Localized Electroporation Research Articles

Transgene delivery via intracellular electroporetic nanoinjection

Development of an effective cytoplasmic delivery technique has remained an elusive goal for decades despite the success of pronuclear microinjection. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In this work we report a cytoplasmic delivery method termed intracellular electroporetic nanoinjection (IEN). IEN is unique in that it manipulates transgenes using electrical forces. The microelectromechanical system (MEMS) uses electrostatic charge to physically pick up transgenes and place them in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronuclei using electrical pulses. Standard electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm for transgene delivery from the cytoplasm to the pronucleus. In this report we describe the principles which allow localized electroporation of the pronuclei including: the location of mouse pronuclei between 21 and 28h post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on electric-field-driven movement of transgenes. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

Delivery of molecules into cells using localized single cell electroporation on ITO micro-electrode based transparent chip

Single cell electroporation is one of the nonviral method which successfully allows transfection of exogenous macromolecules into individual living cell. We present localized cell membrane electroporation at single-cell level by using indium tin oxide (ITO) based transparent micro-electrodes chip with inverted microscope. A focused ion beam (FIB) technique has been successfully deployed to fabricate transparent ITO micro-electrodes with submicron gaps, which can generate more intense electric field to produce very localized cell membrane electroporation. In our approach, we have successfully achieved 0.93μm or smaller electroporation region on the cell surface to inject PI (Propidium Iodide) dye into the cell with 60% cell viability. This experiments successfully demonstrate the cell self-recover process from the injected PI dye intensity variation. Our localized cell membrane electroporation technique (LSCMEP) not only generates reversible electroporation process but also it provides a clear optical path for potentially monitoring/tracking of drugs to deliver in single cell level.

A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits

Since Cajal's early drawings, the characterization of neuronal architecture has been paramount in understanding neuronal function. With the development of electrophysiological techniques that provide unprecedented access to the physiology of these cells, experimental questions of neuronal function have also become more tractable. Fluorescent tracers that can label the anatomy of individual or populations of neurons have opened the door to linking anatomy with physiology. Experimentally however, current techniques for bulk labeling of cells in vitro often affect neuronal function creating a barrier for exploring structure-function questions. Here we describe a new technique for highly localized electroporation within a cell or cell population that enables the introduction of membrane impermeable charged dyes including dextran-conjugated fluorophores, hydrazide tracers, and calcium indicator dyes in vitro. We demonstrate that this technique is highly versatile, allowing for labeling of large or small areas of tissue, allowing for the investigation of both cellular morphology and physiological activity in identified neuronal circuits in acute brain slices. Furthermore, this approach allows subsequent targeted whole-cell patch recording based on well-defined connectivity as well as assessment of physiological activity in targeted circuits on a fast time scale.

Localized electroporation and molecular delivery into single living cells by atomic force microscopy

We present an efficient and fast method for selective and localized electroporation of a single living cell from a population of millions to tens of cells using the modified tip of an atomic force microscope. Electroporation was observed in real time using an inverted microscope. This technique is proposed as a tool for efficient and controlled delivery of biomolecules, proteins, drugs, and genes.

A porous membrane-based culture substrate for localized in situ electroporation of adherent mammalian cells

Abstract We report herein the invention of and proof of function for a porous membrane-based electroporation device that can deliver molecules into spatially restricted and predefined areas of a cell monolayer. The device's cell culture substrate is a microporous alumina membrane (pore size 0.02 μm), with an underlying thin poly-(dimethylsiloxane) (PDMS) film that has one or more holes with diameters in the one-tenth millimeter range. When a transient electric field is generated between the device's two planar electrodes – one of which is placed above the cells and the other below the PDMS layer – the filed condenses only in the volume defined by hole in the PDMS film and therefore localized electroporation can occur. We demonstrate that Lucifer Yellow (LY) and plasmid DNA are selectively introduced into only those HeLa cells located above the holes. Using the device containing a PDMS film with multiple holes, a patterned array of LY-stained cells was resulted. Compared with the operation of and the results obtained from a conventional cuvette electroporation device, our device greatly decreases the necessary operating voltage, can be used with cells attached to a substrate, and increases the number of conditions that can be screened in a single experiment. Finally, since a PDMS film with different sized holes, produces different localized electric field strengths, it is possible to determine the optimum electroporetic conditions in a single experiment.

Microfabrication of Individual 200μm Diameter Transdermal Microconduits Using High Voltage Pulsing in Salicylic Acid and Benzoic Acid

We describe an extension of semiconductor fabrication methods that creates individual approximately 200 microm diameter aqueous pathways through human stratum corneum at predetermined sites. Our hypothesis is that spatially localized electroporation of the multilamellar lipid bilayer membranes provides rapid delivery of salicylic acid to the keratin within corneocytes, leading to localized keratin disruption and then to a microconduit. A microconduit penetrating the isolated stratum corneum supports a volumetric flow of order 0.01 ml per s with a pressure difference of only 0.01 atm (about 10(2) Pa). This study provides a method for rapidly microengineering a pathway in the skin to interface future devices for transdermal drug delivery and sampling of biologically relevant fluids.

A novel hydra matrix metalloproteinase (HMMP) functions in extracellular matrix degradation, morphogenesis and the maintenance of differentiated cells in the foot process.

As a member of Cnidaria, the body wall of hydra is structurally reduced to an epithelial bilayer with an intervening extracellular matrix (ECM). Biochemical and cloning studies have shown that the molecular composition of hydra ECM is similar to that seen in vertebrates and functional studies have demonstrated that cell-ECM interactions are important to developmental processes in hydra. Because vertebrate matrix metalloproteinases (MMPs) have been shown to have an important role in cell-ECM interactions, the current study was designed to determine whether hydra has homologues of these proteinases and, if so, what function these enzymes have in morphogenesis and cell differentiation in this simple metazoan. Utilizing a PCR approach, a single hydra matrix metalloproteinase, named HMMP was identified and cloned. The structure of HMMP was similar to that of vertebrate MMPs with an overall identity of about 35%. Detailed structural analysis indicated some unique features in (1) the cysteine-switch region of the prodomain, (2) the hinge region preceding the hemopexin domain, and (3) the hemopexin domain. Using a bacterial system, HMMP protein was expressed and folded to obtain an active enzyme. Substrate analysis studies indicated that recombinant HMMP could digest a number of hydra ECM components such as hydra laminin. Using a fluorogenic MMP substrate assay, it was determined that HMMP was inhibited by peptidyl hydroxamate MMP inhibitors, GM6001 and matlistatin, and by human recombinant TIMP-1. Whole-mount in situ studies indicated that HMMP mRNA was expressed in the endoderm along the entire longitudinal axis of hydra, but at relatively high levels at regions where cell-transdifferentiation occurred (apical and basal poles). Functional studies using GM6001 and TIMP-1 indicated that these MMP inhibitors could reversibly block foot regeneration. Blockage of foot regeneration was also observed using antisense thio-oligo nucleotides to HMMP introduced into the endoderm of the basal pole using a localized electroporation technique. Studies with adult intact hydra found that GM6001 could also cause the reversible de-differentiation or inhibition of transdifferentiation of basal disk cells of the foot process. Basal disk cells are adjacent to those endoderm cells of the foot process that express high levels of HMMP mRNA. In summary, these studies indicate that hydra has at least one MMP that is functionally tied to morphogenesis and cell transdifferentiation in this simple metazoan.

Identification and characterization of hydra metalloproteinase 2 (HMP2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis.

Several members of the newly emerging astacin metalloproteinase family have been shown to function in a variety of biological events, including cell differentiation and morphogenesis during both embryonic development and adult tissue differentiation. We have characterized a new astacin proteinase, hydra metalloproteinase 2 (HMP2) from the Cnidarian, Hydra vulgaris. HMP2 is translated from a single mRNA of 1.7 kb that contains a 1488 bp open reading frame encoding a putative protein product of 496 amino acids. The overall structure of HMP2 most closely resembles that of meprins, a subgroup of astacin metalloproteinases. The presence of a transient signal peptide and a putative prosequence indicates that HMP2 is a secreted protein that requires post-translational processing. The mature HMP2 starts with an astacin proteinase domain that contains a zinc binding motif characteristic of the astacin family. Its COOH terminus is composed of two potential protein-protein interaction domains: an "MAM" domain (named after meprins, A-5 protein and receptor protein tyrosine phosphatase mu) that is only present in meprin-like astacin proteinases; and a unique C-terminal domain (TH domain) that is also present in another hydra metalloproteinase, HMP1, in Podocoryne metalloproteinase 1 (PMP1) of jellyfish and in toxins of sea anemone. The spatial expression pattern of HMP2 was determined by both mRNA whole-mount in situ hybridization and immunofluorescence studies. Both morphological techniques indicated that HMP2 is expressed only by the cells in the endodermal layer of the body column of hydra. While the highest level of HMP2 mRNA expression was observed at the junction between the body column and the foot process, immunofluorescence studies indicated that HMP2 protein was present as far apically as the base of the tentacles. In situ analysis also indicated expression of HMP2 during regeneration of the foot process. To test whether the higher levels of HMP2 mRNA expression at the basal pole related to processes underlying foot morphogenesis, antisense studies were conducted. Using a specialized technique named localized electroporation (LEP), antisense constructs to HMP2 were locally introduced into the endodermal layer of cells at the basal pole of polyps and foot regeneration was initiated and monitored. Treatment with antisense to HMP2 inhibited foot regeneration as compared to mismatch and sense controls. These functional studies in combination with the fact that HMP2 protein was expressed not only at the junction between the body column and the foot process, but also as far apically as the base of the tentacles, suggest that this meprin-class metalloproteinase may be multifunctional in hydra.