JP2005261323A - Gene transfer method by electroporation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for introducing a gene into a cell and rapidly expressing the introduced gene and an apparatus therefor. <P>SOLUTION: The method for introducing a gene into a cell comprises removing the culture medium of a cell and applying a gene-containing solution to the cell and carrying out electroporation. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a gene introduction method and an electroporation apparatus.

  It is one of the most important techniques in gene manipulation to express an arbitrary protein in a cell.

  Examples of the method for introducing a gene into cultured cells include a calcium phosphate method, a lipofection method, a method using a viral vector, and an electroporation method. Genes can be introduced into cells by these methods, but all of them require several hours to several days to express a protein in the cells.

  Therefore, there has been a demand for a technique capable of performing protein expression more rapidly.

  An object of the present invention is to provide a method for introducing a gene into a cell and quickly expressing the introduced gene and an apparatus therefor.

The present invention is characterized in that a medium is extracted before electroporation so that a high-concentration plasmid DNA containing a gene can directly touch a cell. As a result, the present inventors have found that a protein can be expressed in cultured cells with high efficiency in a very short time, for example, within a few hours, thereby completing the present invention.
1. A method for introducing a gene into cells by applying a solution containing the gene to the cells after removing the cell culture medium and performing electroporation.
2. Item 2. The method according to Item 1, wherein the gene introduced into the cell is applied to the cell as a 20 to 100 µl solution.
3. Item 3. The method according to Item 1 or 2, wherein the length of the electric pulse is 10 to 20 msec and the number of times of applying the electric pulse is 5 to 20.
4). Item 4. The method according to any one of Items 1 to 3, wherein the amount of the medium is 200 to 300 μl per 1 cm ^{2 of the} container containing cells.
5). An electroporation apparatus having at least two electrodes, wherein a part of a second electrode is fixed in a plane surrounded by the first electrode, and a current flowing between the first electrode and a part of the second electrode is a gene. A device characterized by being energized to a cell into which is introduced.
6). Item 6. The device according to Item 5, wherein the first electrode is a circular electrode, and the tip of the second electrode is present near the center of a circle surrounded by the first electrode.

  Hereinafter, the present invention will be described in more detail.

  In the present invention, the gene introduced into the cell is not particularly limited as long as it encodes a protein, and any gene is targeted. The gene may be a heterologous gene for a cultured cell serving as a host, or a host-derived gene may be introduced.

Although the gene itself may be used, it is usually introduced into a cultured cell serving as a host as an expression vector that is incorporated into a vector suitable for the host so as to allow expression. The vector, promoter, enhancer and the like constituting the expression vector may be appropriately selected according to the cultured cell serving as the host.

  Examples of the cell serving as the host include cells adhered to the dish, and specifically include plant cells and animal cells (cells such as mammals, insects, birds, amphibians, reptiles and fish).

  Preferred hosts are mammalian cells, insect cells and plant cells. Examples of mammals include humans, cows, horses, dogs, monkeys, rats, mice, hamsters, etc., preferably human cells such as nerve cells. .

  In the method of the present invention, it is important to remove as much of the cell culture medium as possible. When the cell culture medium (medium) is small, the gene concentration is not diluted when the gene-containing solution is applied to the cells, and the introduction efficiency can be maintained high because the liquid volume is small.

  The total amount of the culture solution and the solution containing the gene to be added is 150 μl or less, preferably 120 μl or less, more preferably 100 μl or less per 400,000 cells.

  Each amount of the solution containing the remaining amount of the culture solution and the added gene is not particularly limited as long as the total amount is within the above range, but the remaining amount of the culture solution is about 50 μl or less per 400,000 cells. The amount of the solution containing the gene to be added is, for example, about 20 to 100 μl per 400,000 cells.

  In the present invention, a gene, for example, a foreign gene is incorporated into a vector and introduced into a cell by electroporation in a state that hardly contains a liquid such as a culture solution. For this reason, for example, as shown in FIG. 1, the end 2a of one electrode 2 (second electrode) is circular, and a part of the other electrode 1 (first electrode), in particular, the tip 1a is the second electrode. An electrode in which two electrodes are fixed to an insulating material 3 (for example, rubber) so as to be near the center of a circle can be used. As shown in FIG. 2, the electrodes 1 and 2 are fixed on a vessel 6 containing cultured cells, and are surrounded by a second electrode 2 having a circular shape 2a and an insulating material 3 that also serves as a support for the electrode. By enclosing the cell 4 in 5 and performing electroporation, the gene can be efficiently introduced into the cell.

  The first electrode and the second electrode are not particularly limited as long as the current can sufficiently flow to the cells with a small amount of solution, but when the first electrode and the insulating support are fixed on the cell culture vessel The cells are confined in a narrow space surrounded by the first electrode, the insulating support, and the container, and a part (particularly the tip) of the second electrode is fixed in the space, so that the cells are efficiently energized. This is illustrated as one of the preferred embodiments.

  The thickness of the second electrode capable of confining cells is about 0.2 to 3 mm, more preferably about 0.5 to 1 mm.

  The distance between the first electrode and a part (tip) of the second electrode is not particularly limited, but is usually about 5 to 20 mm.

  In a preferred embodiment, the insulating material on the second electrode is in contact with the cell so as not to damage the cell, or is fixed at a position close to the cell. The distance between the cells and the insulating material is preferably about 0.5 to 1 mm. When the distance between the cells and the insulating material is shorter, the cells can be efficiently energized.

The voltage used in electroporation can be applied in the range of 10 to 2000 V / cm, and is preferably applied in the range of 50 to 1000 V / cm, more preferably 100 to 500 V / cm in consideration of electrical stimulation to the skin. The energization pattern is not particularly limited, but is preferably an exponential logarithmic pulse or a rectangular pulse, and more preferably a rectangular pulse. Electroporation is effective if it is energized once or more, but the effect increases as the number of times increases. The energization time is preferably about 10 to 20 msec in order to suppress damage to cells due to electrical stimulation.

  After electroporation, gene expression can be performed quickly by adding a medium and incubating.

  According to the present invention, it was observed that the protein as the gene product was expressed within a few hours after gene introduction, preferably within a short time of about 30 minutes to 2 hours.

  By enabling protein expression in such a short time, the function of the protein can be more accurately evaluated.

  In addition, when applied to gene therapy, proteins that are specifically required for affected areas are expressed within tens of minutes for not only chronic diseases but also for sudden illnesses and injuries. Technology can be provided.

Hereinafter, a nerve cell will be described as an example of a cell into which a gene is introduced.
(Materials and methods)
Cell culture
SD rat hippocampus or cortical neurons (E18) were disaggregated in Ca ²⁺ , Mg ²⁺ -free phosphate buffered saline (PBS-). Tissue from embryos was treated with 0.125% trypsin in the same medium for 20 minutes at 37 ° C. After rinsing with DMEM, these tissues were dissociated by gentle dissociation using a disposable plastic pipette. Dissociated neurons on a glass-based dish (Matsunami, Japan) coated with polyethyleneimine with DMEM supplemented with 10% FCS, glucose (10 mM), glutamine (2 mM) and insulin (2 μg / ml) Plated. Neurons were cultured for 10-13 days at 37 ° C. in 5% CO 2/95% air and saturated humidity. Cultures were used for experiments.
Immunohistochemistry Hippocampal neurons grown in glass-based dishes (Matsunami, Japan) were fixed in 4% paraformaldehyde and TBS for 15 minutes at room temperature. The neurons are incubated with anti-rat ferritin polyclonal antibody and then HRP-conjugated anti-rabbit IgG (Panafarm, Japan)
And incubated in PBS. The signal was then visualized by incubation with 3,3'-diaminobenzidine (DAB) and H2O2.
In situ hybridization with cultured hippocampal neurons Cultured neurons were fixed with 4% formaldehyde, treated with 0.2 M HCl for 10 min, 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min, ethanol And dehydrated. Hybridization to 50% formamide, 10 mM Tris (pH 7.6), 200 μg / ml tRNA,
This was carried out at 50 ° C. for 18 hours with a solution containing 1 × Denhardt's solution, 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA and DIG-labeled RNA probe. The 200 bps ferritin heavy chain 3 ′ region was amplified by PCR, subcloned into the pGEM-T vector (PROMEGA, USA), and the probe was synthesized with the DIG RNA labeling kit (Boehringer, Germany). Samples hybridized on slides were washed with 50% formamide in 2x SSC, 2x SSC, 0.2x SSC at 50 ° C. Samples were alkaline phosphatase conjugated anti-DIG Fab fragment (Boehringer,
Germany) and visualized by incubation with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
Plasmid Construction for Translation Analysis A plasmid containing the 5 ′ and 3 ′ UTR sequences of ferritin heavy chain mRNA (Ft5′UTR-d2EGFP-Ft3′UTR) was prepared using pd2EGFP-N1 (Clontech, USA). The 5 ′ UTR of the ferritin H chain was inserted into the NheI and AgeI sites of the plasmid, and the 3′UTR of the ferritin H chain mRNA was inserted into the NotI and BsmI sites. Each insert has the following primers:
NheI, AgeI: 5'-TTGGGGCTAGCCAGACGTTCTCGCCCAGAG3 '(SEQ ID NO: 1), 5'-GGTTACCGGTGATGGCGGCTGGGCAGGG-3' (SEQ ID NO: 2),
NotI, BsmI: Amplified by PCR using 5′-CCTTTGCGGCCGCGCTGACGTCCCCAAGGCCAT-3 ′ (SEQ ID NO: 3), TTTCCGAATGCAGGGTACCAAAATTCTTTATTTGAAG-3 ′ (SEQ ID NO: 4). The plasmid was purified using the QIAprep Spin Mini prep Kit (QIAGEN, Germany) for transfection. Iron Responsive Element (IRE) deficient plasmid (Ft5'UTR (without IRE) -d2EGFP-Ft3'UTR), primers (5'-CCAGACGTTCTCGCCCAGAGGTGCTCGACC-3 '(SEQ ID NO: 5), 5'-CGTCCTTGAAGAAGATGGTGCG -3 ′ (SEQ ID NO: 6)) and purified using the primers (5′-TTGGGGCTAGCCAGACGTTCTCGCCCAGAG-3 ′ (SEQ ID NO: 7), 5′-GGTTACCGGTGATGGCGGCTGGGCAGGG-3 ′ (SEQ ID NO: 8)) Amplified. The PCR product was inserted into the NheI and AgeI sites of the pd2EGFP-N1 plasmid. While observing local translation, maintain the culture dish at 37 ° C and use Tyrode solution (124 mM NaCl, 5 mM KCl, 25 mM Hepes, 1 mM MgCl2, 2 mM CaCl2, 30 mM glucose, pH 7.4) Was continuously poured into the dish at 2 ml / min. As shown in FIG. 5, glutamate (final concentration 100 μM) or xanthine (final concentration 50 μM) and xanthine oxidase (final concentration 0.05 U / ml) after dissociation of the target dendrites in the proximal position using a glass pipette ( Nacalai, Japan) bath-application
To the culture dish. In FIG. 6, glutamic acid (1 mM) was puffed to the target using a glass micropipette (Matsunami, Japan, tip diameter; 1 μm) connected to a pico pump (WPI, USA). Site-directed puff was limited to a 10 μm (radius) region in a control experiment using FITC. After puffing, the pipette was immediately removed to prevent excessive irritation from spilled glutamic acid. The obtained images were analyzed using MetaMorph (Universal Imaging, USA).
Electroporation Electroporation for translation analysis was performed using Square Electroporator CUY21 (BEX, Japan). The culture medium of hippocampal dissociated neurons (10 DIV) was removed by aspiration. Plasmid solution (40 μg / 40 μl PBS-) was added to the neurons and 10 square pulses (300 V, 10 msec) were applied to them. The interval between each pulse was 900 msec.
A Sindbis virus bicistronic vector plasmid (pSinEGdsp) was provided by Dr. Kawamura (Niigata University). The plasmid is derived from pSinRep5 (Invitrogen, USA) and has two subgenomic promoters followed by any gene insertion and a multiple cloning site for EGFP ORF, so the virus can contain any protein in infected cells. EGFP can be produced independently (Kawamura et al., 2003). Ferritin H chain cDNA amplified by RT-PCR using specific primers (5'-TCTAGACGACAGTGCTTGAACGGAACC-3 '(SEQ ID NO: 9), 5'-AGGCCTGGGTACCAAAATTCTTTATTTGAAG-3' (SEQ ID NO: 10)) is Xba1 of the plasmid. Therefore, it was inserted into Stu1 site. The plasmid was cut with Pac1 and mRNA was synthesized in vitro using the mMESSAGE mMACHINE kit (Ambion, USA). Next, Baby Hamster Kidney (BHK) cells were transfected with mRNA and 26S helper mRNA (Invitrogen, USA) by electroporation (1250 V / cm, 50 μF, single pulse) using Gene Pulser2 (BioRad, USA). I did it. Cells were incubated with DMEM supplemented with 10% FCS for 24 hours at 37 ° C., and supernatants were collected as pseudovirion containing solutions.
Cell survival / death assay Neuronal al cell survival was measured by MTT assay according to the original method (Monsmann, 1983; Hansen et al., 1989). Briefly, the tetrazolium salt MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) was added to the culture at a final concentration of 1 mg / ml. After 2 hours incubation at 37 ° C., the assay was stopped by adding 80% by volume lysis buffer (10% SDS in 50% N, N-dimethylformamide, pH 4.7). The absorbance was then measured spectrophotometrically at 570 nm and incubated for 24 hours at 37 ° C. The percentage of viable cells was defined by the following formula:
(A (experiment blank) / A (control blank)) x 100
(Blank values were obtained from wells without cells). Cells undergoing apoptosis were identified by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method according to previous literature (Gavrieli et al., 1992).
Synaptosome preparation Synaptosomes were prepared according to Verity's method (Verity, 1972) with minor modifications. First, day 10 cortical neuron cultures were homogenized with a loose fitted Potter homogenizer (5 ml) in homogenization buffer (0.35 M sucrose, 1 mM MgCl2, 1 mM EDTA, 10 mM Hepes pH 7.4). The homogenate was centrifuged at 2,000 xg for 1 minute and the pellet was discarded.
The supernatant was then centrifuged at 23,000 xg for 5 minutes. 2 ml of the crude mitochondrial pellet obtained
And restratified in a discontinuous gradient (13% and 5% Ficoll in homogenization buffer). The gradient was centrifuged for 40 minutes at 43,000 xg in a Beckman L7 ultracentrifuge equipped with a SW40.1 rotor. Synaptosomes were collected from the 5% -13% interface.
Analysis of reactive oxygen species (ROS) production
Cultured cortical neurons infected with Sindbis virus expressing EGFP and ferritin were harvested and synaptosomal fractions were prepared. The inventor has confirmed that the synaptosomes from ferritin overexpressing neurons have the same protein pattern as the control synaptosomes from neurons infected with Sindbis virus expressing EGFP on SDS-PAGE. The synaptosome fraction was analyzed with an oxyblot kit (Intergen, USA). The carbonyl group of the protein side chain is 2, 4-phenylhydrazine (DNPH)
And derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone). The DNP-derivatized protein was separated by SDS-PAGE and Western blotting using a primary antibody specific for the DNP portion of the protein. The signal was enhanced by the TSA biotin system (Perkin Elmer, USA) and the ECL Plus system (Amersham Bioscience, UK). The chemiluminescent signal was detected and analyzed by LAS-1000 (Fujifilm, Japan) and Image Gauge (Fujifilm, Japan).
(result)
Observation of local translation of ferritin in dendrites To test the hypothesis, the present inventor performed immunohistochemical experiments to observe changes in ferritin protein levels after glutamate stimulation (100 μM, 1 h). Although a slight increase in ferritin signal was observed in the dendrites (FIG. 3), the inventor considered that this was insufficient to conclude that this was solely due to upregulation of ferritin translation levels. This is because the pre-existing ferritin level is unknown. In order to directly confirm ferritin translation in dendrites of cultured neurons, the present inventor has developed a new observation system. In the system, a plasmid containing the sequence of d2EGFP (Clontech, USA) between the 5 ′ and 3 ′ UTRs of ferritin (Ft5′UTR-d2EGFP-Ft3′UTR, FIG. 4a) was constructed and transfected into neurons. It was. Translocation of transcripts to dendrites was confirmed by in situ hybridization using 10-day cultured neurons from rat hippocampus, as shown in our previous report (Ishimoto et al., 2000). (FIG. 4b). Transfected neurons were incubated for 2 hours in the first medium containing cycloheximide, an inhibitor of translation. The transfected neurons were then transferred to a second medium containing the transcription inhibitors actinomycin D and cycloheximide. After incubation for 30 minutes, the transfected neurons were transferred to a third medium containing actinomycin D. In the first medium, the mRNA was transcribed under translational suppression. Actinomycin
In the third medium using D, the fluorescence of d2EGFP was measured as an index for local ferritin translation. Since there was no transcription effect in the third condition, translation effectiveness can be accurately assessed by this system in the third condition (FIG. 4c). After this process, the inventors have confirmed that neurons increase intracellular calcium in response to glutamate application. This means that the neuron maintains viability in the process.

In a control experiment, the present inventors tested whether actinomycin D and cycloheximide inhibit mRNA and protein synthesis. The neurons incubated in the medium without inhibitor 1 hour after electroporation were clearly observed to emit d2EGFP fluorescence (FIGS. 4d, 4e). Incubated with actinomycin D or cycloheximide. The neurons showed no fluorescence even after 6 hours (FIG. 4f,
4g, 4h, 4i). In comparison, neurons incubated for 2 hours in the first medium, 30 minutes in the second medium, and 30 minutes in the third medium showed a significant d2EGFP fluorescence signal (FIG. 4). 4
j, 4k). These signals were considered translation of the d2EGFP protein in the presence of actinomycin D, confirming that this system works properly. Using this system, the inventor tested whether glutamate application induced dendritic ferritin translation. Fluorescence increased significantly in dendrites site-specifically within 20 minutes after application, and the increase was maintained for at least 40 minutes (FIG. 5a). This translational response was similar to glutamate receptor-mediated protein synthesis in subcellular fractions and cultured neurons, but (Weiler., Et al 1993; Scheetz., Et al 2000; Job., Et al 2001) BDNF induction (Aakalu., Et al 2001). In control experiments with mRNA without ferritin UTR, no significant increase in fluorescence intensity was observed at any site (FIG. 5).
b, 5d). In this experiment, the dendrites were cleaved at the proximal point to avoid the effect of d2EGFP synthesized somatically prior to glutamate application. Total fluorescence intensity in Ft5'UTR-d2EGFP-Ft3'UTR transfected dendrites was significantly increased (115% ± 7.7 SD, n = 7), while Ft5'UTR in the presence of cycloheximide (without IRE) -d2EGFP-Ft3'UTR (96.6%
Three types of control experiments with ± 13.1 SD, n = 6), d2EGFP (97.5% ± 6.8 SD, n = 4) and Ft5'UTR-d2EGFP-Ft3'UTR showed no increase (97.5% ± 3.4 SD,
n = 4) (FIG. 5e).
Local ferritin translation induced by local neuronal activity To obtain more direct evidence of local translation at a limited location in response to local synaptic activity, cultured hippocampus treated with inhibitors as in the experiment shown in FIG. Neuronal dendrites were puffed with glutamic acid (1 mM) using a glass micropipette. The fluorescence intensity started to increase within 20 minutes after application at the glutamate puff site (FIG. 6). Such a local increase in intensity was observed in 3.7% (5/136) of the neurons tested.

Direct induction of local ferritin translation by superoxide Reactive oxygen species (ROS) occur after excessive calcium influx into neurons (Atlante et al.
al., 2001) and ROS induces the expression of ferritin (Cairo et al., 1996; Cairo et al., 1998; Orino et al., 2001), indicating that glutamate application at the glutamate synapse site is ROS It suggests that ferritin is induced by cascade. Next, the inventor tested whether ROS directly induced ferritin translation using xanthine / xanthine oxidase, which is known to produce superoxide. Forty minutes after xanthine / xanthine oxidase application, t5′UTR-d2EGFP-Ft3′UTR expressing neurons showed a slight increase in fluorescence in their dendrites. On the other hand, other control dendrites showed a decrease in fluorescence. The decrease in fluorescence may be caused by the destruction of d2EGFP by superoxide.
Effect of Ferritin Overexpression on Neurons To study the biological implications of local ferritin translation, the present inventor designed the following experiment. We tested the effect of overexpressed ferritin protein on excitotoxicity of cultured rat hippocampal neurons using Sindbis virus with two subgenomic promoters for expressing EGFP and ferritin. Since the infected cells simultaneously expressed ferritin and EGFP, the ferritin-expressing neurons were identified by fluorescence microscopy as d2EGFP-expressing neurons. About 65% of neurons were infected with this viral vector. Ferritin expressing neurons were more resistant to glutamate toxicity in four types of studies of neuronal survival, cell viability and apoptotic cell death (FIG. 8). In each experiment, overexpressed ferritin significantly reduced excitotoxicity. This neuroprotective effect of overexpressed ferritin is thought to be caused by suppression of the Fenton reaction that produces hydroxyl radicals, and the present inventors tested whether ferritin overexpression reduces oxidized protein at the synapse. The present inventor has found that there is no difference in infection rate between ferritin-expressing cells (FIG. 9a) and cells expressing only EGFP (FIG. 9b). Using the oxyblot kit, the inventors confirmed a significant decrease in the amount of protein oxidized in the synaptosomes of ferritin-overexpressing neurons (FIG. 9c).
(Discussion)
IRE-dependent rapid induction of ferritin translation in dendrites is initiated by neuronal activity.

The inventor has hypothesized that ferritin translated in dendrites inhibits hydroxyl radical production by capturing iron ions in response to synaptic activity. To test this hypothesis, the present inventors used antibodies to identify increased levels of ferritin protein after glutamate stimulation. Although this result is consistent with the hypothesis, further evidence was needed for the final conclusion. In the next step, the inventor attempted to observe whether local ferritin translation was induced by glutamate stimulation in primary cultures of hippocampal neurons. Aakalu et al. Developed a method to observe translation in dendrites. They found that BDNF application induced CaMK2 translation in hippocampal primary culture (Akalu et al., 2001). The inventors have improved their method using electroporation, cycloheximide and actinomycin D to minimize the effects of pre-existing d2EGFP and changes in transcription efficiency.
In our observation system, d2EGFP fluorescence increased after glutamate stimulation in Ft5′UTR-d2EGFP-Ft3′UTR expressing neurons. Control neurons expressing Ft5′UTR (without IRE) -d2EGFP-Ft3′UTR, d2EGFP showed no increase in fluorescence. Furthermore, neurons transfected with Ft5′UTR-d2EGFP-Ft3′UTR and cultured with cycloheximide did not show any increase.

Since it is well established that ferritin translation is controlled by IRE-IRP interactions, an increase in fluorescence is considered an activity dependent on local ferritin translation. Weiler et al. Reported that mRNA in synaptoneurosomes rapidly binds to ribosomes in response to stimuli to become polysomes (Weiler and Greenough, 1993). Scheetz et al. It was demonstrated to induce CaMK2 protein translation within minutes (Scheetz et al., 2000). Similar rapid local translation was found in dendrites stimulated by (RS) -3,5-dihydroxyphenylglycine 0.5H2O (DHPG) (Job et al., 2001; Eberwine et al., 2001) . Our results have shown that ferritin translation is induced within 20 minutes, so these relatively rapid responses may be a common feature of neurotransmitter-induced local translation, and this local translation Allows synapses to rapidly change their constituent molecules in response to changes in environmental conditions. The inventor has found that the fluorescence in the dendrites is not a uniform increase as shown in FIG. Some restricted sites showed a significant increase compared to others. Similar phenomena have been reported (Akalu et al., 2001; Job et al., 2001; Eberwine et al., 2001), where the site was named a hot spot that correlates with ribosome distribution. In the case of ferritin, the present inventors have found that mRNA is not transported uniformly to dendrites (Ishimoto et al., 2000), so the uneven increase in fluorescence is due to the uneven distribution of ribosomes and ferritin mRNA. Can be explained by both. In FIG. 6, we clearly demonstrated that local stimulation induced local ferritin translation. Although an increase in fluorescence was observed within 20 minutes, the actual time required for translation may be shorter than 20 minutes. This is because GFP emits fluorescence after its folding. Some of the low success rates of translation induction by locally puffed glutamate may be due to stimulation outside the hot spot.
Superoxide produced after neuronal excitation is a direct inducer of ferritin translation.

ROS is produced by neuronal excitation (Atlante et al., 2001) and superoxide and hydrogen peroxide release IRPs from IRE (Cairo et al., 1996; Cairo et al., 1998; Orino et al., 2001) ). The inventor observed that application of xanthine and xanthine oxidase as a superoxide generation system slightly increased the fluorescence in the dendrites, while other control experiments showed a decrease in fluorescence. This result suggests that superoxide and hydrogen peroxide produced by xanthine and xanthine oxidase induced translation. The fluorescence decrease may be due to d2EGFP degradation caused by ROS.
Local ferritin translational function to protect neurons from hydroxyl radical attack Overexpression of ferritin apparently reduced glutamate toxicity. The protective effect was caused by inhibition of hydroxyl radical production after calcium influx, which is considered to be an initiator of glutamate toxicity. The synaptosome fraction from ferritin overexpressing cortical cultures shows a significant decrease in the amount of oxidative protein. These results are consistent with the report that ferritin heavy chain deficient mice show increased oxidized protein in the brain (Thompson et al., 2003).

Since the brain consumes a large amount of oxygen, it tends to generate ROS. There are scavenger systems that contain superoxide dismutase (SOD), glutathione, etc. to suppress their production. However, constitutive high expression of these molecules in neurons adversely affects neuronal or synaptic cell viability or LTP inducibility (Thiels et al., 2000; Jaarsma et al., 2000). In the case of ferritin, constitutive high expression was reported to result in a delay in non-neuronal cell growth (Guo et al., 1998; Cozzi et al., 2000). IRP2-deficient mice that express ferritin in higher amounts than wild type showed motor impairment and neurodegeneration (La Vaute et al., 2001). A relationship between cellular iron levels and Parkinson's disease and Alzheimer's disease has been suggested (Thompson et al., 2001). It is interesting whether local ferritin translation affects the brains of patients with Parkinson's disease or Alzheimer's disease. It may lead to the discovery of the cause and treatment of the disease. The local activity dependence and transient expression of ferritin in response to cellular stimuli is probably a sensible mechanism for maintaining normal synaptic function.
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An example of the electrode used for the electroporation apparatus of this invention is shown. The schematic diagram when a gene is introduce | transduced in a cell with the electroporation apparatus of this invention is shown. Immunostaining of cultured hippocampal neurons after stimulation with glutamate. Glutamate stimulation (100 μM, 1 h), neurons (a) and control neurons (b) were fixed and immunostained with anti-ferritin antibody. Bar indicates 10 μm. Detection system for local protein synthesis in dendrites. (a) mRNA used for the system. The d2EGFP ORF is flanked by the 5 ', 3' UTR of the ferritin heavy chain. (b) In situ hybridization for transgenic mRNA (Ft5'UTR-d2EGFP-Ft3'UTR) in cultured hippocampal neurons (10 DIV). Arrowheads indicate mRNA signals. (c) Observation scheme for local transfection. After transfection, neurons were incubated with cycloheximide for 120 minutes. Observation of fluorescent d2EGFP was started 180 minutes after transfection in the presence of actinomycin D. (d) Control experiment. Cells were incubated with FCS-containing DMEM for 1 hour after transfection. (e) D bright field image. (f) Control experiment. Cells were incubated with cycloheximide-containing medium for 6 hours after transfection. (g) F bright field image. (h) Control experiment. Cells were incubated with actinomycin D-containing medium for 6 hours after transfection. (i) Bright field image of h. (j) Neurons processed by C scheme. Cells were incubated with cycloheximide-containing medium for 2 hours, then with cycloheximide and actinomycin D-containing medium for 30 minutes, and finally with actinomycin D-containing medium for 1 hour. (k) J bright field image. All bars show a length of 10 μm. Local ferritin transfection induced by a bath-application of glutamate. (a) Ft5′UTR-d2EGFP-Ft3′UTR-transfected neurons pseudo-colored fluorescence image. Neurons were processed as shown in Figure 4c. After the dendrite dissection at the proximal location, glutamate was applied at a final concentration of 100 μM. Images were taken at 0, 20, and 40 minutes after application. Bar indicates 10 μm. (b) Control experiment using d2EGFP. (c) Ratio of fluorescence signal at 40 minutes after glutamate application to 0 minute fluorescence signal at each part of the dendrite. Signal ratio increases non-uniformly in dendrites. (d) Fluorescence ratio in a control experiment using d2EGFP. (e) Average profile (± S.D.) of fluorescence change after glutamic acid application. Ft5'UTR-d2EGFP-Ft3'UTR transfected dendrites show a significant increase in fluorescence (n = 7), while Ft5'UTR (without IRE) -d2EGFP-Ft3'UTR (n = 6 ), d2EGFP (n = 4) and cycloheximide together with Ft5'UTR-d2EGFP-Ft3'UTR (n = 4) showed no significant increase. * P <0.05, (Student t-test). Local transfection induced by glutamate application in a limited area. Fluorescence image of Ft5'UTR-d2EGFP-Ft3'UTR transfected neurons. Neurons were processed as shown in Figure 4c. Under the microscope, the dendrites were stimulated by glutamate puff at the site indicated by the arrow. Images were taken at 0, 10, 20, and 40 minutes after stimulation. The image was fake-colored. Bar indicates 10 μm. Local ferritin transfection induced by superoxide. The effect of superoxide on ferritin local transfection was investigated. Xanthine (50 μM) and xanthine oxidase (0.05 u / ml) were added to produce superoxide. (a) Pseudo-colored fluorescence image of Ft5′UTR-d2EGFP-Ft3′UTR transfected neurons. Neurons were processed as shown in Figure 4c. Images were taken at 0, 20, and 40 minutes after application. Bar indicates 10 μm. (b) Control experiment using d2EGFP. (c) Ratio of fluorescence signal at 40 minutes after glutamate application to 0 minute fluorescence signal at each part of the dendrite. (d) Fluorescence ratio in a control experiment using d2EGFP. (e) Average profile of fluorescence change after application of xanthine and xanthine oxidase (± S.D.). Ft5'UTR-d2EGFP-Ft3'UTR transfected dendrites show a slight increase in fluorescence (n = 9), while Ft5'UTR (without IRE) -d2EGFP-Ft3'UTR (n = 5 ), d2EGFP (n = 4) and cycloheximide together with Ft5'UTR-d2EGFP-Ft3'UTR (n = 5) showed no significant increase. * P <0.05, (Student t-test). Protective effect of ferritin against glutamate toxicity. Glutamate toxicity was assayed by four methods. Hippocampal neurons from E18 rats were cultured for 7 days and infected with EGFP alone or Sindbis virus expressing both EGFP and ferritin. After infection, glutamate (100 μM, 1 hour) was applied to the neurons. The assay was performed for 1 hour after application. In each panel, the bar indicates the value of the glutamate-treated culture as a percentage of the untreated culture. (a) Neuronal cell death after glutamate application was measured by counting MAP2-positive cells. (b) Neuronal cell death after glutamate application was measured by counting EGFP positive cells. (c) MTT-reducing activity was measured after glutamate stimulation. (d) TUNEL-positive cells were counted as an index of apoptotic cells. The vertical axis shows the percentage of control experiments that were not given glutamate stimulation. Oxidized protein levels at synaptic sites reduced by ferritin expression. (a) Sindbis virus-infected cells expressing EGFP and ferritin heavy chain. (b) Sindbis virus-infected cells that express EGFP. Bar indicates 10 μm. (c) Synaptosome fractions prepared from virus-infected cultured cortical neurons are analyzed by Western blotting using an oxyblot kit. The vertical axis represents the relative amount of oxidized protein (± S.D.).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st electrode 1a 1st electrode tip 2 2nd electrode 2a Circular part 3 of 2nd electrode 3 Insulating material 4 Cell 5 Space 6 Container

Claims (6)

A method for introducing a gene into cells by applying a solution containing the gene to the cells after removing the cell culture medium and performing electroporation. The method according to claim 1, wherein the gene introduced into the cell is applied to the cell as a 20 to 100 μl solution. The method according to claim 1 or 2, wherein the length of the electric pulse is 10 to 20 msec and the number of application times of the electric pulse is 5 to 20. The method according to any one of claims 1 to 3, wherein the amount of the medium is 200 to 300 µl per 1 cm ^{2 of the} container containing cells. An electroporation apparatus having at least two electrodes, wherein a part of a second electrode is fixed in a plane surrounded by the first electrode, and a current flowing between the first electrode and a part of the second electrode is a gene. A device characterized by being energized to a cell into which is introduced. The apparatus according to claim 5, wherein the first electrode is a circular electrode, and the tip of the second electrode exists near the center of a circle surrounded by the first electrode.