JP2009118745A - Method for transferring gene to eukaryotic cell by utilization of conjugation between living world, eukaryotic cell obtained by the method, and kit for transferring the gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for transferring a gene to an eukaryotic cell by the utilization of a conjugation between living worlds, by which a gene transfer rate using the conjugation between the living worlds can be improved, while conventional gene transfer methods have a problem that gene transfer rates are low. <P>SOLUTION: Provided is the method for transferring the gene, by manipulating the functions of mitochondoria with a mitochondoria function inhibitor such as Antimycin A, Oligomycin or Erythromycin, and improving the gene transfer rate using the conjugation between the living worlds. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a simple and efficient method for introducing a gene into a eukaryotic cell using inter-biojunction, a eukaryotic cell obtained from the method, and a kit for gene transfer thereof.

  Gene transfer refers to introducing a cloned vector into another organism that is not the host of the target gene using an experimental system of the gene and directing the gene to express an effective trait. As a specific method, Examples include a lipofection method or a liposome transfection method, an electroporation method, a method using a viral vector, and a microinjection method.

  The lipofection method or liposome transfection method is one of the transfection methods for introducing genetic material into cells. The genetic material is encapsulated inside a phospholipid bilayer vesicle called a liposome. It passes through the cell membrane. Specifically, a method in which a complex is formed by an electrical interaction between a lipid bilayer vesicle (liposome) composed of positively charged lipid and the like and DNA to be introduced, and is taken into a cell by phagocytosis or membrane fusion. It differs from the liposome method in that DNA is not necessarily encapsulated in the liposome.

  As an advantage of the lipofection method, it is possible to introduce oligonucleotides, polymer DNA, mRNA and double-stranded RNA with high transfection efficiency. Since the operation is completed easily and in a short time, a large number of samples can be processed simultaneously. Moreover, no special equipment / equipment is required. And since it is less toxic to cells than other methods, the cell viability after transfection is high, and it can be used for both transient expression and stable expression. Finally, it can be applied to a wide range of cells. However, as a disadvantage, it is necessary to study optimum conditions such as cell density, DNA amount, liposome amount, culture time, and culture medium for transfection, and the range of optimum conditions is narrow. In general, the reagent is expensive.

  Next, the electroporation method is a method in which DNA is incorporated by transiently destabilizing the cell membrane structure of the lipid bilayer by applying a high voltage pulse to the cells. The efficiency of transfection depends on the conditions of voltage, electric pulse length, temperature, cell and DNA concentration, and buffer composition. The advantages of this method are that it is easy to operate and has high gene transfer efficiency. However, it requires an expensive machine, and only one sample can be transferred with a single electric pulse. There are drawbacks such as time-consuming processing.

  The viral vector method uses the method of infecting cells with viruses, and the viral vector envelope adsorbed by cell membrane fusion fuses with the cell membrane of the cell, and the vector inside the virus is sent into the cell and the gene It is a method to introduce.

  Furthermore, although there is a microinjection method, a method for introducing a sample into one cell through a fine glass injection needle. Although the principle is simple, it requires advanced technology. In order to minimize the damage to the cells, it is necessary to use the finest needle possible and inject the required amount in a very short time. The advantage of this method is that it can be introduced only into specific cells and separately into the nucleus and cytoplasm. In addition, the type of molecules (nucleic acid, protein, etc.) and size of the solution are not limited, and there are advantages such as high introduction efficiency and a very small amount of sample solution. However, special devices and techniques are required, and application to suspension cells is difficult. And there is a disadvantage that the number of target cells is limited.

  However, in all of the gene introduction methods described above, plasmid DNA must be extracted and adjusted from normal Escherichia coli and the like, and there are problems that many operation steps are required and time and cost are excessive.

  Therefore, there is a biogenetic junction method as a gene introduction method that does not require extraction and adjustment of plasmid DNA from E. coli or the like and does not require extra time and cost.

Biological junction is the first phenomenon reported by Heinenmann and Sprague in 1989 using Escherichia coli and budding yeast.
. This phenomenon is that the plasmid transfer system, which is essentially a gene transfer system between bacteria, can transmit the plasmid from bacteria to eukaryotes with a low frequency. From the late 1980s to the early 1990s, research was carried out to apply this phenomenon to eukaryotic gene transfer methods, but the gene transfer efficiency could not be increased to a practical level. The gene transfer method using the above has been replaced by the one using the Agrobacterium T-DNA transfer system. In recent years, Waters has reported successful gene transfer from Escherichia coli to animal cultured cells by inter-organism junctions. Although the potential for its potential application has been shown, it has become a dormant technique.

So far, research on the junction between organisms has been limited mainly to the modification of gene transfer vectors, and has not yet reached the level of practical gene transfer efficiency.
Heinemann, JA and Sprague GF Jr. Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast.Nature 340, 205-209 (1989). Waters, VL Conjugation between bacterial and mammalian cells. Nat. Genet., 29, 375-376 (2001). Kawai, S., Pham, TA, Nguyen, HT, Nankai, H., Utsumi, T., Fukuda, Y. and Murata, K. Molecular insights on DNA delivery into Saccharomycescerevisiae. Biochem. Biophys. Res. Commun. 317,100- 107 (2004). Ito, H., Fukuda, Y., Murata, K. and Kimura, A. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168 (1983). Nishikawa, M., Suzuki, K. and Yoshida, K. DNA integration into recipient yeast chromosomes by trans-kingdom conjugation between Escherichia coliand Saccharomycescerevisiae. Curr. Genet., 21,101-108 (1992).

  Therefore, it is a problem that the gene transfer rate by the junction between organisms is lower than the conventional gene transfer method, and it is a problem to improve the gene transfer rate. Furthermore, in order to establish the inter-biojunction as a practical gene transfer method, gene transfer efficiency must be increased.

  The present invention has been made in view of the above problems, and the object thereof is obtained from a method for amplifying the efficiency of gene transfer into a eukaryotic cell using a junction between organisms as a practical gene transfer method and the method. Another object of the present invention is to provide a eukaryotic cell and its gene transfer kit.

  The function of accepting eukaryotic cells to reject conjugation has not been studied so far, searching for genes that function to refuse conjugation, destroying them, or inhibiting the expression or function of these genes with drugs, etc. I thought that this could increase the efficiency of gene transfer.

  Thus, as a result of intensive studies in view of the above problems, the present inventors have found that the efficiency of gene introduction by inter-organism binding can be improved by manipulating mitochondrial functions for eukaryotic cells, and the present invention. It came to complete. That is, the present invention includes the following industrial fields useful invention.

  (1) A gene introduction method using inter-organism junctions, characterized in that in the method of gene introduction from E. coli to eukaryotic cells, the function of mitochondria of eukaryotic cells is manipulated.

  (2) The gene introduction method using junction between organisms according to claim 1, wherein the operation of the function of mitochondria according to (1) is performed using a mitochondrial function inhibitor.

  (3) The gene introduction method using the junction between organisms according to claim 2, wherein the mitochondrial function inhibitor according to (2) is Antimycin A, Oligomycin and Erythromycin.

  (4) The gene transfer method using inter-organism junction according to any one of claims 1 to 3, wherein the eukaryotic cell according to (1) is yeast.

  (5) A eukaryotic cell into which a gene has been introduced by the method according to any one of (1) to (4).

  (6) The eukaryotic cell according to (5) is a yeast, wherein the gene is introduced by the method according to any one of claims 1 to 4.

  (7) A gene introduction rate amplification kit for introducing a gene by the method according to any one of (1) to (4).

  The method for improving the gene transfer rate according to the present invention is a method for improving the gene transfer rate by inter-biojunction as described above, and is characterized by manipulating the function of mitochondria in eukaryotic cells.

  Therefore, by manipulating the mitochondrial function of eukaryotic cells, the rate of gene transfer for inter-organism junctions can be improved, and the number of commonly used E. coli steps can be reduced.

Manipulating the mitochondrial function more specifically has the effect of increasing the gene transfer rate due to the junction between organisms by inhibiting the mitochondrial function of eukaryotic cells using a mitochondrial function inhibitor.
That is, by using the method of the present invention, a useful gene can be directly and efficiently introduced into a eukaryote such as yeast from a biological material that is used for cloning of a useful gene in biotechnology, such as E. coli. It becomes possible, and it has the effect of amplifying the efficiency of gene transfer into eukaryotic cells using the junction between organisms as a practical gene transfer method.

Hereinafter, the present invention will be described in detail. However, the scope of the present invention is not limited to these descriptions, and modifications other than the following examples can be made as appropriate without departing from the spirit of the present invention. Moreover, all the well-known literatures described in this specification are used as reference in this specification.

<1. Definition of terms of the present invention>
In the present invention, a eukaryotic cell refers to a cell having a structure called a cell nucleus in the cell and separated from the other part of the cell by a membrane (nuclear membrane), such as yeast, fungi, plant and Refers to animal cells.

  In the present invention, gene transfer refers to introducing a cloned vector into another organism that is not the host of the target gene and directing the gene to express an effective trait, and a method for gene transfer The term “method” refers to a method in which a vector obtained by cloning a target gene into another organism that is not a host is introduced, and the gene is directed to the direction where an effective trait can appear.

  Further, in the present invention, mitochondria are organelles contained in almost all eukaryotic cells, and are organs that produce energy by β-oxidation of fatty acids or oxidative phosphorylation by an electron transport system. It is.

  And manipulating mitochondrial function means function inhibition by mitochondrial function inhibitors, culture under anaerobic conditions, introduction of mutation into mitochondrial genome, introduction of mutation into mitochondrial protein gene, induction of expression of mitochondrial protein gene This refers to the destruction of transcriptional regulator gene.

  In addition, mitochondrial function inhibitors that inhibit the function of the organelles of the mitochondria include Antimycin A, Oligomycin, Erythromycin, Chloramphenicol, Mitomycin C, Rutamycin, Ethidiumbromide, 5-iododeoxyuridine, Trichosporin-B class, Enniatin A, Valinomycin , Gramicidin, Nigericin, Dianemycin, Mikamycin.

  In the present invention, the junction between organisms is an interesting phenomenon in which Escherichia coli, which is a prokaryote, and eukaryotes join and transduce genes across the organism.

  Then, by using the technique of the present invention, a step of preparing plasmid DNA as compared with conventional gene transfection methods such as lipofection method or liposome transfection method, electroporation method, viral vector method and microinjection method. The gene can be directly introduced from E. coli into eukaryotic cells without requiring extra time and cost.

  That is, it is possible to introduce a gene simply by mixing E. coli and eukaryotic cell suspension.

  Furthermore, the gene transfer rate amplification kit is characterized by including the gene transfer method using the junction between organisms of the present invention, and the kit of the present invention is a gene transfer method by junction between organisms that improves gene transfer efficiency. Any other configuration may be included as long as the configuration can be implemented. For example, a buffer containing a function inhibitor of the inter-organism junction suppressor gene such as a mitochondria function inhibitor of eukaryotic cells, a pack containing an oxygen absorber for anaerobic culture, a mitochondrial function gene by RNAi or antisense RNA, etc. Inhibition of gene expression that suppresses junctions between organisms.

<2. Selection method of strain with increased bioa conjugation efficiency of the present invention>
In the present invention, the biosphere conjugation reaction is carried out by using a donor plasmid containing an oriT sequence of an Inc Q-type plasmid such as pAY205, or a mob gene group, or a donor plasmid having an oriT sequence of an Inc P-type plasmid, for example, Pre-cultured in E. coli containing a tra gene group such as a strain in the genome or E. coli holding a helper plasmid containing a tra gene group such as pRH210, pRH220.

  Then, the pre-cultured E. coli is reacted with each budding yeast knockout mutant in TNB buffer.

  Non-essential gene knockout mutant kit (Yeast Deletion Clones (Mat-Alpha Complete Set), manufactured by Invitrogen) made from α-type budding yeast BY4742 or BY4739, or a-type budding yeast BY4741 or BY4730 Using the non-essential gene knockout mutant kit (Yeast Deletion Clones (Mat-a Complete Set), manufactured by Invitrogen), genome-wide screening of inter-organism junction inhibition genes is performed.

  Screening is performed three times in the selection of the strain with increased bioa conjugation efficiency of the present invention.

  In the first screening, the reaction mixture was spotted on a selective medium (SC-Ura) plate after the inter-organism junction reaction, and the number of yeast colonies whose URA3 gene was complemented by the transfer of pAY205 and the ability to synthesize uracil was recovered. Thus, a mutant strain in which an increase in the number of colonies of 4 times or more compared to the control is observed is obtained.

  Next, in the second screening, the mutant strain obtained in the first screening is further subjected to two conjugation reactions, and the average value of the total three conjugation reactions is 8 times more colonies than the budding yeast strain. A mutant strain in which an increase was observed was obtained.

  In the third screening, the mutant obtained in the second screening is subjected to three more biological junction reactions, and half the amount of the conjugation reaction solution is selected in a selective medium (SC-Ura) plate, 1/5000. The diluted solution corresponding to the amount was spotted on a complete medium (YPD + Chloramphenicol) plate supplemented with antibiotics for sterilization of Escherichia coli, and the number of transconjugant and recipient cells was counted to determine the interconjugation efficiency between organisms.

  In the second screening, the third screening, or either, a mutant strain with increased bioa conjugation efficiency in which an increase of 16 times or more is observed as compared with the control budding yeast is obtained.

  We also succeeded in obtaining mutant strains with higher efficiency than 1 transconjugant / 500 recipient yeast cells (Fig. 2).

  In a complementation experiment by producing a diploid with a control budding yeast, all mutations were complemented, and the result returned to the wild-type conjugation efficiency. That is, from the above results, it was proved that the disrupted strain of the inter-organism junction inhibiting gene is a useful recipient strain by the gene transfer method using inter-organism junction.

<3. Method for Increasing Biological Junction Efficiency Using Mitochondrial Function Inhibition of the Present Invention>
According to the method for selecting strains with increased bioa conjugation efficiency of the present invention in 2, the mutant strains with increased bioa conjugation efficiency selected include a mitochondrial protein gene deficient strain of the nuclear code.

  Therefore, in order to manipulate the function of mitochondria, mitochondrial function inhibitors Antimycin A, Oligomycin, Erythromycin, Chloramphenicol, Mitomycin C, Rutamycin, Ethidiumbromide, 5-iododeoxyuridine, Trichosporin-B, Enniatin A, Valinomycin, Gramicidin, Nigericin, Dianemycin Add Mikamycin (.

  An increase in the efficiency of conjugation between biospheres was observed in yeasts of α type (BY4742) and a type (BY4741), which are budding yeast (FIG. 3).

  Antimycin A was pre-cultured on a YPD plate at 28 ° C. for 2 days. Saccharomyces cerevisiae is preferably planted on an YPD plate supplemented with Antimycin A in a range of 2.5 μM to 40 μM, more preferably 5 μM to 20 μM, and even more preferably 7.5 μM to 15 μM, and further at 28 ° C. Culture was performed for 1 day. 2.5 μM or less is not preferable because mitochondrial function is insufficiently inhibited, and 40 μM or more is not preferable because the influence of cytotoxicity by the drug becomes strong. And it planted to the YPD plate which added Antimycin A, and also culture | cultivated at 28 degreeC for 1 day.

  The biosphere conjugation reaction was carried out using yeast after antimycin A treatment, and the interorganism conjugation efficiency was determined in the same manner as in the third screening. As a result, the biological junction efficiency can be increased compared to the control at the set treatment concentration.

  Oligomycin was precultured on a YPD plate at 28 ° C. for 2 days. Oligomycin was added in the range of preferably 2 mg / ml to 32 mg / ml, more preferably 4 mg / ml to 16 mg / ml, and even more preferably 6 mg / ml to 12 mg / ml. It was planted on a YPD plate and further cultured at 28 ° C. for 2 days. 2 mg / ml or less is not preferable because mitochondrial function is insufficiently inhibited, and 32 mg / ml or more is not preferable because the effect of cytotoxicity by drugs becomes strong. Then, it was planted on a YPD plate supplemented with Oligomycin and further cultured at 28 ° C. for 2 days.

  Bioligation reaction was carried out using yeast after Oligomycin treatment, and the efficiency of bioligation was determined by the same method as 3rd screening. As a result, an increase in the efficiency of budding yeast between the organisms was observed compared to the control at the set treatment concentration.

  Erythromycin was pre-cultured on a YPD plate at 28 ° C. for 2 days. Erythromycin was added in the range of preferably 0.2 mg / ml to 12.8 mg / ml, more preferably 0.4 mg / ml to 6.4 mg / ml, and even more preferably 0.8 mg / ml to 3.2 mg / ml. It was planted on a YPD plate and further cultured at 28 ° C. for 2 days. 0.2 mg / ml or less is not preferable because of insufficient mitochondrial function inhibition, and 12.8 mg / ml or more is not preferable because the effect of cytotoxicity by drugs becomes strong. Then, it was planted on a YPD plate supplemented with erythromycin and further cultured at 28 ° C. for 2 days.

  Biological conjugation reaction was performed using yeast after erythromycin treatment, and bioconjugation efficiency was determined by the same method as 3rd screening. As a result, an increase in the efficiency of budding yeast between the organisms was observed compared to the control at the set treatment concentration.

  As described above, it has been proved that the addition of a function inhibitor to an inter-organism junction-inhibiting gene is a useful method for increasing gene introduction efficiency in gene introduction using inter-organism junction.

<4. Gene transfer efficiency of the present invention over conventional gene transfer>
The introduction efficiency of the simple PEG method, which is the simplest gene introduction method, has been found among the 5000 strain mutants that can be introduced with 102 transformants / μg DNA / 107 recipient yeast cells.
. Naturally, this system requires DNA extraction from E. coli.

  On the other hand, in the present invention, the mutant strain having the highest conjugation efficiency can be converted to 2 × 10 4 transformants / 3.75 × 10 7 donor E. coli cells / 107 recipient yeast cells when converted to the above efficiency standard, and it is clear that it is excellent.

On the other hand, although the operation is more complicated than the simple PEG method, the most commonly used lithium acetate method has a relatively high gene transfer efficiency, and the transfer efficiency is about 4 × 10 2 transformants / μg DNA / 106 recipient yeast cells.
. This system also requires DNA extraction from E. coli.

  On the other hand, by using the inter-organism junction for the yeast pretreated with the mitochondrial function inhibitor of the present invention, the gene transfer efficiency is higher than that of the conventional method even in a yeast that is not a mutant with an increased interorganism junction efficiency. That is, about 2 μg of plasmid DNA can be obtained from a culture solution of about 2 ml of a high-copy plasmid (generally used) in E. coli. This culture solution contains at least 2.5 × 10 8 E. coli cells. From this, the amount of plasmid DNA that can be extracted from the 3.75 × 10 6 E. coli cells that are usually used for gene transfer by inter-biojunction is converted to about 0.03 μg. Therefore, when converted in terms of μg DNA, our mutants with increased biology conjugation efficiency are about 6.7 × 10 4 transformants / μg DNA / 3.3 × 107 recipient yeast cells. If treated well, it would be about 7x103 transformants / μg DNA / 3.3x107 recipient yeast cells.

  That is, by using the present invention, simplicity, multi-sample operability, and transformation efficiency are established, and it is shown that the method is comprehensively superior to conventional gene transfer methods.

Although this invention is demonstrated more concretely based on an Example and FIGS. 1-5, this invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

<Method for selecting strain with increased biological efficiency of the present invention>
Biological junction between E. coli and yeast
Was used as a model system (FIG. 1).

  The inter-organism conjugation reaction was performed by dispensing 3.75 × 10 6 cells of E. coli HB101 carrying the helper plasmid pRH210 and the donor plasmid pAY205 into a 96-well microtiter plate, and pre-cultured on a YPD plate (28 ° C., 2 days). Saccharomyces cerevisiae knockout mutant 5 × 105 or more and 10 × 105 or less mixed with cells and in 28 μl of TNB buffer (80 mM Tris-HCl (pH 7.5), 0.05% NaCl) at 28 ° C. for 1 hour I did it.

  Using a non-essential gene knockout mutant kit (Yeast Deletion Clones (Mat-Alpha Complete Set), manufactured by Invitrogen) consisting of 4828, produced from the α-type budding yeast BY4742 Genome-wide screening for inhibitory genes was performed.

  In the first screening, the reaction mixture was spotted on a selective medium (SC-Ura) plate after the inter-organism conjugation reaction, and the number of yeast colonies whose URA3 gene was complemented by the transfer of pAY205 and the uracil synthesis ability was recovered was counted. did. In comparison with control BY4742, 341 mutant strains were observed in which an increase in the number of colonies of 4 times or more was observed.

  In the second screening, the mating strain obtained in the first screening was further subjected to two conjugation reactions, and an increase in the number of colonies of 8 times or more of BY4742 was observed in the average value of the total three conjugation reactions. 99 mutant strains were obtained.

  In the third screening, the mutant obtained in the second screening is subjected to three more biological junction reactions, and one-half of the conjugation reaction solution is added to a selective medium (SC-Ura) plate, 1/5000. The diluted solution corresponding to the amount was spotted on a complete medium (YPD + Chloramphenicol) plate supplemented with antibiotics for sterilization of Escherichia coli, and the number of transconjugant and recipient cells was counted to determine the interconjugation efficiency between organisms. In either the second screening, the third screening, or any of them, 22 strains (22 genes) in which the increase in inter-organism junction efficiency is observed, which is observed to be 16 times or more higher than the control BY4742 strain, are obtained, It succeeded in obtaining the mutant which shows high efficiency more than 1 transconjugant / 500 recipient yeast cells (FIG. 2).

  In a complementation experiment by producing a diploid with a-type yeast (BY4741), all 22 mutations were complemented and returned to wild-type conjugation efficiency.

  From the above results, it was proved that the disrupted strain of the inter-organism junction-inhibiting gene is a useful recipient strain by the gene transfer method using inter-organism junction.

<Method for Increasing Biological Junction Efficiency Using Mitochondrial Function Inhibition of the Present Invention>
Among the 22 strains with increased biological junction efficiency, 8 nuclear-encoded mitochondrial protein gene-deficient strains were included. An increase in the inter-organism conjugation efficiency of both type (BY4742) and type a (BY4741) yeast was observed. The details are described below.

  For antimycin A, BY4742 and BY4741 strains precultured at 28 ° C for 2 days on YPD plates were planted on YPD plates supplemented with Antimycin A in the range of 40 µM or less, and 1 at 28 ° C. Cultures were performed for days.

  Biological conjugation reaction was carried out using yeast after antimycin A treatment, and the conjugation efficiency between the biological kingdoms was obtained by the same method as 3rd screening. As a result, as shown in FIG. 3, at the treatment concentration of 10 μM, an increase in the biojunction efficiency was observed for BY4742 by 4.9 times and BY4741 by 3.9 times compared to 0 mg / ml.

  For Oligomycin, BY4742, BY4741 strains that had been pre-cultured on a YPD plate for 2 days at 28 ° C were planted on an YPD plate supplemented with Oligomycin in a range of 8 µg / ml or less. Cultures were performed for days.

  Bioligation reaction was performed using yeast after treatment with Oligomycin, and the efficiency of bioligation was determined by the same method as 3rd screening. As a result, as shown in FIG. 4, an increase in biojunction efficiency was observed 4.7 times for BY4742 and 2.8 times for BY4741 compared to 0 mg / ml at a treatment concentration of 8 μg / ml.

  For erythromycin, BY4742, BY4741 strains precultured at 28 ° C for 2 days on YPD plates were planted on YPD plates supplemented with erythromycin in the range of 6.4 mg / ml or less. Cultures were performed for days.

  Biological conjugation reaction was performed using yeast after erythromycin treatment, and bioconjugation efficiency was determined by the same method as 3rd screening. As a result, as shown in FIG. 5, an increase in the biojunction efficiency was observed for BY4742 by 6.8 times and BY4741 by 6.6 times compared to 0 mg / ml at a treatment concentration of 1.6 mg / ml.

  As described above, it has been proved that the addition of a function inhibitor to an inter-organism junction-inhibiting gene is a useful method for increasing gene introduction efficiency in gene introduction using inter-organism junction.

  As described above, in the present invention, since the gene transfer rate can be improved by inhibiting the function of mitochondria in eukaryotic cells, a useful gene can be directly and simply introduced with high efficiency.

  Gene transfer is performed in all fields of medicine, pharmacy, agriculture, fermentation engineering, and general biology, regardless of basic / application fields. Therefore, the present invention can be used for gene transfer into eukaryotic cells. It may be widely used as an inexpensive and simple method.

  Furthermore, there is no need for plasmid DNA preparation, especially when gene introduction of a large number of samples is required, such as when genome-wide gene introduction is performed, and thus there is a possibility that the gene introduction method is easy and can be automated quickly.

It is the figure which showed typically that a gene is introduce | transduced by the junction between biological spheres from E. coli to a eukaryotic cell. It is the figure which showed the biojunction conjugation efficiency of 22 strains with an increase in biojunction efficiency. It is the figure which showed the result of having investigated the BY4742 stock | strain and BY4741 stock | stump which were processed with the YPD culture medium containing each drug of each density | concentration each increase in the conjugation efficiency by addition of the biophysical joint inhibition gene function inhibitor Antimycin A. . It is the figure which showed the result of having examined the BY4742 stock | strain and BY4741 stock | strain which were processed for 2 days with the YPD culture medium containing each drug of each density | concentration increase of the conjugation efficiency by addition of Oligomycin. It is the figure which showed the result of having examined the BY4742 strain | stump | stock and BY4741 strain | stump | stock which were processed with the YPD culture medium containing each chemical | medical agent of each density | concentration each increase in the conjugation efficiency by addition of the biophysical junction inhibition gene function inhibitor Erythromycin.

Claims (7)

In the method of gene transfer from E. coli to eukaryotic cells,
Manipulating the function of mitochondria in eukaryotic cells,
Gene transfer method using junction between organisms.
Manipulation of the function of the mitochondria,
It is performed using a mitochondrial function inhibitor,
A gene transfer method using the junction between organisms according to claim 1.
The mitochondrial function inhibitor is
Antimycin A, Oligomycin and Erythromycin,
A gene transfer method using the junction between organisms according to claim 2.
The eukaryotic cell is yeast;
The gene transfer method using the junction between biological kingdoms of any one of Claims 1 thru | or 3.
A eukaryotic cell into which a gene has been introduced by the method according to any one of claims 1 to 4.
The yeast into which a gene has been introduced by the method according to any one of claims 1 to 4, wherein the eukaryotic cell is yeast.
A gene introduction rate amplification kit for introducing a gene by the method according to any one of claims 1 to 4.