Method for introducing nucleic acid and nucleic acid introduction device

Abstract

To provide a method for introducing foreign nucleic acids including genes into host cells under simple control conditions, while reducing the risk of nuclear damage.SOLUTION: This method for introducing nucleic acids includes step (a) of preparing host cells, step (b) of preparing a first solution containing foreign nucleic acids, step (c) of irradiating the host cells with ultraviolet rays having a principal wavelength belonging to a range of 200 to 235 nm, and step (d) of contacting the first solution with the host cells during the execution of step (c) or after the execution of step (c).SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a method for introducing nucleic acids, and particularly to a method for introducing nucleic acids into cells using ultraviolet light. The present invention also relates to a nucleic acid introduction device using ultraviolet light.

Background Art

[0002] Conventionally, a technique is known in which a laser is used to create pores in the cell membrane of a host cell, and a foreign gene is introduced into the host cell through these pores (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] The technique disclosed in Patent Document 1 is to cause a part of the laser light to be absorbed by proteins and phospholipids, and use the energy derived from the laser light to create pores in the cell membrane.

[0005] However, according to this method, a part of the energy of the laser light may reach the nucleus inside the cell, and the nucleus may be damaged. If such a situation occurs, the host cell may die.

[0006] This may be because when the diameter of the pores formed in the cell membrane is large, the laser light incident through the pores reaches the nucleus. Another reason may be that a part of the laser light having high energy penetrates proteins and phospholipids and reaches the nucleus.

[0007] In view of the above problems, as a means for introducing a foreign gene into a host cell while suppressing damage to the nucleus of the host cell, a method of controlling the output and irradiation time of laser light (hereinafter collectively referred to as "irradiation conditions") within a range where no damage to the nucleus occurs is conceivable. However, in order to adopt this method, it is necessary to appropriately set the irradiation conditions according to the type and size of the host cell and the foreign gene. However, since there are a wide variety of variations regarding the type and size of the host cell and the foreign gene, it is not easy to derive the optimal irradiation conditions.

[0008] In particular, since laser light has high energy, there is a problem that it is difficult to finely control the amount of energy irradiated.

[0009] In view of the above problems, an object of the present invention is to provide a method for introducing a foreign nucleic acid such as a gene into a host cell with simple control content while suppressing the risk of causing damage to the nucleus. Another object of the present invention is to provide a nucleic acid introduction device suitable for the implementation of such a method.

Means for Solving the Problems

[0010] The method for introducing a nucleic acid according to the present invention comprises: step (a) of preparing a host cell; step (b) of preparing a first solution containing a foreign nucleic acid; step (c) of irradiating the host cell with ultraviolet light whose main wavelength belongs to the range of 200 nm to 235 nm; and step (d) of bringing the first solution into contact with the host cell during or after the execution of step (c).

[0011] In this specification, "nucleic acid" is a concept that includes DNA and RNA belonging to natural nucleic acids, as well as artificial nucleic acids. Artificial nucleic acids are substances obtained, for example, by modifying the backbone of natural nucleic acids, DNA or RNA, into different chemical structures.

[0012] In this specification, the "dominant wavelength" refers to the wavelength that exhibits an intensity of 50% or more with respect to the peak intensity in the spectrum. Naturally, the peak wavelength in the spectrum corresponds to the "dominant wavelength".

[0013] Cells have a cell membrane on their outer edge. The cell membrane is composed of phospholipids and proteins. Here, the protein has a relatively high absorption rate for ultraviolet rays in the wavelength range of 200 nm to 235 nm compared to the absorption rate for ultraviolet rays with a wavelength of 240 nm or more.

[0014] Figure 1 is a graph showing the relationship between the absorption coefficient of a protein and the wavelength. It can be seen that the absorption coefficient of the protein becomes significantly higher as the wavelength becomes shorter in the wavelength range shorter than 240 nm.

[0015] That is, when the host cell is irradiated with ultraviolet rays whose dominant wavelength belongs to the range of 200 nm to 235 nm, most of the ultraviolet rays are absorbed by the cell membrane of the host cell, and hardly reach the nucleus inside it. And when ultraviolet rays are absorbed by the cell membrane, damage caused by the energy derived from the ultraviolet rays is given to the cell membrane, and pores are opened.

[0016] Therefore, by bringing a first solution containing foreign nucleic acid into contact with the host cell during or after irradiation with ultraviolet rays, the foreign nucleic acid contained in the first solution can be introduced through the pores generated in the cell membrane of the host cell. And since most of the ultraviolet rays irradiated as described above are absorbed by the cell membrane of the host cell, the possibility of causing cytotoxicity (cell damage) to the host cell is extremely low.

[0017] Note that the step (c) of irradiating the host cell with ultraviolet rays is usually performed in an air atmosphere. Ultraviolet rays with a wavelength of less than 200 nm generate ozone when absorbed by oxygen in the air. If a large amount of ozone is generated, there is a risk of damaging the host cell by this ozone. From this perspective, it is preferable that the dominant wavelength of the ultraviolet rays irradiated to the host cell is 200 nm or more.

[0018] Note that the main wavelength of the ultraviolet rays irradiated in step (c) is preferably in the range of 200 nm to 235 nm, more preferably in the range of 206 nm to 230 nm, and particularly preferably in the range of 222 nm to 224 nm.

[0019] Also, the peak wavelength of the ultraviolet rays irradiated in step (c) preferably belongs to the range of 200 nm to 235 nm, and preferably has a half-value width of less than 20 nm. Examples of light sources that emit such ultraviolet rays include KrCl excimer lamps (with a peak wavelength near 222 nm), KrBr excimer lamps (with a peak wavelength near 207 nm), etc. Also, examples of other light sources include LEDs.

[0020] The step (d) may be carried out in a state where the irradiation of the ultraviolet rays is stopped after the execution of the step (c).

[0021] According to the above, in the time zone when the first solution containing the foreign nucleic acid comes into contact with the host cell, since the irradiation of the ultraviolet rays is stopped, the foreign nucleic acid is not irradiated with the ultraviolet rays. As a result, damage to the foreign nucleic acid is suppressed.

[0022] The step (a) includes a step of preparing a culture vessel containing a culture region, and a step of introducing the host cell into the culture region containing the culture solution. Before the step (c), there is a step (e) of removing the culture solution contained in the culture vessel. The step (d) may include a step of supplying the first solution to the culture region.

[0023] The step (c) may be a step of irradiating the ultraviolet rays with an irradiation dose set according to the type of the host cell and the type of the foreign nucleic acid.

[0024] Specifically, on the light source side that emits ultraviolet light, methods such as adjusting the output of the light source (the illuminance of ultraviolet light on the window surface), adjusting the separation distance between the region where the host cell is contained (e.g., a culture vessel) and the light source, and adjusting the irradiation time can be utilized. Also, it is possible to control the irradiation dose of ultraviolet light by combining the methods listed here.

[0025] In particular, compared with the case of irradiating laser light, by controlling the irradiation time in units of seconds to tens of seconds, it is possible to easily adjust the irradiation dose of ultraviolet light. Furthermore, by adjusting the output of the light source or the separation distance between the light source and the culture vessel, etc., by intentionally lowering the illuminance of the ultraviolet light irradiated on the culture vessel, the controllability of the irradiation dose of ultraviolet light to the culture vessel by adjusting the irradiation time is improved.

[0026] Furthermore, a light attenuation member may be provided on the window surface of the light extraction window from which ultraviolet light is extracted from the light source, or at a position between the light extraction window and the region where the host cell is contained with respect to the optical axis direction of the ultraviolet light. When irradiating the host cell with ultraviolet light through such a light attenuation member, the controllability of the irradiation dose of ultraviolet light irradiated on the host cell by adjusting the irradiation time is further improved.

[0027] The nucleic acid introduction device according to the present invention is a nucleic acid introduction device for introducing a foreign nucleic acid into a host cell accommodated in a culture vessel including a culture region, a light source that emits ultraviolet light with a main wavelength belonging to the range of 200 nm to 235 nm toward the culture region, a control unit that performs lighting control of the light source, and an injection mechanism configured to be able to inject a first solution containing the foreign nucleic acid into the culture region, wherein the control unit executes control to turn on the light source before the injection of the first solution into the culture region is started from the injection mechanism after the host cell is placed in the culture region.

[0028] According to the above configuration, it is possible to introduce foreign nucleic acid into a host cell while suppressing the induction of cytotoxicity in the host cell. In particular, compared with the conventional method of introducing foreign nucleic acid into a host cell using a laser beam, the controllability during introduction is improved.

[0029] The control unit may be configured to execute control to turn off the light source before the injection of the first solution into the culture region from the injection mechanism is started after executing control to turn on the light source.

[0030] The nucleic acid introduction device includes a light source device on which the light source is mounted. The light source device may have a position adjustment mechanism capable of adjusting the height position of the light source.

[0031] The position adjustment mechanism may be configured to perform control to adjust the height position of the light source to a height position specified based on information regarding the type of the host cell and the type of the foreign nucleic acid.

[0032] The nucleic acid introduction device includes a light source device on which the light source is mounted. The light source device has a light extraction window for extracting the ultraviolet light, and may have a light attenuation member that can be disposed downstream of the light extraction window with respect to the optical path direction of the ultraviolet light and that reduces the intensity of the ultraviolet light.

[0033] The light source device has a plurality of the light attenuation members having different light attenuation rates, and one of the light attenuation members selected based on information regarding the type of the host cell and the type of the foreign nucleic acid may be configured to be installable on the optical path of the ultraviolet light.

Advantages of the Invention

[0034] According to the present invention, it is possible to introduce foreign nucleic acid into a host cell by a simple method while suppressing the risk of causing damage to the nucleus.

Brief Description of the Drawings

[0035]

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Embodiments for Carrying Out the Invention

[0036] Embodiments of the method for introducing a nucleic acid and the nucleic acid introduction device according to the present invention will be described below with appropriate reference to the drawings. However, the following drawings are schematically shown, and the dimensional ratios on the drawings do not match the actual dimensional ratios. Also, the dimensional ratios do not necessarily match among the respective drawings. Further, the number of elements shown on the drawings does not necessarily match the actual number.

[0037] FIG. 2 is a flowchart showing an example of the procedure of the method for introducing a nucleic acid of the present embodiment. In the following description, the step numbers shown in FIG. 2 are referred to as appropriate. FIG. 3 is a block diagram schematically showing the configuration of the nucleic acid introduction device of the present embodiment. The nucleic acid introduction device 1 of the present embodiment includes an injection mechanism 3, a suction mechanism 8, and a light source device 30.

[0038] (Step #1) A host cell to which a foreign nucleic acid is to be introduced is prepared. As a more detailed example, first, a culture vessel 10 in which a plurality of wells 11 are arranged in a matrix as schematically shown in FIG. 4 is prepared. The well 11 corresponds to the "culture region". The culture vessel 10 is, for example, a microplate.

[0039] Next, using the injection mechanism 3, a culture solution 13 containing host cells 12 is dispensed into the plurality of wells 11 of the culture vessel 10 (see FIG. 5). FIG. 5 is a drawing schematically showing the cross-sectional structure of the well 11 in a state where the culture solution 13 containing the host cells 12 is injected. As the injection mechanism 3, a known dispenser can be used.

[0040] By executing Step #1, the host cells 12 are seeded in the plurality of wells 11 of the culture vessel 10.

[0041] In FIG. 4, among the numerous wells 11, 11,... provided in the culture vessel 10, a state where host cells 12 are not seeded in some of the wells is schematically illustrated. However, this is merely an example for illustration purposes, and host cells 12 may be seeded in all of the wells 11, 11,....

[0042] Step #1 corresponds to step (a).

[0043] (Step #2) Prepare the foreign nucleic acid to be introduced into the host cell. More specifically, a liquid (first solution) containing the foreign nucleic acid is prepared. As this liquid, for example, a serum-free medium can be used. The foreign nucleic acid is one or more belonging to the group consisting of DNA, RNA, and artificial nucleic acids. As an example, the foreign nucleic acid is a plasmid.

[0044] Step #2 corresponds to step (b). Note that the execution order of Step #1 and Step #2 is arbitrary, and they can also be executed simultaneously.

[0045] (Step #3) While a culture solution 13 containing host cells 12 is dispensed into the plurality of wells 11 of the culture vessel 10, wait for a predetermined time (e.g., 1 day to several days). By this waiting, the host cells 12 adhere to the bottom surface of the wells 11. During this waiting period, it is preferable to close the opening surfaces of all the wells 11, 11,... of the culture vessel 10 with a lid (not shown).

[0046] After the elapse of the waiting period, the culture solution 13 contained in the wells 11, 11,... is aspirated, for example, by an aspiration mechanism 8 (see FIG. 3). As described above, when the upper surface of the culture vessel 10 is closed with a lid during the waiting period, the culture solution 13 may be aspirated with the lid removed. When the aspiration mechanism 8 is a different device from the injection mechanism 3, as shown in FIG. 3, the culture vessel 10 may be conveyed to the installation location of the aspiration mechanism 8 using a conveyance mechanism 4. Note that the aspiration mechanism 8 may be configured as the same device as the injection mechanism 3.

[0047] Since the standby period has elapsed, even after the culture solution 13 has been aspirated, host cells 12 remain fixed in the plurality of wells 11, 11,... of the culture vessel 10.

[0048] Step #3 corresponds to step (e). Note that the execution order of step #2 and step #3 is arbitrary.

[0049] (Step #4) When step #3 is executed, ultraviolet light L1 is irradiated from the light source device 30 onto the culture vessel 10 in a state where the culture solution 13 has been extracted and the host cells 12 are exposed within the wells 11, 11,... (see Fig. 3). In this case as well, the culture vessel 10 may be conveyed to the irradiation region of the ultraviolet light L1 irradiated from the light source device 30 using the conveyance mechanism 4 as necessary.

[0050] Fig. 6 is a plan view schematically showing the state in which the culture vessel 10 is irradiated with the ultraviolet light L1, following Fig. 4. Fig. 7 is a drawing schematically showing the state in which the well 11 where the host cells 12 are fixed is irradiated with the ultraviolet light L1, following Fig. 5.

[0051] Fig. 8 is a block diagram schematically showing the configuration of the light source device 30. Fig. 9 is a side view schematically showing an example of the light source device 30.

[0052] The light source device 30 of the present embodiment includes a light source 31, a power supply unit 32 for energizing the light source 31, and a control unit 33 for performing energization control of the power supply unit 32.

[0053] The light source 31 emits ultraviolet light L1 whose main wavelength belongs to the range of 200 nm to 235 nm. As a specific example, the light source 31 is a KrCl excimer lamp, a KrBr excimer lamp, or an LED. The ultraviolet light L1 emitted from the light source 31 is irradiated onto the culture vessel 10 through the light extraction window 34.

[0054] More specifically, as shown in FIG. 9, the culture vessel 10 may be irradiated with ultraviolet light L1 while being placed on the upper surface of the pedestal 51. In this case, the culture vessel 10 may be transported to the location of the pedestal 51 by the transport mechanism 4 (see FIG. 3). Furthermore, it is also possible to use the transport mechanism 4 itself as the pedestal 51.

[0055] In the example shown in FIG. 9, the light source 31 is housed in the light source housing unit 35, and this light source housing unit 35 is fixed to the holding member 37 via the position adjustment mechanism 38. The position adjustment mechanism 38 is configured to be able to adjust the height position of the light source housing unit 35. For example, the position adjustment mechanism 38 and the holding member 37 are screwed together, and by moving the position adjustment mechanism 38 along the axial direction d1 of the holding member 37 with the screw loosened, the height position of the light source 31 can be adjusted.

[0056] FIG. 10 is a schematic perspective view of the light source device 30 as viewed from an angle different from that of FIG. 9. The light source device 30 shown in FIGS. 9 to 10 includes a light attenuation member 41 disposed downstream of the light extraction window 34 with respect to the optical path direction of the ultraviolet light L1. As a more detailed example, the light source device 30 has a light attenuation member adjustment unit 43, and by operating the light attenuation member adjustment unit 43, it is possible to switch between a state where the light attenuation member 41 is installed and a state where it is not installed on the optical path of the ultraviolet light L1. Furthermore, as shown in FIG. 10, the light source device 30 may be provided with a plurality of light attenuation members 41 (41a, 41b) having different light attenuation rates, and by adjusting the light attenuation member adjustment unit 43, it may be possible to switch the light attenuation member 41 installed on the optical path of the ultraviolet light L1.

[0057] The light attenuation member 41 has a function of transmitting a part of the ultraviolet light L1 and shielding the progress of a part of the ultraviolet light L1. The light attenuation member 41 is, for example, a sheet-like member made of a resin such as PTFE, PE, or PP.

[0058] By adjusting the height position of the light source housing unit 35 or adjusting the installation / non-installation of the light attenuation member 41, the illuminance of the ultraviolet ray L1 irradiated to the culture vessel 10 can be adjusted. Further, by controlling the energization time from the power supply unit 32 to the light source 31 by the control unit 33, the irradiation time of the ultraviolet ray L1 is adjusted. By controlling at least one of the illuminance of the ultraviolet ray L1 irradiated to the culture vessel 10 and the time during which the culture vessel 10 is irradiated with the ultraviolet ray L1, the irradiation dose of the ultraviolet ray L1 irradiated to the culture vessel 10, that is, the irradiation dose of the ultraviolet ray L1 irradiated to the host cells 12 fixed in the wells 11 can be controlled.

[0059] FIG. 11 is a drawing schematically showing a state in which the host cells 12 are irradiated with the ultraviolet ray L1. The cells are covered with a cell membrane 21 at the outer edge, and have a nucleus 22 and cytoplasm 23 inside thereof. The cell membrane 21 is composed of a phospholipid and a protein. Further, the cytoplasm 23 also contains a protein. As described above with reference to FIG. 1, the protein has a relatively high absorption rate for ultraviolet rays in the wavelength range of 200 nm to 235 nm as compared with the absorption rate for ultraviolet rays of 240 nm or more. For this reason, when the ultraviolet ray L1 whose main wavelength belongs to the range of 200 nm to 235 nm emitted from the light source 31 irradiates the host cells 12, most of the ultraviolet ray L1 is absorbed by the cell membrane 21 of the host cells 12. As a result, as schematically shown in FIG. 12, pores 26 are formed in the cell membrane 21. On the other hand, as a result of most of the ultraviolet ray L1 being absorbed by the cell membrane 21, the ultraviolet ray L1 hardly reaches the nucleus 22 existing inside the cell membrane 21.

[0060] However, when the intensity of the ultraviolet ray L1 is extremely high or when the irradiation time of the ultraviolet ray L1 is extremely long, it is considered that the intensity of the ultraviolet ray L1 reaching the nucleus 22 increases. From such a viewpoint, it is preferable that the control unit 33 performs control to stop the irradiation of the ultraviolet ray L1 from the light source 31 when the scheduled irradiation time has elapsed.

[0061] Step #4 corresponds to step (c). The execution order of step #2 and step #4 is arbitrary.

[0062] (Step #5) By executing Step #4, the host cell 12 with the hole 26 is contacted with the first solution containing the foreign nucleic acid prepared in Step #2. Specifically, the first solution is injected into each well 11, 11,... of the culture vessel 10. When injecting, the same injection mechanism 3 as used in Step #1 can be used.

[0063] Thereafter, a culture solution is injected into each well 11, 11,... of the culture vessel 10. Then, it is waited for a predetermined time. During this waiting period, the foreign nucleic acid in the first solution is introduced into the host cell 12 through the hole 26.

[0064] Note that when executing Step #5, it is preferably that the irradiation of the ultraviolet ray L1 is stopped. That is, Step #5 is preferably executed after Step #4 is completed. Thereby, since the foreign nucleic acid is not irradiated with the ultraviolet ray L1, the damage to the foreign nucleic acid by the ultraviolet ray L1 is suppressed.

[0065] However, the present invention does not exclude the case of executing Step #5 during the execution of Step #4.

[0066] Step #5 corresponds to step (d). After the end of Step #5, subsequent processing is executed as necessary.

[0067] [Verification 1] An attempt was made to introduce the CMV-EGFP plasmid as a foreign nucleic acid into Cos7 cells as the host cell 12 using the method described above. As the light source 31, a KrCl excimer lamp (peak wavelength 222 nm) was used. FIG. 13 is a drawing schematically showing the structure of the CMV-EGFP plasmid.

[0068] [Verification method] Verification was performed according to the following procedure.

[0069] (Step #1) As the culture vessel 10, a microplate (manufactured by Corning, Multiple Well Plate 96 Well Flat Bottom 3596) having 96 wells 11 was prepared. The volume of each well 11 was 0.32 mL. Then, 1×10 4 cells of Cos7 cells were seeded in each well 11. Specifically, a culture solution with a density of 1×10 5 cells / mL in which Cos7 cells were mixed was injected into each well 11 in an amount of 100 μL. As the culture solution, a mixed solution of DMEM Basic medium (manufactured by Cosmo Bio Co., Ltd., MG-30a), 10% FCS (fetal bovine serum: manufactured by Corning, 35-079-CV), and a penicillin-streptomycin solution (100-fold dilution) (manufactured by Fujifilm Wako Pure Chemical Corporation, 168-23191) as an antibiotic was used.

[0070] (Step #2) A serum-free medium (Opti-MEM (registered trademark)) mixed with the CMV-EGFP plasmid as the foreign nucleic acid was prepared. This mixed solution corresponds to the "first solution".

[0071] (Step #3) With the culture vessel 10 on which Step #1 was performed covered, it was left standing for 1 day. Then, the lid was removed, and the culture solution was aspirated from each well 11 to expose the Cos7 cells.

[0072] (Step #4) After the execution of Step #3, ultraviolet light L1 was irradiated from the light source 31 composed of a KrCl excimer lamp onto the culture vessel 10. At this time, a plurality of samples were obtained by varying the irradiation conditions (the distance between the light source 31 and the culture vessel 10, the illuminance of the window surface at the light extraction window 34 of the light source 31). The irradiation time was made uniform at 5 seconds.

[0073] (Step #5) After the execution of Step #4, immediately, 10 μL of the first solution prepared in Step #2 was injected into each well 11. Thereafter, after waiting for 1 hour with the culture vessel 10 covered, 90 μL of the culture solution was injected into each well 11. As the culture solution, reduced serum medium (manufactured by Gibco, Opti-MEM (registered trademark): Reduced Serum Media, 31985062) was used.

[0074] Thereafter, after further waiting for 24 hours with the culture vessel 10 covered, the culture vessel 10 was observed with a fluorescence microscope. When the CMV-EGFP plasmid is introduced into the host cell 12, within the host cell 12, green fluorescent protein (GFP) that emits green light when irradiated with light in the near-ultraviolet region is generated.

[0075] <Results> Figure 14 is a graph showing the relationship between the irradiation dose of the ultraviolet ray L1 irradiated in Step #4 and the number of Cos7 cells into which the plasmid was introduced. In Figure 14, for comparison, the data when Step #4 was not executed (irradiation dose 0) is also shown.

[0076] Figures 15A to 15B are captured images of the state where the culture vessel 10 is irradiated with light in the near-ultraviolet region. In Figures 15A to 15B, the descriptions of 10 cm, 20 cm, 50 cm, and 100 cm correspond to the separation distances between the light source 31 and the culture vessel 10, respectively.

[0077] According to the results of Figure 14 and Figures 15A to 15B, when the irradiation dose of the ultraviolet ray L1 is 0.069 mJ / cm 2 it can be seen that the amount of plasmid introduced into the cells is relatively small. Also, when the irradiation dose of the ultraviolet ray L1 is 5 mJ / cm 2 cell death of the cells themselves was confirmed. On the other hand, when the irradiation dose of the ultraviolet ray L1 is between 0.069 mJ / cm 2 and 5 mJ / cm 2 values, namely 0.268 mJ / cm 2 and 1.5 mJ / cm 2In this case, it was confirmed that a large number of plasmids were introduced into the cells.

[0078] From this result, it can be seen that there is a preferable range of the irradiation dose of ultraviolet ray L1 when introducing foreign nucleic acid into the host cell. Therefore, if information regarding the range of the preferable irradiation dose is experimentally obtained in advance, the lighting of the light source 31 can be controlled by the control unit 33, and the separation distance between the light source 31 and the culture vessel 10 can be adjusted by the position adjustment mechanism 38 as necessary, or a light attenuation member 41 can be arranged, so that it can be understood that a sufficient amount of foreign nucleic acid can be introduced into the host cell.

[0079] In particular, by adjusting the separation distance between the light source 31 and the culture vessel 10 or arranging the light attenuation member 41, the adjustment range of the irradiation dose to the culture vessel 10 by controlling the light emission output and irradiation time of the light source 31 is expanded, and the degree of freedom of control is increased.

[0080] [Verification 2] The viability of the host cells was evaluated based on the presence or absence of irradiation with ultraviolet ray L1. Specifically, the change in the relative value of the number of host cells in which step #4 was executed was compared based on the number of cells of the host cells in which step #4 was not executed. The comparison results are shown in FIG. 16.

[0081] According to FIG. 16, when irradiated with ultraviolet ray L1 at a relatively high irradiation dose as in the case of an irradiation dose of 5 mJ / cm 2 and the case of 30 mJ / cm 2 , it can be seen that the cells tend to die. On the other hand, when the irradiation dose is lower than 5 mJ / cm 2 , i.e., 0.069 mJ / cm 2 , 0.268 mJ / cm 2 , 1.5 mJ / cm 2 , the proportion of cells that died was low, and the viable cell rate showed around 100%.

[0082] From this result, it is confirmed that no cytotoxicity occurs in the host cells after the foreign nucleic acid is introduced if the irradiation dose of ultraviolet ray L1 is set within an appropriate range.

[0083] Note that according to FIG. 16, when the irradiation dose is 0.069 mJ / cm 2 , and in the case of 0.268 mJ / cm 2 , the number of cells has increased compared to the case where the ultraviolet ray L1 was not irradiated. Although the reason for this is not clear, it is conceivable that the host cells were stimulated by the irradiation of the ultraviolet ray L1 and cell division occurred.

[0084] [Alternative Embodiment] Hereinafter, an alternative embodiment will be described.

[0085] 〈1〉Before the ultraviolet ray L1 is irradiated in step #4, it is not necessary to extract the culture solution in step #3. However, since the culture solution may absorb the ultraviolet ray L1 and the illuminance of the ultraviolet ray L1 irradiated to the host cell 12 may decrease, in this case, it is necessary to increase the light emission intensity of the light source 31 as necessary.

[0086] 〈2〉In the above embodiment, the case where the light source device 30 includes the position adjustment mechanism 38 has been described. However, in the present invention, whether the light source device 30 includes the position adjustment mechanism 38 is arbitrary.

[0087] 〈3〉In the above embodiment, the case where the light source device 30 includes a plurality of light attenuation members 41 having different light attenuation rates has been described. However, the light source device 30 may include a single light attenuation member 41. Further, the light source device 30 may not include the light attenuation member 41.

[0088] 〈4〉The present invention is not limited to the above-described embodiment and includes various modifications. For example, the above-described embodiment has been described in detail for better understanding of the present invention and is not necessarily limited to the one having all the configurations described. The scope of the present invention is indicated by the claims, and it is intended that all changes within the meaning and scope equivalent to the claims are included.

Explanation of Reference Numerals

[0089] 1: Nucleic acid introduction device 3: Injection mechanism 4: Transfer mechanism 8: Suction mechanism 10: Culture vessel 11: Well 12: Host cell 13: Culture medium 21: Cell membrane 22: Nucleus 23: Cytoplasm 26: Hole 30: Light source device 31: Light source 32: Power supply unit 33: Control unit 34: Light extraction window 35: Light source housing unit 37: Holding member 38: Position adjustment mechanism 41: Light attenuation member 43: Light attenuation member adjustment part 51: Pedestal L1: Ultraviolet ray

Claims

1. Step (a) of preparing a host cell, Step (b) of preparing a first solution containing a foreign nucleic acid, Step (c) of irradiating the host cell with ultraviolet light whose main wavelength belongs to the range of 200 nm to 235 nm, A method for introducing a nucleic acid, comprising: step (d) of bringing the first solution into contact with the host cell during or after the execution of step (c).

2. The method for introducing a nucleic acid according to claim 1, wherein step (d) is performed in a state where the irradiation with ultraviolet light is stopped after the execution of step (c).

3. Step (a) includes a step of preparing a culture vessel including a culture region and a step of introducing the host cell into the culture region containing a culture solution, Before step (c), there is step (e) of removing the culture solution contained in the culture vessel, The method for introducing a nucleic acid according to claim 1 or 2, wherein step (d) includes a step of supplying the first solution to the culture region.

4. The method for introducing a nucleic acid according to claim 1 or 2, wherein step (c) is a step of irradiating the ultraviolet light with an irradiation dose set according to the type of the host cell and the type of the foreign nucleic acid.

5. A nucleic acid introduction device for introducing a foreign nucleic acid into a host cell contained in a culture vessel including a culture region, comprising: A light source that emits ultraviolet light whose main wavelength belongs to the range of 200 nm to 235 nm toward the culture region, A control unit that performs lighting control of the light source, An injection mechanism configured to be able to inject a first solution containing the foreign nucleic acid into the culture region, The nucleic acid introduction device, wherein the control unit performs control to turn on the light source after the host cell is placed in the culture region and before the injection of the first solution into the culture region is started from the injection mechanism.

6. The nucleic acid introduction device according to claim 5, wherein the control unit performs control to turn off the light source after performing control to turn on the light source and before the injection of the first solution into the culture region is started from the injection mechanism.

7. Comprising a light source device on which the light source is mounted, The nucleic acid introduction device according to claim 5 or 6, wherein the light source device has a position adjustment mechanism capable of adjusting the height position of the light source.

8. The nucleic acid introduction device according to claim 7, wherein the position adjustment mechanism performs control to adjust the height position of the light source to a height position specified based on information regarding the type of the host cell and the type of the foreign nucleic acid.

9. comprising a light source device on which the light source is mounted, the light source device has a light extraction window for extracting the ultraviolet light, and a light attenuation member that can be disposed downstream of the light extraction window with respect to the optical path direction of the ultraviolet light and that reduces the intensity of the ultraviolet light, the nucleic acid introduction device according to claim 5 or 6.

10. the light source device has a plurality of the light attenuation members having mutually different light attenuation rates, and one of the light attenuation members selected based on information regarding the type of the host cell and the type of the foreign nucleic acid is configured to be installable on the optical path of the ultraviolet light, the nucleic acid introduction device according to claim 9.

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