents by electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stage embryos

Genome edited animals can now be easily produced using the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Traditionally, these animals have been produced by the introduction of endonucleases into pronuclear-stage embryos. Recently, a novel electroporation method, the “Technique for Animal Knockout system by Electroporation (TAKE),” has been established as a simple and highly efficient tool to introduce endonucleases into embryos instead of methods such as microinjection. Use of frozen-warmed pronuclear-stage embryos in this method has further contributed to efficient production of genome edited animals. However, early developmental stage embryos, including pronuclear-stage embryos, especially those of rats, sometimes show low resistance to physical damage by vitrification and introduction of endonucleases during microinjection. In this study, we propose an ethanol-free, slow-freezing method to reduce physical damage to pronuclear-stage embryos followed by the TAKE method. All mouse and rat frozen embryos were survived after electroporation, and 18% and 100% of offspring were edited target gene, respectively. The resulting protocol is an efficient method for producing genome edited animals.     

Improved mRNA electroporation method for Xenopus neurula embryos

Electroporation has led to new approaches to the analysis of gene regulation of the chick embryonic system. However, application of this method to Xenopus, another model organism of embryology, has left many difficulties to be overcome. The specially devised electrodes, the examination of luciferase activities expressed, and the direct visualization of green fluorescence protein allow us to optimize the conditions of electroporation for Xenopusembryos. The use of mRNA rather than DNA improved the expression efficiency 120 times more than for the case of plasmid DNA, and the effect emerged more immediately after electroporation. The noncontact electroporation adopted here caused less damage to cells and tissues than with the needle type electrode, making it practical for efficient application to early embryos. Furthermore, the mRNA electroporation technique is applicable for other systems in which the DNA electroporation has not had any significant effect because of its low expression efficiency.     

Spatially and temporally controlled electroporation of early chick embryos

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.     

The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator

Technique for Animal Knockout system by Electroporation (TAKE) is a simple and efficient method to generate genetically modified (GM) mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems. To reinforce the versatility of electroporation used for gene editing in mice, the electric condition was optimized for vitrified-warmed mouse embryos, and applied to the fresh embryos from widely used inbred strains (C57BL/6NCr, BALB/cCrSlc, FVB/NJcl, and C3H/HeJJcl). The electric pulse settings (poring pulse: voltage, 150 V; pulse width, 1.0 ms; pulse interval, 50 ms; number of pulses, +4; transfer pulse: voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; number of pulses, ±5) were optimal for vitrified-warmed mouse embryos, which could efficiently deliver the gRNA/Cas9 complex into the zygotes without zona pellucida thinning process and edit the target locus. These electric condition efficiently generated GM mice in widely used inbred mouse strains. In addition, electroporation using the electrode with a 5 mm gap could introduce more than 100 embryos within 5 min without specific pretreatment and sophisticated technical skills, such as microinjection, and exhibited a high developmental rate of embryos and genome-editing efficiency in the generated offspring, leading to the rapid and efficient generation of genome editing mice. The electric condition used in this study is highly versatile and can contribute to understanding human diseases and gene functions by generating GM mice more easily and efficiently.     

Improved Transfection Efficiency of Chicken Gonadal Primordial Germ Cells for the Production of Transgenic Poultry

Electroporation is a common method of DNA transfection for many types of eukaryotic cells, but has not been attempted in avian primordial germ cells (PGCs). DNA uptake in chicken primordial germ cells (PGCs) was tested using electroporation with and without dimethyl sulfoxide (DMSO). Gonadal tissue and chicken embryonic fibroblasts (CEFs) were isolated from 6-day-old embryos (stage 29), transfected with pCMV carrying the bacterial lacZ gene, and cultured for 24 h. Gonadal primordial germ cells (gPGCs) were purified from culture using a Ficoll gradient. The addition of DMSO significantly increased the transfection efficiency of gPGCs but had no effect on chicken embryonic fibroblasts. Electroporation of gPGCs resulted in an 80% transfection efficiency, compared with about 17% observed with liposomes. Approximately 200 transfected gPGCs were injected into 2.5-day-old (stage 17) recipient embryos and the eggs were incubated for an additional 3.5 days, 7.5 days or to ...     

Gene transfer by electroporation into intact scutellum cells of wheat embryos

Summary Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-F capacitor and with 100 g DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MES 2-N-morpholinoethane sulfonic acid     

Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor

Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 10³ cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.     

A novel electroporation method using a capillary and wire-type electrode

Electroporation is widely used to achieve gene transfection. A common problem in electroporation is that it has a lower viability than any other transfection method. In this study, we developed a novel electroporation device using a capillary tip and a pipette that was effective on a wide range of mammalian cells, including cell lines, primary cells, and stem cells. The capillary electroporation system considerably reduced cell death during electroporation because of its wire-type electrode, which has a small surface area. The experimental results also indicated that the cell viability was dependent on the change in pH induced by electrolysis during electroporation. Additionally, the use of a long and narrow capillary tube combined with simple pipetting shortened the overall time of the electroporation process by up to 15 min, even under different conditions with 24 samples. These results were supported by comparison with a conventional electroporation system. The transfection rate and the cell viability were enhanced by the use of the capillary system, which had a high transfection rate of more than 80% in general cell lines such as HeLa and COS-7, and more than 50% in hard-to-transfect cells such as stem or primary cells. The viability was approximately 70-80% in all cell types used in this study.     

'Shocking' developments in chick embryology: electroporation and in ovo gene expression

Efficient gene transfer by electroporation of chick embryos in ovo has allowed the development of new approaches to the analysis of gene regulation, function and expression, creating an exciting opportunity to build upon the classical manipulative advantages of the chick embryonic system. This method is applicable to other vertebrate embryos and is an important tool with which to address cell and developmental biology questions. Here we describe the technical aspects of in ovo electroporation, its different applications and future perspectives.     

Transient expression of GUS and anthocyanin constructs in intact maize immature embryos following electroporation

Electroporation was used for the delivery and subsequent expression of GUS and anthocyanin reporter genes into intact maize immature embryos. The optimal conditions consisted of culturing immature embryos for 4 days on N6 1-100-25-Ag medium prior to electroporation (375 V/cm; 960 µF capacitance) in EPR buffer containing DNA and 0.07 M sodium glutamate at room temperature (22°C) after a 10 min heat shock at 37°C. Under these conditions, over 40 spots of GUS transient activity were observed per immature embryo. Transient gene expression after electroporation was further demonstrated using an anthocyanin construct, which is specific for expression in plant cells.     

Electroporation of cell membranes.

Electric pulses of intensity in kilovolts per centimeter and of duration in microseconds to milliseconds cause a temporary loss of the semipermeability of cell membranes, thus leading to ion leakage, escape of metabolites, and increased uptake by cells of drugs, molecular probes, and DNA. A generally accepted term describing this phenomenon is "electroporation." Other effects of a high-intensity electric field on cell membranes include membrane fusions, bleb formation, cell lysis... etc. Electroporation and its related phenomena reflect the basic bioelectrochemistry of cell membranes and are thus important for the study of membrane structure and function. These phenomena also occur in such events as electric injury, electrocution, and cardiac procedures involving electric shocks. Electroporation has found applications in: (a) introduction of plasmids or foreign DNA into living cells for gene transfections, (b) fusion of cells to prepare heterokaryons, hybridoma, hybrid embryos... etc., (c) insertion of proteins into cell membranes, (d) improving drug delivery and hence effectiveness in chemotherapy of cancerous cells, (e) constructing animal model by fusing human cells with animal tissues, (f) activation of membrane transporters and enzymes, and (g) alteration of genetic expression in living cells. A brief review of mechanistic studies of electroporation is given.     

Transformation ofHaemophilus influenzae following electroporation with plasmid and chromosomal DNA

Naturally transformable species, such asHaemophilus influenzae, have evolved systems for the efficient uptake and integration of DNA from the surrounding environment. We compared this competence-dependent DNA uptake system to electroporation, which has been widely used in the past few years to introduce DNA into cells, for transformingHaemophilus influenzae. Electroporation improved transformation efficiency when noncompetent cells were used and when DNA lackingHaemophilus-specific uptake sequences was used for transformation of competent cells. An increase in plasmid-to-chromosome recombination was seen when plasmid DNA containing chromosomal inserts was introduced into competent cells by electroporation, as observed previously for natural transformation.     

Transferring genes into cultured mammalian embryos by electroporation

Mammalian whole embryo culture (WEC) was developed long before transgenic and gene targeted animals are widely used. Electroporation (EP) into cultured rodent embryos has expanded the potential to analyze gene functions in mammalian embryos by transferring exogenous plasmid vectors or small nucleotides in region- and stage-specific ways. This method is quite simple, and therefore enables us to analyze gene functions more quickly than genetic manipulation. In this review, we introduce combinatorial methods of WEC and EP, and summarize various applications in developmental neurobiology.     

Simple Genome Editing of Rodent Intact Embryos by Electroporation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE) method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN) comprised the edited targeted gene as a knockout (67% of mice and 88% of rats) or knock-in (both 33%). The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.     

Electroporation of Haemophilus influenzae is effective for transformation of plasmid but not chromosomal DNA.

Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.     

High Efficiency Production of germ-line Transgenic Japanese Medaka (Oryzias latipes) by Electroporation with Direct Current-shifted Radio Frequency Pulses

Although there have been several studies showing the production of transgenic fish through electroporation techniques, success rates have been low and few studies show germ-line integration and expression. When electroporation has been successful, the device used is no longer commercially available. The goal of this experiment was to find an alternative efficient method of generating transgenic Japanese medaka (Oryzias latipes) using a commercially available electroporation device. The Gene Pulser II and RF module (Bio-Rad Laboratories, USA), along with two reporter gene constructs, were used. In contrast to other electroporation devices, which are based on a single pulse with exponential decay or square wave technology, the Gene Pulser II incorporates a direct current (DC)-shifted radio frequency (RF) signal. With this technique, over 1000 embryos can be electroporated in less than 30 min. The plasmid pCMV-SPORT--gal (Invitrogen, USA) was used in the supercoiled form to optimize parameters for gene transfer into single-celled embryos, and resulted in up to 100% somatic gene transfer. Similar conditions were used to generate fish transgenic for both the pCMV-EGFP plasmid (Clontech, USA) and a cytomegalovirus (CMV) driven phytase-EGFP construct. The conditions used were a voltage of 25 V, a percent modulation of 100%, a radio frequency of 35 kHz, a burst duration of 10 ms, 3 bursts, and a burst interval of 1.0 s. Seventy percent of the embryos electroporated with the pCMV-EGFP construct survived to sexual maturity, and of those, 85% were capable of passing the transgene on to their offspring. Transgenic second generation back-crossed (BC₂) fry were subjected to Southern blot analysis, which confirmed germ-line integration, and observation for green fluorescence protein, which confirmed protein expression. DC-shifted RF pulses are effective and efficient in the production of transgenic medaka, and germ-line integration and expression can be achieved without linearization of the transgene vector.     

Electroporation-mediated transient gene expression in isolated scutella of Hordeum vulgare

Electroporation was used to introduce DNA into excised scutella of immature embryos of Hordeum vulgare L. cvs Golden Promise and Delita. Using the firefly luciferase gene as reporter, parameters were analyzed for high transient gene expression while maintaining tissue viability. Enzymatic wounding was necessary for DNA uptake. The optimized protocol involves use of linearized DNA and addition of 15% (w/v) polyethylene glycol at a field strength of 950 V cm⁻¹ and approximately 56 ms pulse length. A one-day preculture was required for obtaining callus after electroporation. Transient gene expression was further demonstrated using the β-glucuronidase gene. Blue spots were detected at the abaxial scutellar surface, indicating that cells competent for somatic embryogenesis are also amenable to transfection by electroporation.     

The effective method of gene transfer into mammalian cells cultured in vitro

Efficiency of transformation by a number of vectors with the different selective markers of a set of cell lines has been studied for three different methods based on using calcium phosphate, polybrene, or electroporation. Electroporation is shown to be the most efficient one. Using this method with the system rat2k-cells-pAGO vector we have obtained the frequencies of transformation up to 2-3.10(-3). We suggest to use this system as a model for investigation of homologous recombination in the framework of the gene therapy project.     

Electroporation of the vasculature and the lung

Electroporation has proven to be a highly effective technique for the in vivo delivery of genes to a number of solid tissues. In most of the reported methods, DNA is injected into the target tissue and electrodes are placed directly on or in the tissue for application of the electric field. While this works well for solid tissues, there are many tissues and organs that are not amenable to such an approach. In this review I will focus on the development of electroporation protocols for two such tissues: the vasculature and the lung. Several methods for in vivo electroporation of the vasculature have been developed in recent years that deliver DNA to vessel segments from either the inside or outside of the vessel. The advantages and disadvantages of each are discussed, as are the applications for which they have been used. In more recent work, our laboratory has developed a novel method to deliver genes to the rodent lung that results in high level, uniform, gene expression throughout all cell types of the lung. Most importantly, this technique is safe, and causes no inflammatory response or alterations in normal physiology of the organs. Taken together, these studies demonstrate the utility of electroporation for gene transfer to non injectible tissues.     

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.