A low-cost, high-adaptability consumable kit for a high-pressure gas gene gun and a method for high-pressure gas gene gun bombardment using the same

Abstract

The present application belongs to the technical field of high-pressure gas gene gun, and particularly relates to a low-cost and high-adaptability consumable kit for a high-pressure gas gene gun and a method for high-pressure gas gene gun bombardment by using the same. The consumable kit provided by the present application comprises: a breakable film assembly composed of polyethylene terephthalate film; a blocking and dispersing assembly composed of stainless steel woven mesh; a sample bearing assembly composed of polyimide film; and a gene gun metal particle assembly composed of tungsten powder. The consumable kit provided by the present application is low in cost, high in flexibility of material parameter adjustment, and high in universality. When the consumable kit is used for introduction of exogenous genes, the gene transfection efficiency is equivalent to that of a conventional high-pressure gas gene gun.

Description

Technical Field

[0001] This invention belongs to the field of high-pressure gas gene gun technology, specifically relating to a low-cost, highly adaptable consumable kit for a high-pressure gas gene gun and a method for using the kit to bombard with a high-pressure gas gene gun. Background Technology

[0002] High-pressure gas gene guns (gene guns) are core equipment for genetic manipulation in the field of biotechnology. Their principle involves using high-pressure gas to accelerate micron-sized gold powder (BIORAD Cat.#1652262 0.6 μm Gold Microcarriers) coated with DNA, allowing it to penetrate the cell wall or cell membrane and thus introduce foreign genes. Currently, domestically produced gene guns, such as the Ningbo Xinzhi GJ-1000, are widely used in transgenic research on plants, microorganisms, and small animal cells due to their high cost-effectiveness.

[0003] The reliable operation of this device is highly dependent on a series of special consumables, mainly including: (1) a ruptureable membrane, which is mostly a polyester film of a specific thickness (such as 25 μm or 50 μm) for precise control of gas release pressure; (2) a barrier mesh / metal mesh membrane for intercepting large particles, sample carrier membranes, and dispersing microparticle streams to avoid excessive damage to target cells; (3) a sample carrier membrane / flying membrane for carrying micron-sized gold powder particles coated with DNA to be transfected; and (4) nano gold powder particles, which serve as carriers of DNA and penetrate target cells through physical acceleration.

[0004] The existing technology and consumables system has the following core defects: First, the cost is high. Original manufacturer-specific consumables (such as the rupture membrane, flight membrane, metal mesh, and BIORAD 0.6 μm gold powder that come with the GJ-1000) are expensive. As a commonly used gene manipulation technique in molecular biology experiments, the cost of consumables for a single experiment, excluding high-pressure gas, is as high as 80 yuan, and the cost of purchasing a complete set of consumables for a single experiment is nearly 10,000 yuan.

[0005] Second, the fixed material parameters limit experimental flexibility. Original manufacturer-supplied rupture membranes typically offer only a few fixed thicknesses, corresponding to limited rupture pressure thresholds. However, different target cells (such as yeast and fungi with tough cell walls versus fragile animal cells) require significantly different bombardment pressures. Existing consumables with fixed parameters cannot achieve precise and continuous pressure adjustment, limiting the gene gun's adaptability to different biological samples.

[0006] Third, poor versatility leads to significant resource waste. Consumables for different brands or models of gene guns are incompatible. During equipment replacement or shared use, laboratories often experience substantial idleness and waste due to incompatible consumable specifications. Summary of the Invention

[0007] In view of this, the present invention provides a low-cost, highly adaptable consumable kit for a high-pressure gas gene gun and a method for high-pressure gas gene gun bombardment using the same kit. The consumable kit provided by the present invention is low in cost, highly flexible with adjustable material parameters, and highly versatile. Furthermore, when using the consumable kit provided by the present invention for the introduction of exogenous genes, the gene transfection efficiency is comparable to that of traditional high-pressure gas gene guns.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a low-cost, highly adaptable consumable kit for a high-pressure gas gene gun, comprising: Ruptureable membrane assembly: composed of polyethylene terephthalate film; Barrier and dispersion components: made of stainless steel woven mesh; Sample carrier component: composed of polyimide film; Gene gun metal microparticle assembly: composed of tungsten powder.

[0009] Preferably, the thickness of the polyethylene terephthalate film is 150~200 μm.

[0010] Preferably, the stainless steel woven mesh has a mesh count of 20 to 30; and the stainless steel wire constituting the stainless steel woven mesh has a diameter of 0.2 to 0.3 mm.

[0011] Preferably, the thickness of the polyimide film is 0.05 mm.

[0012] Preferably, the tungsten powder has a particle size of 0.4~0.8 μm and a purity of ≥99.9%.

[0013] The present invention also provides a method for high-pressure gas gene gun bombardment using the above-described consumable kit, comprising the following steps: (1) After washing, the tungsten powder is resuspended in an aqueous glycerol solution to obtain a pretreated tungsten powder suspension; (2) The tungsten powder suspension was mixed with DNA solution, calcium chloride solution and spermidine solution in sequence. The resulting mixture was incubated on ice and washed in sequence, and then resuspended in alcohol to obtain DNA-tungsten powder complex suspension. (3) Place the polyethylene terephthalate film at the outlet end of the gene gun gas accelerator tube and tighten the seal to put it in a taut state; (4) Place the stainless steel mesh flat in the mesh slot downstream of the polyethylene terephthalate film; (5) Fix the polyimide film in the sample slot of the gene gun; (6) The DNA-tungsten powder complex suspension was dropped onto the central region of the polyimide film and dried to obtain a carrier film with DNA sample attached; (7) Place the carrier membrane with the DNA sample attached, along with the sample tank, upside down into the metal mesh membrane pressure ring, and place them sequentially on the upstream of the metal mesh membrane, then tighten the gene gun nut. (8) After fixing the sample to be transfected on the bottom plate of the gene gun sample chamber, the gene is introduced by bombardment.

[0014] Preferably, in step (1), the cleaning is performed sequentially by an alcohol aqueous solution and a sterile purified water.

[0015] Preferably, the specific steps of the alcohol-water cleaning are as follows: mix tungsten powder with 70% vol alcohol-water solution, vortex at room temperature for 3-5 minutes and let stand for 15 minutes, then centrifuge the mixture, discard the supernatant, and obtain tungsten powder cleaned with alcohol. The sterile purified water washing step is as follows: mix the tungsten powder washed with alcohol with sterile purified water, vortex for 1 minute, let stand for 1 minute, centrifuge for 5-8 seconds, discard the supernatant, and obtain the tungsten powder washed with sterile purified water; the sterile purified water washing is performed 3 times. The step of resuspending in glycerol aqueous solution is as follows: mix tungsten powder washed with sterile purified water with glycerol aqueous solution and vortex for 2-3 minutes; the concentration of glycerol aqueous solution is 50% vol.

[0016] Preferably, the concentration of the DNA solution is 0.1~2.0 μg / μL; the concentration of the CaCl2 solution is 2.5M; and the concentration of the spermidine solution is 0.1M.

[0017] Preferably, the volume ratio of the tungsten powder suspension to the DNA solution, CaCl2 solution, and spermidine solution is 5:6:10:4; and the incubation time on ice is 10 min.

[0018] Preferably, the distance between the bottom plate of the silo and the barrier net is 3 to 10 cm.

[0019] Compared with the prior art, the technical solution provided by the present invention has the following advantages: First, the cost is significantly reduced. The polyethylene terephthalate film, polyimide film, stainless steel mesh, and commercially available tungsten powder used are all industrial standard parts produced on a large scale. The material cost of a single experiment has been reduced from tens of yuan to less than one yuan, which significantly lowers the application threshold of gene gun technology.

[0020] Second, the pressure adjustment range is wide and continuous, providing high experimental flexibility. The rupture pressure can be adjusted by changing the thickness of the polyethylene terephthalate film, thereby optimizing the bombardment parameters.

[0021] Third, it breaks the dependence on the supply chain and achieves "on-demand production." The alternative materials are all general-purpose industrial materials with clear specifications and parameters, and are widely available and readily accessible. Laboratories do not need to stockpile expensive specialized consumables for extended periods.

[0022] Fourth, it has good biological and chemical compatibility. Polyethylene terephthalate film, polyimide film, stainless steel and tungsten powder are all inert materials, non-toxic and non-allergenic, and resistant to common surface disinfectants such as ethanol, and can be directly applied to sterile biological experiments. Detailed Implementation

[0023] This invention provides a low-cost, highly adaptable consumable kit for a high-pressure gas gene gun, comprising: Ruptureable membrane assembly: composed of polyethylene terephthalate film; Barrier and dispersion components: made of stainless steel woven mesh; Sample carrier component: composed of polyimide film; Gene gun metal microparticle assembly: composed of tungsten powder.

[0024] In this invention, the thickness of the polyethylene terephthalate film is 150~200 μm; the shape of the polyethylene terephthalate film is a circular sheet with a diameter of 16 mm.

[0025] In this invention, the stainless steel woven mesh is a circular woven mesh sheet with a diameter of 24-26 mm, preferably 25 mm; the stainless steel wire (preferably 304 stainless steel wire) has a diameter of 0.2-0.3 mm; the stainless steel woven mesh has a mesh count of 20-30, preferably 24. In this invention, the edges of the stainless steel woven mesh can be flattened or edge-bound.

[0026] In this invention, the polyimide film can be a circular sheet with a diameter of 25 mm and a thickness of 0.05 mm, which can ensure its dimensional stability under high-pressure gas impact.

[0027] In this invention, the tungsten powder has a particle size of 0.4~0.8 μm, preferably 0.5 μm; a purity of ≥99.9%; the tungsten powder is spherical or near-spherical particles; the tungsten powder particles are uniform, without agglomeration or with slight agglomeration and are dispersible.

[0028] The present invention also provides a method for bombarding the above-mentioned consumable kit with a high-pressure gas gene gun, comprising the following steps: (1) After washing, the tungsten powder is resuspended in an aqueous glycerol solution to obtain a pretreated tungsten powder suspension; (2) The tungsten powder suspension was mixed with DNA solution, calcium chloride solution and spermidine solution in sequence. The resulting mixture was incubated on ice and washed in sequence, and then resuspended in alcohol to obtain DNA-tungsten powder complex suspension. (3) Place the polyethylene terephthalate film at the outlet end of the gene gun gas accelerator tube and tighten the seal to put it in a taut state; (4) Place the stainless steel mesh flat in the mesh slot downstream of the polyethylene terephthalate film; (5) Fix the polyimide film in the sample slot of the gene gun; (6) The DNA-tungsten powder complex suspension was dropped onto the central region of the polyimide film and dried to obtain a carrier film with DNA sample attached; (7) Place the carrier membrane with the DNA sample attached, along with the sample tank, upside down into the metal mesh membrane pressure ring, and place them sequentially on the upstream of the metal mesh membrane, then tighten the gene gun nut. (8) After fixing the sample to be transfected on the bottom plate of the gene gun sample chamber, the gene is introduced by bombardment.

[0029] In this invention, tungsten powder is washed and then resuspended in an aqueous glycerol solution to obtain a pretreated tungsten powder suspension.

[0030] In this invention, the cleaning can be a sequential process of alcohol aqueous solution cleaning and sterile purified water cleaning.

[0031] In this invention, the specific steps of the alcohol-water cleaning are as follows: tungsten powder and 70% vol alcohol-water solution are mixed, vortexed at room temperature for 3-5 minutes, and then allowed to stand for 15 minutes. The mixture is then centrifuged, and the supernatant is discarded to obtain the alcohol-cleaned tungsten powder. In this invention, the centrifugation speed can be 2000 rpm, and the time can be 1 minute.

[0032] In this invention, the sterile purified water washing step involves mixing the tungsten powder washed with alcohol with sterile purified water, vortexing for 1 minute, letting it stand for 1 minute, centrifuging for 5-8 seconds, discarding the supernatant, and obtaining the tungsten powder washed with sterile purified water; the sterile purified water washing can be performed 3 times.

[0033] In this invention, the step of resuspending in glycerol aqueous solution is as follows: tungsten powder washed with sterile purified water is mixed with glycerol aqueous solution and vortexed for 2-3 min; the concentration of the glycerol aqueous solution is 50% vol.

[0034] After obtaining the tungsten powder suspension, the present invention mixes the tungsten powder suspension with DNA solution, calcium chloride solution and spermidine solution in sequence. The resulting mixture is then incubated on ice, washed and resuspended in alcohol to obtain DNA-tungsten powder complex suspension.

[0035] In this invention, the concentration of the DNA solution can be 0.1~2.0 μg / μL; the concentration of the CaCl2 solution can be 2.5 M; the concentration of the spermidine solution can be 0.1 M; the volume ratio of the tungsten powder suspension to the DNA solution, CaCl2 solution, and spermidine solution can be 5:6:10:4; in this invention, the tungsten powder suspension is mixed with the DNA solution, CaCl2 solution, and spermidine solution by vortexing, wherein the vortexing time is 3 s when mixing with the DNA solution, 2~3 s when mixing with the CaCl2 solution, and 3~5 s when mixing with the spermidine solution. In this invention, the incubation time on ice can be 10 min. In this invention, the washing consists of washing with a 70% vol alcohol aqueous solution pre-cooled to -20℃ and washing with 100% alcohol pre-cooled to -20℃.

[0036] In this invention, a polyethylene terephthalate (PET) film is placed at the outlet end of the gas accelerator tube of a gene gun and sealed tightly to put it under tension; a stainless steel mesh is placed flat in the mesh slot downstream of the PET film; a polyimide film is fixed in the sample holder of the gene gun; and the DNA-tungsten powder composite suspension is dropped onto the central region of the polyimide film and dried to obtain a carrier membrane with DNA sample attached.

[0037] In this invention, the drying can be done at room temperature; the drying time at room temperature can be 5 to 10 minutes; the drying can be done until there are no obvious liquid marks on the surface, so that the DNA-tungsten powder complex is firmly attached to the film surface.

[0038] In this invention, a carrier membrane with attached DNA sample is inverted and inserted into a metal mesh pressure ring along with a sample tank, and then placed sequentially upstream of the metal mesh membrane. The gene gun nut is then tightened. After the sample to be transfected is fixed on the bottom plate of the gene gun sample compartment, it is bombarded to complete the gene introduction.

[0039] In this invention, when tightening the gene gun nut, it is sufficient to tighten it until it is no longer loose, but not so tight as to deform it.

[0040] In this invention, the distance between the bottom plate of the silo and the barrier net is 3 to 10 cm, preferably 6 to 8 cm.

[0041] In this invention, the bombardment operation is as follows: The gene gun switch is turned on, a vacuum is drawn, and the high-voltage trigger is pressed and held. High-pressure gas (helium) first causes the polyethylene terephthalate (PET) film to burst. The shock wave generated by the burst carries the DNA-coated metal particles and the sample carrier component (polyimide membrane / flying membrane) together towards the blocking and dispersing component (stainless steel mesh). The particles are uniformly dispersed and continue to accelerate as they pass through the metal mesh, while the carrier membrane is intercepted by the stainless steel mesh. Finally, the DNA-coated metal particles penetrate the sample to be transfected located on the substrate, completing the gene introduction.

[0042] In this invention, when the thickness of the polyethylene terephthalate film is 200 μm, the bursting pressure is 7~8 MPa; when the thickness of the polyethylene terephthalate film is 150 μm, the bursting pressure is 5.0 MPa.

[0043] In summary, this invention utilizes inexpensive, mass-produced industrial materials such as polyethylene terephthalate (PET) film, polyimide film, stainless steel mesh, and tungsten powder, instead of specialized injection molding materials or imported polymer materials and pre-treated precious metal particles, thus reducing consumable costs. The wide and continuous adjustable pressure range stems from using a 150-200 μm thick PET film as the rupture membrane. By selecting different thicknesses or stacking layers, the rupture threshold can be controlled (approximately 1.5 MPa pressure can be obtained when the PET film thickness is 50 μm; approximately 3.5 MPa pressure can be obtained when the PET film thickness is 100 μm; approximately 5.0 MPa pressure can be obtained when the PET film thickness is 150 μm; and approximately 7.0-8 MPa pressure can be obtained when the PET film thickness is 200 μm). The advantage of selectable particle size stems from the use of tungsten powder with a particle size of 0.4~0.8 μm (optimal 0.5 μm), replacing the original manufacturer's fixed-size heavy metal particles. The advantage of breaking supply chain dependence comes from standardized material specifications (thickness, mesh count, diameter, particle size), all of which are industry-standard, not equipment-specific. The advantage of uniform bombardment and minimal damage comes from using a 24-mesh stainless steel mesh as a blocking and dispersing element; its porosity and pore size work synergistically to optimize airflow and particle dynamics. The advantage of good biocompatibility comes from the chemical inertness of the materials themselves (PET, polyimide, stainless steel, tungsten), and the high thermal stability of the polyimide film, which prevents debris shedding under high-pressure gas impact.

[0044] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1 Ruptureable membrane assembly: 200 μm thick, 16 mm diameter circular polyethylene terephthalate film (purchased from Dongguan Jiuzhou Packaging Materials Co., Ltd.), with a smooth surface and no visible defects; single layer; Blocking and dispersing components: 24-mesh stainless steel mesh, 25 mm in diameter; Sample support component: 0.05 mm thick, 25 mm diameter circular polyimide film (purchased from Shenzhen Changlongxin Plastic Materials Co., Ltd.); Gene gun metal microparticle assembly: pretreated tungsten powder with a particle size of 0.5 μm; Pretreatment steps: Place 200 mg of 0.5 μm tungsten powder in a 1.5 mL centrifuge tube, add 1 mL of 70% vol ethanol, vortex at 12000 rpm for 5 min at room temperature, let stand at room temperature for 15 min, then centrifuge at 2000 rpm for 1 min, discard the supernatant to obtain ethanol-washed tungsten powder; add 1 mL of sterile purified water to the ethanol-washed tungsten powder, vortex for 1 min, let stand for 1 min, centrifuge at 3000 rpm for 8 s, discard the supernatant, repeat the sterile purified water washing process 3 times to obtain sterile purified water-washed tungsten powder; add 1 mL of 50% vol glycerol to the sterile purified water-washed tungsten powder in a clean bench, vortex for 3 min, and store at -20 ℃ for later use (pretreated tungsten powder suspension); DNA-Tungsten Powder Complex: The pretreated tungsten powder suspension was vortexed for 5 s. 25 μL of the tungsten powder suspension was added to 30 μL of paromomycin anti-plasmid DNA solution (0.5 μg / μL). After vortexing for 3 s, 50 μL of pre-cooled 2.5 M CaCl2 solution was added and vortexed for 3 s. Then, 20 μL of pre-cooled 0.1 M spermidine solution was added and gently vortexed for 5 s. The mixture was then placed on ice for 10 min, centrifuged briefly (5000 rpm for 5 s), and the supernatant was discarded. 200 μL of 50% vol ethanol pre-cooled to -20 °C was added to the resulting solid phase. Tungsten powder on the tube wall was blown to the bottom of the tube using a pipette tip and mixed thoroughly. The mixture was centrifuged (5000 rpm for 5 s) and the supernatant was discarded. Another 200 μL of pre-cooled ethanol was added to the resulting solid phase. 100% alcohol at ℃ was used to blow tungsten powder from the tube wall to the bottom of the tube with a pipette tip. The mixture was then blown and mixed. After centrifugation (5000 rpm for 5 s), the supernatant was discarded. 10 μL of pre-cooled 100% alcohol was added to the obtained solid phase and the mixture was vortexed for 2 s to obtain the DNA-tungsten powder complex.

[0046] Place the polyethylene terephthalate (PET) film at the outlet end of the gas accelerator tube of the gene gun and tighten it with the sealing cap (the installation torque should be such that the sealing cap is tightened to the point of no gaps) to ensure it is under tension. Place the stainless steel mesh in the metal mesh base slot downstream of the PET film, ensuring it falls completely into the slot, flat and without wrinkles. Fix the polyimide film in the sample slot of the gene gun, ensuring the film is flat and taut. Add 10 μL of DNA-tungsten powder complex suspension evenly to the center area of ​​the polyimide film, and then dry it at room temperature in a clean bench for 10 min until there are no obvious liquid marks on the surface, so that the DNA-tungsten powder complex is firmly attached to the film surface. After drying, the sample-attached sample-attached sample-carrying membrane is inverted and placed into the metal mesh pressure ring, and then placed sequentially upstream of the metal mesh. After loading, tighten the gene gun nut.

[0047] 10 mL of Tetrahymena cells (approximately 1 × 10⁻⁶) cultured to the plateau phase were used. 7 Centrifuge at 600 g for 3 min, resuspend in 10 mL of sterile 10 mM Tris-HCl solution, repeat centrifugation at 600 g for 3 min, then resuspend in 10 mL of sterile 10 mM Tris-HCl solution in a culture dish and incubate overnight (8 h) at 30°C. The next day, before transfection, centrifuge the overnight cultured cells at 600 g for 3 min and resuspend in 1 mL of sterile 10 mM Tris-HCl solution to prepare the transfection sample.

[0048] In a clean petri dish, sterile filter paper was moistened with 1 mL of 10 mM Tris-HCl. The sample to be transfected was then laid flat on the moistened filter paper and fixed to the bottom plate of the gene gun sample compartment, with the bottom plate 6 cm away from the blocking mesh. The gene gun was turned on, and after evacuation, the high-pressure trigger was pressed and held. The high-pressure gas first caused the ruptureable membrane component (polyethylene terephthalate film) to burst, generating an impact force of approximately 7 MPa. This shock wave carried the DNA-coated metal particles and the sample carrier component (polyimide membrane / flying membrane) together towards the blocking and dispersing component (stainless steel metal mesh). The particles were uniformly dispersed and continued to accelerate as they passed through the metal mesh, while the carrier membrane was intercepted by the metal mesh. Finally, the DNA-coated metal particles penetrated the sample on the bottom plate, completing the bombardment. After the bombardment, the sample was transferred to 30 °C for recovery culture for 6 h, and then paromomycin at a final concentration of 120 μg / mL was added for screening. The samples were then aliquoted into 96-well plates for further culture.

[0049] Experimental results: Transfection efficiency (baromomycin positivity rate): 100% (n=1).

[0050] Cost of consumables per experiment (ruptureable membrane assembly, barrier and dispersion assembly, sample carrier assembly and tungsten powder): approximately 0.3 yuan.

[0051] Example 2 The only difference from Example 1 is that when the thickness of the polyethylene terephthalate film is 50 μm, the high-pressure gas causes the ruptureable membrane module (polyethylene terephthalate film) to burst, generating a pressure of about 1.5 MPa.

[0052] Example 3 The only difference from Example 1 is that when the thickness of the polyethylene terephthalate film is 100 μm, the high-pressure gas causes the ruptureable membrane module (polyethylene terephthalate film) to burst, generating a pressure of about 3.5 MPa.

[0053] Example 4 The only difference from Example 1 is that when the thickness of the polyethylene terephthalate film is 150 μm, the high-pressure gas causes the ruptureable membrane module (polyethylene terephthalate film) to burst, generating a pressure of about 5.0 MPa.

[0054] For Tetrahymena transfection, the required high-pressure gas pressure is usually 4.0~7.0 MPa. Therefore, the suitable thickness range of the ruptureable membrane selected in this invention is 150~200 μm.

[0055] Example 5 The only difference from Example 1 is that the thickness of the polyimide film in the sample carrier component is 0.025 mm, and the transfection efficiency (n=1) is 8.75%.

[0056] Example 6 The only difference from Example 1 is that the thickness of the polyimide film in the sample carrier component is 0.075 mm, and the transfection efficiency (n=1) is 11.54%.

[0057] For the same plasmid, all three thicknesses of polyimide films in Examples 1 and 5-6 successfully underwent high-pressure gas gene gun bombardment. However, the 0.025 mm thick film was too soft, had strong surface static electricity, was difficult to assemble, and could not provide sufficient support; the 0.075 mm thick film was too tough, making it inconvenient to handle during gene gun assembly, resulting in very low transfection efficiency. In contrast, the 0.05 mm thick polyimide film combined toughness and tensile strength while ensuring high transfection efficiency.

[0058] Example 3 Using paromomycin resistance plasmids carrying different target genes, the operation in Example 1 was repeated 28 times, and the average transfection efficiency of the 28 transfections was 97.60%.

[0059] Comparative Example 1 The only difference from Example 1 is that the gene gun is composed of: Rupture diaphragm: Ningbo Xinzhi GJ-1000 original factory-matched rupture diaphragm (nominal pressure 7.0 MPa setting). Barrier mesh: Ningbo Xinzhi GJ-1000 original factory matching metal mesh membrane Sample carrier film: Ningbo Xinzhi GJ-1000 original factory matching flight film Gene gun metal microparticles: Gold powder (BIORAD Cat.#1652262 0.6 μm Gold Microcarriers) Other conditions are the same as in Example 1.

[0060] Experimental results: Average transfection efficiency (baromomycin positivity rate): 98.95% (n=2); Cost of consumables per experiment: approximately 80 yuan.

[0061] Compared with Example 3, the transfection efficiency of the original consumables (Comparative Example 1) was 98.95%, while the scheme in Example 3 was 97.60%, showing comparable transfection efficiencies. However, the cost of consumables per use in this invention (approximately 0.3 yuan) is significantly lower than the cost of the original consumables (approximately 80 yuan). Furthermore, the polyethylene terephthalate film (200 μm thick), polyimide film (0.05 mm thick), stainless steel mesh (24 mesh), and tungsten powder (0.5 μm particle size) used in this invention are all general-purpose industrial materials with clearly defined specifications, widely available and readily accessible. The thickness, mesh size, and particle size of each component can be selected within a certain range according to experimental needs, making the process more convenient.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A low-cost, highly adaptable consumable kit for a high-pressure gas gene gun, characterized in that, include: Ruptureable membrane assembly: composed of polyethylene terephthalate film; Barrier and dispersion components: made of stainless steel woven mesh; Sample carrier component: composed of polyimide film; Gene gun metal microparticle assembly: composed of tungsten powder.

2. The low-cost, highly adaptable consumable kit as described in claim 1, characterized in that, The thickness of the polyethylene terephthalate film is 150~200 μm.

3. The low-cost, highly adaptable consumable kit as described in claim 1, characterized in that, The stainless steel woven mesh has a mesh count of 20-30; the stainless steel wires constituting the stainless steel woven mesh have a diameter of 0.2-0.3 mm.

4. The low-cost, highly adaptable consumable kit as described in claim 1, characterized in that, The polyimide film has a thickness of 0.05 mm.

5. The low-cost, highly adaptable consumable kit as described in claim 1, characterized in that, The tungsten powder has a particle size of 0.4~0.8 μm; Purity ≥ 99.9%.

6. A method for high-pressure gas gene gun bombardment using the low-cost, highly adaptable consumable kit according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) After washing, the tungsten powder is resuspended in an aqueous glycerol solution to obtain a pretreated tungsten powder suspension; (2) The tungsten powder suspension was mixed with DNA solution, calcium chloride solution and spermidine solution in sequence. The resulting mixture was incubated on ice and washed in sequence, and then resuspended in alcohol to obtain DNA-tungsten powder complex suspension. (3) Place the polyethylene terephthalate film at the outlet end of the gene gun gas accelerator tube and tighten the seal to put it in a taut state; (4) Place the stainless steel mesh flat in the mesh slot downstream of the polyethylene terephthalate film; (5) Fix the polyimide film in the sample slot of the gene gun; (6) The DNA-tungsten powder complex suspension was dropped onto the central region of the polyimide film and dried to obtain a carrier film with DNA sample attached; (7) Place the carrier membrane with the DNA sample attached, along with the sample tank, upside down into the metal mesh membrane pressure ring, and place them sequentially on the upstream of the metal mesh membrane, then tighten the gene gun nut. (8) After fixing the sample to be transfected on the bottom plate of the gene gun sample compartment, the gene is introduced by bombardment.

7. The method as described in claim 6, characterized in that, In step (1), the cleaning is performed sequentially by an alcohol aqueous solution and a sterile purified water. The specific steps of the alcohol-water cleaning are as follows: mix tungsten powder with 70% vol alcohol-water solution, vortex for 3-5 minutes at room temperature, let stand for 15 minutes, then centrifuge the mixture, discard the supernatant, and obtain tungsten powder cleaned with alcohol. The sterile purified water washing step is as follows: mix the tungsten powder washed with alcohol with sterile purified water, vortex for 1 min, let stand for 1 min, centrifuge for 5-8 s, discard the supernatant, and obtain the tungsten powder washed with sterile purified water; the sterile purified water washing is performed 3 times. The step of resuspending in glycerol aqueous solution is as follows: mix tungsten powder washed with sterile purified water with glycerol aqueous solution and vortex for 2-3 minutes; the concentration of glycerol aqueous solution is 50% vol.

8. The method as described in claim 6, characterized in that, The concentration of the DNA solution is 0.1~2.0 μg / μL; the concentration of the CaCl2 solution is 2.5 M; and the concentration of the spermidine solution is 0.1 M.

9. The method as described in claim 6, characterized in that, The volume ratio of the tungsten powder suspension to the DNA solution, CaCl2 solution, and spermidine solution is 5:6:10:4; the incubation time on ice is 10 min.

10. The method as described in claim 6, characterized in that, The distance between the bottom plate of the silo and the barrier net is 3~10 cm.