Method for introducing nucleic acids and method for introducing nucleic acids to the periphery of pancreatic islets

Abstract

To provide a method for introducing nucleic acids into the periphery of pancreatic islets without performing intraparenchymal injection into the pancreas. [Solution] A method for introducing nucleic acids, comprising an injection step of injecting a nucleic acid-containing solution into the pancreatic duct through the ampulla of Vater of an animal (excluding humans).

Description

Technical Field

[0001] The present invention relates to a nucleic acid introduction method and a method for introducing nucleic acid into the pancreatic islet periphery.

Background Art

[0002] The pancreas has pancreatic islets (endocrine part) including β cells that secrete insulin, α cells that secrete glucagon, δ cells that secrete somatostatin, etc., and an exocrine part that secretes pancreatic juice into the duodenum. Less than 5% of the volume of the pancreas is the pancreatic islets (endocrine part), and 95% or more of the volume of the pancreas is the exocrine part. In the periphery of the pancreatic islets (pancreatic islet periphery), immature β cells, α cells, and δ cells are heterogeneously present, and mature β cells are present in the central part (nuclear part).

[0003] In a high-fat diet diabetes model or the like, it is known that β cells in the pancreatic islet periphery are converted into non-β cells such as α cells. From the viewpoint of developing a treatment method for reverting dedifferentiated β cells back to functional β cells, etc., a method for specifically introducing nucleic acid into the pancreatic islet periphery is desired. So far, a method for introducing nucleic acid has been reported by injecting a plasmid mixture into the pancreas by parenchymal injection at a depth of 3 to 4 mm using a cannula and then performing electroporation (Non-Patent Document 1). However, with this method, nucleic acid can be introduced into the pancreatic islets only with extremely low efficiency, and furthermore, parenchymal injection into the pancreas causes pancreatitis.

[0004] Therefore, a method for introducing nucleic acid into the pancreatic islet periphery without performing parenchymal injection into the pancreas has not yet been provided, and its prompt provision is strongly demanded.

Prior Art Documents

Patent Documents

[0005]

Non-Patent Document 1

[0006] The present invention aims to solve the aforementioned problems of the conventional approach and achieve the following objectives. Specifically, the present invention aims to provide a method for introducing nucleic acids to the periphery of pancreatic islets without performing intraparenchymal injection into the pancreas. [Means for solving the problem]

[0007] As a result of diligent research conducted by the inventors to achieve the above objective, they have discovered that it is possible to provide a method for introducing nucleic acids to the periphery of pancreatic islets without performing intraparenchymal injection into the pancreas.

[0008] The present invention is based on the aforementioned findings by the inventors, and the means for solving the aforementioned problems are as follows: <1> This method for introducing nucleic acids is characterized by including an injection step in which a nucleic acid-containing solution is injected into the pancreatic duct through the ampulla of Vater of an animal (excluding humans). <2> The aforementioned <1> This method involves introducing nucleic acids into the periphery of pancreatic islets, characterized by using the nucleic acid introduction method described above. [Effects of the Invention]

[0009] According to the present invention, a method for introducing nucleic acids into the periphery of pancreatic islets without performing intraparenchymal injection into the pancreas is provided. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a schematic diagram showing a method for injecting nucleic acid-containing solution into the pancreatic duct through the ampulla of Vater in animals (excluding humans). [Figure 2] Figure 2 is a schematic diagram illustrating an example of a method for applying electrical pulses to the pancreas. [Figure 3]Figure 3 is a diagram showing the results of immunostaining of the sections prepared in Examples 1 to 4. [Figure 4] Figure 4 is a graph showing the YFP positive rate in the sections prepared in Examples 1 to 4. [Figure 5] Figure 5 is a diagram showing the results of immunostaining of the section prepared in Example 5.

Mode for Carrying Out the Invention

[0011] (Nucleic Acid Introduction Method) The nucleic acid introduction method includes an injection step, and may further include other steps.

[0012] <Injection Step> The injection step is a step of injecting a nucleic acid-containing solution from the Vater papilla of an animal (excluding humans) into the pancreatic duct.

[0013] -Nucleic Acid-Containing Solution- The nucleic acid-containing solution contains nucleic acid and may further contain other components.

[0014] The injection amount of the nucleic acid-containing solution is not particularly limited and can be appropriately selected according to the purpose. However, in order to avoid the risk of death due to dilution of body fluid, when the animal's weight is up to 20 g, it is preferably 100 μL or less, when it is up to 25 g, it is preferably 130 μL or less, and when it is up to 30 g, it is preferably 160 μL or less.

[0015] The solvent of the nucleic acid-containing solution is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include PBS, physiological saline, and the like.

[0016] --Nucleic Acid-- The nucleic acid is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include DNA, RNA, and the like. The nucleic acid may be a vector such as a plasmid vector, a viral vector, or an artificial chromosome vector.

[0017] The concentration of the nucleic acid in the nucleic acid-containing solution is not particularly limited and can be appropriately selected according to the purpose. However, it is preferably 0.1 μg / μL or more and 50 μg / μL or less, more preferably 1 μg / μL or more and 10 μg / μL or less, still more preferably 2 μg / μL or more and 6 μg / μL or less, and particularly preferably 3 μg / μL or more and 5 μg / μL or less.

[0018] --Other components-- The other components are not particularly limited and can be appropriately selected according to the purpose. Examples include nuclear translocation promoters, coloring agents, and the like.

[0019] The nuclear translocation promoter is not particularly limited and can be appropriately selected according to the purpose. However, a water-soluble nuclear translocation promoter is preferred in terms of introducing the nucleic acid into the pancreatic islet marginal region without performing parenchymal injection into the pancreas, and trans-cyclohexane-1,2-diol (TCHD) is more preferred.

[0020] The nuclear translocation promoter may be a compound having an affinity for the nuclear envelope complex. By using the nuclear translocation promoter, the nucleic acid can penetrate the nuclear envelope even in non-dividing cells. By using the nuclear translocation promoter, nucleic acids can be introduced into non-dividing cells with high efficiency and expressed.

[0021] The lower limit of the concentration of the TCHD in the nucleic acid-containing solution is not particularly limited and can be appropriately selected according to the purpose. However, in terms of introducing the nucleic acid into the pancreatic islet marginal region without performing parenchymal injection into the pancreas, it is preferably 0.1% or more, more preferably 0.2% or more, still more preferably 0.4% or more, particularly preferably 0.6% or more, and most preferably 0.8% or more. The upper limit of the concentration of the TCHD in the nucleic acid-containing solution is not particularly limited and can be appropriately selected according to the purpose. However, in terms of avoiding cytotoxicity, it is preferably 5% or less, more preferably 3% or less, still more preferably 2% or less, particularly preferably 1.5% or less, and most preferably 1.2% or less. Furthermore, it is preferable that the lower limit and upper limit of the numerical range be one of the numbers indicated as the lower limit and one of the numbers indicated as the upper limit. Among these, a concentration of 0.1% to 5% is preferred, 0.2% to 3% is more preferred, 0.4% to 2% is even more preferred, 0.6% to 1.5% is particularly preferred, and 0.8% to 1.2% is most preferred, in terms of introducing nucleic acids to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas and avoiding cytotoxicity.

[0022] There are no particular restrictions on the aforementioned coloring agents, and they can be appropriately selected depending on the purpose. For example, Fast Green is one such coloring agent.

[0023] -Animals (excluding humans)- The aforementioned animals (excluding humans) are not particularly limited as long as they have a pancreas, and can be appropriately selected according to the purpose. Examples include mammals such as mice, rats, dogs, and monkeys. The animals mentioned above are preferably living animals, and more preferably animals that are alive under anesthesia. For example, if the nucleic acid in the nucleic acid-containing solution has a Cre recombinase gene sequence, a mouse in which the loxP-STOP-loxP-YFP gene has been knocked into the Rosa locus (ROSA-loxP-STOP-loxP YFP) can be used. The aforementioned ROSA-loxP-STOP-loxP YFP is commercially available from Jackson Laboratory Japan Co., Ltd.

[0024] -injection- As for the injection method, there are no particular restrictions as long as the nucleic acid-containing solution is injected from the ampulla of Vater into the pancreatic duct, and it can be appropriately selected according to the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without performing an intraparenchymal injection into the pancreas, as shown in Figure 1, it is preferable to insert the tip of the injection needle from the outside of the duodenum (covered by a double layer of muscle, the inner circular muscle and the outer longitudinal muscle) into the closed center of the sphincter, and then insert it into the pancreatic duct (pancreatic duct) from the ampulla of Vater to inject the nucleic acid-containing solution. It is even more preferable to clip the common bile duct on the liver side, then insert the tip of the injection needle from the outside of the duodenum (covered by a double layer of muscle, the inner circular muscle and the outer longitudinal muscle) into the closed center of the sphincter, insert it into the pancreatic duct (pancreatic duct) from the ampulla of Vater, clip the ampulla of Vater, and then inject the nucleic acid-containing solution. Pancreatic duct cells have a physiological mechanism to add bicarbonate ions and water to pancreatic juice, and the water is transported by paracellular secretion through intercellular passages between pancreatic duct cells. In other words, even if nucleic acids are introduced into the ducts and an electric field is applied, if the cells have tight junctions with each other, the nucleic acids will only be introduced into the duct cells themselves. However, in the case of pancreatic duct cells, due to their unique characteristics, the nucleic acid-containing fluid can move beyond the pancreatic duct cells through intercellular passages and reach the area around the periphery of the pancreatic islets, thus enabling the transport of nucleic acids via the pancreatic ducts.

[0025] The ampulla of Vater is covered by a ring-shaped sphincter that opens when releasing enzymes and closes when release is complete (the ring-shaped sphincter is normally contracted, so the opening is closed within the duodenal lumen, but physiologically the digestive hormone (cholecystokinin) causes the sphincter to relax and open, creating a physiologically leak-free system). By inserting the tip of an injection needle into the sphincter, nucleic acids can be introduced into the duct (pancreatic duct) without leakage or autodigestion.

[0026] The ampulla of Vater is the opening of the pancreatic duct and common bile duct that open into the duodenum (the part where the common duct, formed by the confluence of the pancreatic duct and common bile duct, opens into the descending part of the duodenum), and is also called the duodenal papilla or major duodenal papilla. The pancreatic duct is a tubular organ that connects the pancreas to the common bile duct and transports pancreatic juice, which aids digestion through exocrine pancreatic secretion, towards the common bile duct.

[0027] <Other processes> The aforementioned other steps are not particularly limited and can be appropriately selected depending on the purpose. Examples include abdominal incision, electroporation, and abdominal closure.

[0028] -Laparotomy process- The aforementioned abdominal opening step is a step of opening the abdominal wall of the target animal and exposing the abdominal cavity before the injection step. The aforementioned abdominal incision procedure is preferably performed under anesthesia.

[0029] -Electroporation Process- The electroporation step is a step in which electroporation is performed after the injection step.

[0030] There are no particular restrictions on the conditions for the electroporation described above, and they can be appropriately selected depending on the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without performing intraparenchymal injection into the pancreas, conditions in which a first electrical pulse is applied once, followed by two or more second electrical pulses, are preferred.

[0031] As long as the number of the second electrical pulses is two or more, there is no particular limit, and it can be appropriately selected depending on the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without performing intraparenchymal injection into the pancreas, three or more pulses are preferred, four or more pulses are more preferred, and five or more pulses are even more preferred.

[0032] There are no particular restrictions on the interval between each of the second electrical pulses, and it can be appropriately selected depending on the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas, an interval of 100 milliseconds to 2000 milliseconds is preferred, 500 milliseconds to 1500 milliseconds is more preferred, 700 milliseconds to 1300 milliseconds is even more preferred, 900 milliseconds to 1100 milliseconds is particularly preferred, and 950 milliseconds to 1050 milliseconds is most preferred.

[0033] There are no particular restrictions on the voltage of the second electrical pulse, and it can be appropriately selected depending on the purpose. However, it is preferable that the voltage of the second electrical pulse be lower than that of the first electrical pulse in order to introduce nucleic acids to the periphery of the pancreatic islets without performing intraparenchymal injection into the pancreas.

[0034] There are no particular restrictions on the voltage of the first electrical pulse, and it can be appropriately selected depending on the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas, a voltage of 30V to 60V is preferred, 40V to 50V is more preferred, 42V to 48V is even more preferred, 43V to 47V is particularly preferred, and 44V to 46V is most preferred.

[0035] There are no particular restrictions on the voltage of the second electrical pulse, and it can be appropriately selected depending on the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas, a voltage of 10V to 40V is preferred, 20V to 30V is more preferred, 22V to 28V is even more preferred, 23V to 27V is particularly preferred, and 24V to 26V is most preferred.

[0036] There are no particular restrictions on the current of the second electrical pulse, and it can be appropriately selected depending on the purpose. However, it is preferable that the current of the second electrical pulse be lower than that of the first electrical pulse, in order to introduce nucleic acids to the periphery of the pancreatic islets without performing intraparenchymal injection into the pancreas.

[0037] There are no particular restrictions on the current of the first electrical pulse, and it can be appropriately selected depending on the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas, a current of 150 mA to 350 mA is preferred, 200 mA to 300 mA is more preferred, 220 mA to 280 mA is even more preferred, 230 mA to 270 mA is particularly preferred, and 240 mA to 260 mA is most preferred.

[0038] There are no particular restrictions on the current of the second electrical pulse, and it can be appropriately selected depending on the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas, a current of 20 mA to 220 mA is preferred, 70 mA to 170 mA is more preferred, 90 mA to 150 mA is even more preferred, 100 mA to 140 mA is particularly preferred, and 110 mA to 130 mA is most preferred.

[0039] There are no particular restrictions on the duration of the second electrical pulse, and it can be appropriately selected depending on the purpose. However, it is preferable that the duration of the second electrical pulse be the same as that of the first electrical pulse, in order to introduce nucleic acids to the periphery of the pancreatic islets without performing intraparenchymal injection into the pancreas.

[0040] There are no particular restrictions on the duration of the first electrical pulse, and it can be appropriately selected depending on the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas, a duration of 1 millisecond to 500 milliseconds is preferred, 5 milliseconds to 200 milliseconds is more preferred, 10 milliseconds to 100 milliseconds is even more preferred, 20 milliseconds to 70 milliseconds is particularly preferred, and 40 milliseconds to 50 milliseconds is most preferred.

[0041] There are no particular restrictions on the duration of the second electrical pulse, and it can be appropriately selected depending on the purpose. However, in order to introduce nucleic acids to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas, a duration of 1 millisecond to 500 milliseconds is preferred, 5 milliseconds to 200 milliseconds is more preferred, 10 milliseconds to 100 milliseconds is even more preferred, 20 milliseconds to 70 milliseconds is particularly preferred, and 40 milliseconds to 50 milliseconds is most preferred.

[0042] In the electroporation described above, there are no particular restrictions on the location where the electrical pulses are applied, and they can be appropriately selected according to the purpose. However, the pancreas is preferred in that nucleic acids are introduced to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas, the head or tail of the pancreas is more preferred, the head and tail of the pancreas are even more preferred, and two locations in the head of the pancreas and one location in the tail of the pancreas are particularly preferred.

[0043] As shown in Figure 2, when applying electrical pulses to the pancreas, it is preferable to apply the electrical pulses while the pancreas is sandwiched between gauze moistened with physiological saline solution.

[0044] The electroporation described above can be performed using commercially available equipment such as the BEX Pulse Generator CUY21Vivo-SQ.

[0045] -Abdominal closure process- The abdominal closing step is the step of closing the abdomen of the target animal that has been opened after the electroporation step.

[0046] The aforementioned nucleic acid introduction method enables clonal analysis of highly heterogeneous pancreatic islet endocrine cells derived from single cells. When combined with slice culture, it allows for real-time imaging analysis of the proliferation and differentiation of cell lineage-labeled cells, as well as functional assays of β-cells. When combined with BrdU labeling, it also enables cell lineage analysis of divided daughter cells.

[0047] Specifically, by introducing an expression vector (Rat insulin promoter-Cre:Rip-Cre) in which the Cre recombinase gene is linked downstream of the rat insulin promoter into mice (ROSA-loxP-STOP-loxP YFP) in which the loxP-STOP-loxP-YFP gene has been knocked into the Rosa locus, β-cell lineage analysis becomes possible. By introducing the Rip-Cre plasmid into Rosa 26R-YFP (ROSA-loxP-STOP-loxP YFP);floxed PDX1 mice (mice in which the PDX1 gene exon is flanked by flox-flox and the PDX1 gene is inactivated by Cre recombinase activity), PDX1 KD (knockdown) experiments become possible. Furthermore, by introducing the CAG-loxP-STOP-loxP-EGFP vector into Rip-Cre mice (Rat insulin By introducing promoter-Cre into transgenic mice (in which the mouse genome has been inserted), β-cell lineage analysis becomes possible. Similarly, by introducing the CAG-loxP-STOP-loxP-EGFP vector into GCG-Cre mice (mice in which a DNA sequence encoding the Cre recombinase gene is knocked into the glucagon locus, causing Cre to express in response to glucagon promoter activation), α-cell lineage analysis becomes possible. Furthermore, it is also possible to co-introduce knock-in vectors and forced expression vectors along with these. Furthermore, by introducing the CRISPR-CAS9 vector, gene modification and genome editing become possible, and by introducing a reporter construct that monitors signal activity, it becomes possible to visualize signals such as the notch signal.

[0048] (A method for introducing nucleic acids into the peripheral regions of the pancreatic islets) The method for introducing nucleic acids to the pancreatic islet margins is the nucleic acid introduction method described above. In other words, as a result of performing the nucleic acid introduction method described above, it becomes possible to introduce nucleic acids to the periphery of the pancreatic islets.

[0049] The pancreas contains exocrine glands that secrete digestive enzymes such as pancreatic juice amylase into the duodenum, as well as endocrine glands called islets of Langerhans. More than 90% of the pancreas is made up of exocrine glands, which are clusters of endocrine cells floating in the secretory fluid like islands. The pancreatic islets (islets of Langerhans) are spherical endocrine gland tissues scattered throughout the pancreas of many vertebrates.

[0050] With the nucleic acid introduction method described above, applying an electric field after nucleic acid introduction causes the nucleic acid to enter from the periphery of the pancreatic islets. Electroporation encounters resistance at the first layer of the cell, preventing it from penetrating deeper cell layers (it enters the surface). Therefore, when nucleic acid enters from the surface of the pancreatic islets, it does not enter the central part (core), but can be specifically introduced to the peripheral part (periphery of the pancreatic islets).

[0051] Whether or not nucleic acids were successfully introduced into the pancreatic islet margins can also be determined by the same method used to confirm whether or not nucleic acids were successfully introduced into immature β-cells. Examples of the aforementioned immature β-cells include cells that are weakly insulin-positive and glut2-negative or weakly positive.

[0052] (Animals into which nucleic acids have been introduced into the periphery of the pancreatic islets) The animals into which nucleic acids have been introduced into the periphery of the pancreatic islets are produced by the nucleic acid introduction method described above or by the method of introducing nucleic acids into the periphery of the pancreatic islets. [Examples]

[0053] The following describes embodiments of the present invention, but the present invention is not limited in any way to these embodiments.

[0054] <Nucleic acid introduction> (Example 1) 1. Mice in which the loxP-STOP-loxP-YFP gene was knocked into the Rosa locus (ROSA-loxP-STOP-loxP YFP) were anesthetized with isoflurane and underwent laparotomy. The anesthesia equipment used included the KN-1071 NARCOBIT-E (for isoflurane), KN-1010 induction anesthesia box set, KN-1013 color flow meter, KN-1019 Natsume-type anesthesia mask, and KN-1050-R anesthetic gas processing device (Type R), all manufactured by Natsume Seisakusho Co., Ltd.

[0055] 2. I identified the location of the ampulla of Vater, which is the opening of the pancreatic duct and common bile duct into the duodenum. 3. The common bile duct on the liver side is secured in two places with clips (KN-353 Disposable clips for animal experiments (non-sterile), manufactured by Natsume Seisakusho Co., Ltd.), and a nucleic acid-containing solution (DNA total 4 μg / μL, 0.1% Fast Green) containing an expression vector (Rip-Cre: pRIP.Cre.GH) (J Biol Chem. 1999 274:305-15, Genbank file pRIP-Cre-GH.gp) with the Cre recombinase gene linked downstream of the rat insulin promoter is filled into a 33G injection needle (TASK Steriject PRE-33013-100). This solution contains 180-240 μL of nucleic acid-containing solution (DNA purified using the Qiagen GIGA Endfree kit and dissolved in distilled water, to which 1 / 10 of 10× PBS, 1 / 10 of 1% Fast Green, and 1 / 10 of 10% TCHD are added, and finally adjusted to 1× with distilled water). The tip of a 33G 0.24×13mm needle was inserted from the outside of the duodenum (covered by a double layer of muscle, the inner circular muscle and the outer longitudinal muscle) into the closed center of the sphincter, and then into the pancreatic duct through the ampulla of Vater. The ampulla of Vater was also secured with a clip (KN-353 disposable clip for animal experiments (non-sterile), manufactured by Natsume Seisakusho Co., Ltd.), and the nucleic acid-containing solution (100-160 μL) was slowly injected.

[0056] 4. After removing the needle and clip, the pancreas was quickly sandwiched between gauze moistened with physiological saline solution, and electroporation was performed at a constant voltage using a BEX Pulse Generator CUY21Vivo-SQ (1st electrical pulse 45V 45ms, 1 time: approximately 250mA; 2nd electrical pulse 25V 45ms (ON time), 5 times: approximately 120mA (the interval between each pulse was 999ms (OFF time))) at a total of 3 locations: 2 in the head of the pancreas (duodenal side) and 1 in the tail of the pancreas (spleen side). 5. The abdomen was closed.

[0057] After 6.7 days, the pancreas was perfused and fixed, removed, washed with PBS, replaced with 30% sucrose, embedded in OCT compound, and frozen. Sections 20 μm thick were then prepared. The sections were permeabilized with PBS containing 1% triton-100 for 30 minutes, and then immunostained with primary antibodies (Guinea pig-anti-insulin antibody (Dako Envision FLEX-Insulin, Agilent Technologies, Inc.) and Mouse-anti-GFP antibody (recognizes YFP)) (11 814 460 001 Roche) and secondary antibodies (Alexa647-anti-Guinea pig IgG(L+H) (706-606-148 Jackson Immuno Research) and Alexa488-anti-Mouse IgG(H+L)) (715-546-151 Jackson Immuno Research), followed by nuclear staining with DAPI. The results of observing the aforementioned section with a Zeiss LSM710 are shown in the first row of Figure 3. In Figure 3, from right to left, the combined staining results for each antibody are shown: the staining results for anti-insulin antibody, the staining results for anti-GFP antibody, and the DAPI staining results. The YFP positivity rate is shown in Figure 4.

[0058] By introducing an expression vector (Rip-Cre) in which the Cre recombinase gene is linked downstream of the rat insulin promoter into mice (ROSA-loxP-STOP-loxP YFP) in which the loxP-STOP-loxP-YFP gene has been knocked into the Rosa locus, Cre is expressed in cells where insulin gene expression is activated. The STOP codon flanked by the loxP sequence is then removed by Cre recombinase, resulting in the expression of the YFP gene. Since Rip-Cre is introduced only into the periphery of the pancreatic islets, YFP positivity is observed in cells where insulin gene expression has been activated in the periphery of the pancreatic islets.

[0059] (Example 2) Nucleic acid introduction was carried out in the same manner as in Example 1, except that trans-cyclohexane-1,2-diol (TCHD) was added to the nucleic acid-containing solution to a concentration of 0.25%. The results are shown in the second row of Figure 3 and in Figure 4.

[0060] (Example 3) Nucleic acid introduction was carried out in the same manner as in Example 1, except that trans-cyclohexane-1,2-diol (TCHD) was added to the nucleic acid-containing solution to a concentration of 0.5%. The results are shown in the third row of Figure 3 and in Figure 4.

[0061] (Example 4) Nucleic acid introduction was carried out in the same manner as in Example 1, except that trans-cyclohexane-1,2-diol (TCHD) was added to the nucleic acid-containing solution to a concentration of 1%. The results are shown in the fourth row of Figure 3 and in Figure 4.

[0062] In this example, Rip-Cre is used as the assay method for introducing nucleic acids into the periphery of the pancreatic islets, rather than an expression vector that can be expressed anywhere, such as CAG-RFP. Therefore, Figure 4 shows the percentage of cells that not only received nucleic acid introduction but also expressed Rip-Cre in insulin promoter-activated cells, undergoing recombination and expressing YFP. For example, in Example 4, the 12% positive rate for YFP is based on the number of cells in the section counted by the nucleus of DAPI (including many β cells in the core). Considering the proportion of β cells in the periphery of the pancreatic islets (10-20% of the total), the number of cells introduced is considerable.

[0063] (Example 5) Nucleic acid was introduced and sections were prepared in the same manner as in Example 4. The sections were permeabilized with PBS containing 1% triton-100 for 30 minutes, and then immunostained with primary antibodies (Guinea pig anti-insulin antibody, Rabbit anti-Glut2 antibody (#07-1402-I, Merck KGaA), and Rat anti-GFP antibody (recognizes YFP)) and secondary antibodies (Alexa647 anti-Guinea pig IgG (L+H) (706-606-148, Jackson Immuno Research), Alexa594 anti-Rabbit IgG (H+L) (711-586-152, Jackson Immuno Research), and Alexa488 anti-Rat IgG (H+L) (712-546-153, Jackson Immuno Research)), followed by nuclear staining with DAPI. The results of observing the aforementioned section with a Zeiss LSM710 are shown in Figure 5. In Figure 5, from right to left, the combined staining results for each antibody are shown: the staining results for anti-insulin antibody, anti-Glut2 antibody, anti-GFP antibody, and DAPI.

[0064] Figure 5 shows that immature β-cells in the periphery of the pancreatic islets that are weakly insulin-positive and glut2-negative or weakly positive express YFP (e.g., cell A in Figure 5), while cells that appear to be β-cells in the core of the pancreatic islets that are strongly insulin and glut2-positive do not express YFP (e.g., cell B in Figure 5). Furthermore, more than two weeks after electroporation, YFP-positive β cells appeared in the core of the pancreatic islets, suggesting that immature YFP-positive β cells from the islet periphery matured and migrated.

[0065] Based on these results, it was found that nucleic acids can be introduced to the periphery of the pancreatic islets without intraparenchymal injection into the pancreas by a nucleic acid introduction method that includes an injection step of a nucleic acid-containing solution into the pancreatic duct from the ampulla of Vater.

[0066] Examples of embodiments of the present invention include the following: <1> This method for introducing nucleic acids is characterized by including an injection step in which a nucleic acid-containing solution is injected into the pancreatic duct through the ampulla of Vater of an animal (excluding humans). <2> The nucleic acid-containing solution contains a nuclear translocation promoter, <1> This is the nucleic acid introduction method described in [reference]. <3> The nuclear translocation promoter is trans-cyclohexane-1,2-diol (TCHD), <2> This is the nucleic acid introduction method described in [reference]. <4> The concentration of the TCHD in the nucleic acid-containing solution is 0.1% or more and 5% or less. <3> This is the nucleic acid introduction method described in [reference]. <5> The injection step is followed by an electroporation step, which includes an electroporation step. <1> This is the nucleic acid introduction method described in [reference]. <6> In the electroporation process, one first electrical pulse with a voltage of 30V or more and 60V or less is applied, and two or more second electrical pulses with a voltage of 10V or more and 40V or less are applied. <5> This is the nucleic acid introduction method described in [reference]. <7> In the electroporation process, one first electrical pulse with a current of 150 mA or more and 350 mA or less is applied, and two or more second electrical pulses with a current of 20 mA or more and 220 mA or less are applied. <5> This is the nucleic acid introduction method described in [reference]. <8> The duration of the first electrical pulse is 1 millisecond or more and 500 milliseconds or less, and the duration of the second electrical pulse is 1 millisecond or more and 500 milliseconds or less, <6> This is the nucleic acid introduction method described in [reference]. <9> In the electroporation process, electrical pulses are applied to two locations in the head of the pancreas and one location in the tail of the pancreas. <5> This is the nucleic acid introduction method described in [reference]. <10> The aforementioned <1> from <9> This method involves introducing nucleic acids into the periphery of pancreatic islets, characterized by using one of the nucleic acid introduction methods described in any of the above.

Claims

1. A method for introducing nucleic acids, characterized by including an injection step of injecting a nucleic acid-containing solution into the pancreatic duct through the ampulla of Vater of an animal (excluding humans).

2. The nucleic acid introduction method according to claim 1, wherein the nucleic acid-containing solution contains a nuclear translocation promoter.

3. The nucleic acid delivery method according to claim 2, wherein the nuclear translocation promoter is trans-cyclohexane-1,2-diol (TCHD).

4. The nucleic acid introduction method according to claim 3, wherein the concentration of TCHD in the nucleic acid-containing solution is 0.1% or more and 5% or less.

5. The nucleic acid introduction method according to claim 1, further comprising an electroporation step performed after the injection step.

6. The nucleic acid introduction method according to claim 5, wherein in the electroporation step, one first electrical pulse with a voltage of 30V or more and 60V or less is applied, and two or more second electrical pulses with a voltage of 10V or more and 40V or less are applied.

7. The nucleic acid introduction method according to claim 5, wherein in the electroporation step, one first electrical pulse with a current of 150 mA or more and 350 mA or less is applied, and two or more second electrical pulses with a current of 20 mA or more and 220 mA or less are applied.

8. The nucleic acid introduction method according to claim 6, wherein the duration of the first electrical pulse is 1 millisecond or more and 500 milliseconds or less, and the duration of the second electrical pulse is 1 millisecond or more and 500 milliseconds or less.

9. The nucleic acid introduction method according to claim 5, wherein in the electroporation step, electrical pulses are applied to two locations in the head of the pancreas and one location in the tail of the pancreas.

10. A method for introducing nucleic acids to the periphery of pancreatic islets, characterized by using the nucleic acid introduction method described in any one of claims 1 to 9.

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