JPH07504575A - Pre-treatment of ultrafine particles before using them in a particle gun - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] name of invention Background of the invention: Pre-treatment of ultrafine particles before use in a particle gun The present invention is aimed at improving methods for transporting biological substances such as nucleic acids into the cytoplasm of living cells. related. With rapid advances in recombinant DNA technology, biologists are increasingly transport from one cell to another, and transport synthetic biological substances to living cells. Increasingly, there is a need to activate biological materials. dyes and proteins (antibodies and enzymes), and in the most usual sense (RNA and DNA). A) includes nucleic acid genetic material.

Many of the technologies used are extremely slow and the methods used can only transport biological substances to at most a few cells at a time. . More recently, 5tanford et al. published the magazine “J” in 1987. Oural of Particle 5science and Techn As published in ``Deliv. ery of 5 ubstances int.

Ce1ls and Ti5sues Using AParticle Gu n'', a particle bombardment method using a particle gun has been developed.These descriptions are for reference only. For use in this disclosure.

Targeting earlier inventions by Dwight Tomes, one of the co-inventors This is an improvement to the particle gun that uses a gun with a rifled barrel. particle gun attack east For information on methods of transporting biological substances such as DNA and DNA into living cells, see 19. U.S. Patent Application No. 07/351 filed May 12, 1989. Specification No. 075 (Invention Author: Tomeselal, and title of invention: Improved Particle Gun). Ru. These descriptions are also used in this disclosure by reference.

Needless to say, the effectiveness of particle transport depends on the ability of the transport particle to be inserted and pick up biological material. In addition, the ability of biological cells to express traits can also be influenced by numerous conditions. It also depends on the situation. If trait expression is low, transport is not going well. Moreover, if the expression of biological cells is successful, it will be possible to pick up transport biological substances. In addition, if the range of trait expression is wide, it can be said that the nucleic acid insertion experiment was successful. That is.

In the case of particle sequencing, the biological material (such as DNA) is mixed with the carrier. Typically This carrier is essentially a metal insert.

It is the human body and takes the form of microbeads that act as microscopic particles. And these minute The particles are about 1 micron to about 4 microns in diameter. The material of these beads is ungsten, palladium, platinum or gold, or even alloys of these good. From an economic point of view, tungsten is preferable; If the metal is not treated in advance, the results will be poor for the transportation and presentation of the gold. . Bead diameters generally range from about 1 micron to about 1.5 microns.

The optimal method involves mixing the beads with a small amount of biological material, such as DNA or RNA. . This is mixed with calcium chloride and a predetermined amount of polyamine is added.

Generally speaking, the range of each of these components should be as follows.

Add 25 μl of tungsten particles to 2 ml of sterile water at a concentration of 5 to 400 mg; Transfer the mixture to a 10 ml sterile centrifuge tube and stir to suspend the beads. beads The preferred concentration of is 375 mg/2 ml. suspended tungsten steel Add 25 µI of the enzyme at a concentration of 1 µg/µI to an Esopendorf tube. This amount of beads is A force that can be adjusted between 1 μm and 20 μl (the preferred amount is 10 μm. DNA/ Bead mixture and 25/μm calcium chloride solution at 1°0-4.0M, preferably are mixed at a concentration of 2.5M.

Adding spermidine to biological material/microparticle complexes has been done for some time. However, more broadly speaking, when adjusting ultrafine particles, tungsten beads and Transformation rates can be increased by adding various polyamines to a mixture of DNA or RNA. This is the first time that a significant improvement has been found. controlled by theory Polyamines can modify the binding of cellular material to microbeads. improve the transport of biological substances into cells. Seen so far Suitable polyamines that have been published include spermine, spermidine, caldine (Cal dine), thermine, etc. A favorable Boria Mistake: It turns out that spermine is superior to the previously disclosed spermidine. 9 Therefore, at this point the concentration is 0. O’5M to 0.5M, preferably 0.1M Add the polyamine, preferably spermine, in a volume of 10 μl, then use your fingers to Vortex to mix. Let this mixture stand for 10 minutes, then 9, OO for 1-2 minutes. Centrifuge in Orpm. You can use centrifugation, but it is not necessary. Not at all. This ultrafine particle mixture becomes a pellet at the bottom of the Eppendorf tube.

Prior to use, remove the supernatant to obtain 30 μl of interest.

The DNA/bead mixture is briefly sonicated to suspend the microparticles before use. Ru. Suspended ultrafine particles carrying biological substances are 1 to 5μ11, preferably 1.5μ Transfer to the front end of the ultra-large particles by a micropipette in the form of a l aliquot.

Juushi: There are some difficulties when using ultrafine particles, especially when the particles contain large amounts of DNA. May aggregate. This not only makes separation difficult, but also causes the particles to or have a stronger tendency to kill cells.

Therefore, this method that does not kill cells during particle bombardment allows for low-cost treatment. By the method of utilizing ungsten beads, and such low cost beads by a method that can achieve a high degree of transformation and expression despite using There is a need to develop ultrafine particle bombardment methods to improve the transport of biological materials into living cells. still exists. The primary purpose of the present invention is to satisfy this need. Ru.

Disclosure of invention The present invention enables biological cells to be produced by bombarding living cells with particles coated with biological substances. Concerning improvements in methods for successfully transporting physical substances into living cells. The preferred particles are According to the method of the present invention, tungsten, which is more economical than gold, can be used. can be used. The object of the present invention can be achieved if treated with an inorganic strong acid.

Detailed description of the invention According to the method of the present invention, prior to coating ultrafine beads, these beads are first Pre-treat with strong acid. Strong inorganic acids include nitric acid, sulfuric acid, hydrochloric acid, phosphoric acid, or a mixture of these acids. Select from the group consisting of compounds. A preferred strong acid is nitric acid.

The inorganic strong acid concentration is not critical. Regarding the lower limit, 0.01 mole is sufficient. Can be used. Additionally, the upper limit is determined by economic factors of the process. Immediately However, even if excessive amounts are used, there will be no negative effect on the metallurgical properties of the beads, or in other words, on the surface properties. A volume that does not produce any There is no reason to use higher concentrations. Generally speaking, the practical upper limit is approximately 1 ．． It is about 0 mole. The preferred concentration range of the inorganic strong acid is 0. 1 mol to about 0.5 mol It is about 1000 yen.

Processing time is also not critical; a typical range is from about 5 minutes to about 60 minutes, with a preferred or about 10 minutes to about 20 minutes. Important things in time GL beads and nitric acid The contact time should be sufficient. Stirring the beads during pretreatment will ensure sufficient contact can be achieved. Continuous stirring is also preferable, but ultrasonication stirring is more suitable. be. When treated with nitric acid, if the particles are clean following ultrasonic treatment, There is no need for them to settle out of suspension. If the ultrasonic treatment is 20 minutes, the cleaning particles The particles are in suspension - i.e. the cleaning time is sufficient to clean the tungsten particles. It was found that the tungsten was left in suspension for 20 minutes before the nitric acid cleaning. The precision of the beads used is also not critical, as they are precipitated from tungsten and palladium. , when the bead material is selected from the group consisting of platinum, gold, or alloys of these metals. The method according to the invention shows significant advantages. Bead diameter is approximately 1 micron to approximately 1.5 micron Preferably up to . The beads of choice are tungsten beads. Because the book By using an inventive pre-treatment method, high-cost metals such as platinum, palladium and gold can be This is because tungsten can be used as effectively as tungsten.

After application of the pretreatment method according to the invention, various conventional steps can be used. B Wash the acid out of the case, but in this case, wash it with an alcohol such as ethanol. You can wash the acid off using a water bottle. Next 1: Air dry the beads as usual. However, this may also be carried out by rapid vacuum drying. After this, biology such as DNA Add target substance to ultrafine particles and mix properly. Biological substances such as calcium chloride Known additives can be mixed with a predetermined amount of amines such as polyamines. Ru. Next, plant tissues are bombarded to transport biological substances into living cells and intracellularly. Activate with.

The present invention will be explained below with reference to examples, but the present invention is not limited to these examples. Ma In addition, as the examples demonstrate, in the case of the pretreatment of the present invention, tungsten beads are more effective than gold beads.

Example 1 In this example, nitric acid was used as a means to suppress flocculent precipitation and strengthen DNA binding. After cleaning the particle surface using a particle gun, tungsten was and test gold This purpose was achieved by using nitric acid prior to rapid vacuum drying of the tungsten and gold particles. Evaluating the effect of cleaning treatments on these particles using standard targets It is in.

Using a DNA embryonic corn suspension as the genotypic material, this corn Add Murashige salt and Skoog salt to the Physi suspension medium.Reference: Physi ol.

Murashig in PI ant, 15: 473-497 (1962). Paper published by E, T., and Skoog, F. “A Revised Mett (ad for Rapid Growth and Bioassyswit h　Tabacco　Ti5sue　Cu1tures''. However, in this document , 2. 2,4-D, which is 0 mg/L of 2゜4-dichlorophenoxy-acetic acid, and and 3% sucrose. Additionally, this medium contains the following components: Add corn callus medium to 9, A"1L and 700mg/L Prolis 0.75 mg/L of 2.4-D and 3% of Soufroan References: Cereal Ti5sue and CeII Cu1tureS N1j noff and Junek, Amsterdam, 1985 Tomes, D. .

“Ce1l CultureS Somatic Embryiog” announced by enesis and Plant Regeneration in Mai ze, Rice, Sorghum, and Millets”.

For particle gun treatment, the particle gun described in the specification by Tomes et al. The beads used were 1.8 layer tungsten made by GTE and 1.8 layer thick tungsten made by GE. 2 layer m tungsten manufactured by Engelhard Industries (French) - Cress) using gold 1. In addition, use 25 mg of tissue per Petri dish. - At night, after pre-treatment with 0.25M mannitol, p One shock treatment was performed using DP460 DNA - for pDP460 , will be explained in Example 2 below.

The shock treatment was carried out as follows, and after 1 day of subculturing, the suspended cells were used. After using a 710um screen and sieving it, the corn suspension Medium 0.25M? Nitol at a concentration of 50 mg/ml (1 g in 20 ml medium) Resuspend the tissue in 9-night shaker at 1 m in preparation for particle bombardment. Two layers of 61 l of corn suspension medium + 0.25 M mannitol were added. 77 Pipette 0.5 ml of aliquot onto the center of Tutman filter paper. Concentrate cells in the center of each Petri dish to maximize shock processing efficiency and process all nine particles The treatment was repeated 6 times for each, and the standard positive control was also repeated 6 times. The experiment was repeated twice independently using a total of 26 samples. End the experiment To clean the tungsten particles and gold particles, 2 ml of 0.1M nitric oxide was added. Weighed 375 mg of particles in the acid, then in order to keep the particles in suspension, Sonicate on ice for 20 minutes with the sonicator set to its lowest setting, or Then, the particles were washed twice using sterile deionized water and finally washed with 1 ml of 95% etO Wash with H. After that, perform quick vacuum drying. All samples were subjected to one impact treatment using a particle gun. Following this particle gun treatment, the top filter paper of each sample was placed in corn callus medium. Transfer 9: Incubate cells at 28°C for 48 hours in the dark, then transfer to intermediate G. Evaluation of US activity GUS cytochemical evaluation was performed on all samples 48 hours after treatment during t:o. Compare particle seed plate, particle cleanliness, and particle source based on GUS activity between From the dispersion analysis of nine experiments, there was a difference between the types of ultrafine particles (tungsten λ gold) and Although we found that there were meaningful differences between microparticle cleaning treatments, there were significant differences between these variables. Overall, no statistically significant interaction was observed and nine t-tests were used. , the type and treatment of ultrafine particles were analyzed separately, and the only one recognized was Significance is for GTE tungsten and GTE tungsten and GE tan. This was as expected since there was a big difference in performance between the two and Gusten. T-Island: After nitric acid treatment, the performance of tungsten from different sources is almost the same; The data of this test is shown below.

Treatment at 36Pa: β-glucuronidase m chemical analysis of m's gene expression Genotype: 54-68-5, Maize Seichogoj~-night DNA: pPHI 460 Sanjino 146km once Particles Particles GUS inclusion group Type Processing DNA N Minimum average Maximum GET EtOH460122913 7342GET Nitric acid 460 12 62 217 460 Significance = 0.11 1 GTET EtOH46011381169GTET Nitric acid 460 12 3 9 187 440 Significance = 0. 023 F gold EtOH46012453122F gold nitric acid 460 12 8 100 259 significance = 0.119 GET:GE Tungsten GTET: GTE tungsten F gold: (flakeless) gold From the data in Table 1, it can be seen that particles not treated with nitric acid, treated gold particles, and untreated gold particles In comparison, pretreatment of the tungsten particle surface suppresses flocculation and It can be seen that this is an effective means of strengthening bonds.

Example 2 In this example, the method of the present invention using tungsten pretreated with nitric acid was Comparing with tungsten beads that were not pretreated, we also found that DuPont recommends Tungsten was not treated with nitric acid according to standard published protocols. Comparison with cell bombardment treatment using ultrafine particles in a particle gun. Weigh 60 mg of particles and suspend in 1 ml of 100% ethanol. 10.　 Centrifuge for 1 minute at 000 rpm to pellet particles. Ethanol supernatant Collect and replace with 1 ml of sterile deionized water. Perform sonication. 10. 00 Centrifuge for 1 minute at 0 rpm. Following the final wash, rinse in 1 ml deionized water. Resuspend.

Store in the freezer. After preparation, the particles are coated with DNA.

DNA-coated particles are prepared in the form of small aliquots in microtubes.

(Each aliquot of the pan is sufficient for three impact treatments). a large number of aliquots can be made at once. For one aliquot (ensure resuspension) ) Put 25 μl of the washed particles into a microtube, then add (μg/μl) Add 2.5 μm of DNA (1.1), mix by swirling with your fingers 5 times, and add 2.5 μm of DNA (2.5 μm). 5 Add Ca CI 2, stir by making a vortex 5 times with your fingers, and add 10μI of Add permidine (0.1 M = free base) and mix by vortexing with your fingers 5 times. Approximately 1 Leave for o minutes. Spin sediment for 3 minutes and discard 40-50 μl of supernatant (this (this will thicken the particles enough for three bombardments). remaining aliquots Repeat this method for all parts.

In this example, the DuPont protocol was modified to include 1.8μ tungsten particles. Comparison with the protocol of the present invention prepared by the method described below. Weigh 375mg of 1.8μ tungsten and suspend it in 2ml of 0.1M nitric acid. . Sonicate for 20 minutes on ice. 10. Centrifuge for 1 minute at OOOrpm and removes nitric acid. Add 2 ml of sterile deionized water, sonicate briefly, then , perform centrifugation, and repeat the water washing twice. Remove the supernatant water and add 2 ml of 10 Add 0% ethanol. The particles are resuspended by sonication and Remove a 25 μl aliquot for each treatment. Add 1.5ml of aliquot Put it in the Luff tube. The tungsten/ethanol suspension is quickly dried in vacuum. particles Store at room temperature and cover.

The Pioneer tungsten/DNA precipitation method is as follows. 10μI DN Add A (at 1 μg/μl) to the vacuum quick-dried tungsten and mix with a pipettor. Ru.

2. of 25 μm. Add 5MCaCl2 to the tungsten/DNA suspension, Mix with pipettor. 10μl of 0. 1M spermine with tungsten/D Add to the N A / Ca CI 2 suspension and stir by creating a vortex with your fingers. at room temperature Let the suspension stand for 15 minutes, then collect 15 μl of supernatant. Sonicate the suspension After treatment, a 1.5 μl aliquot is bound to the microparticles.

Using embryogenic corn suspension 2-122-4 (W23xB73): a The culture medium and particle bombardment method are as outlined in Example 1.

Continuously sample the particle preparation prepared on the first day and use the particles from the first day. , the experiment was conducted on the first, second, third, fourth, and fifth day using each method. The DNA/particle mixing was carried out daily. In this example, 16 samples were each mixed. Of those used, 14 were treated with pDP460 DNA and added MidopDP460 also contains nucleotides 421 to +2 of Ca M V 35 S. Gard-enhanced promoter [R, C, Gardner et al, Nucleic Ac1ds Res, 9.2871 (1981), Tobacco Mosaic Wi 79-bpHind from pJlllol spanning the 5° leading sequence of Rus l-8al I [D, R, Ga1lie.

D, E, 5leatSJ, W, Watts, P, C, Turner, T, M, A , Wison, Nucleic Ac1dsRes, 15.　3257　 (1987) spanning the first intron from maize Adh I-3. 579-bp7 fragment [E, S. Dennis et al., 1bid, 12.3983 (1987)], from pRAJ275 spanning the GUS sequence. An 1870-bp fragment of [R, Jefferson.

S, Burgess, D, Hirsh, Proc, Natl, Acad, Sc i, U, S, A, , 83.8447 (1986)], and pUc18 [C , Yan i sc h - P e r r o n, J, V i e i r aSJ, M e s si n g, G e n e 33.103 (19 85)] to Agrobacterium tumefaciens genes [M-Bevan, W, M, Barnes, M, D, Ch　I　　t　on　n, Sc　c　　　e　n　c　eII, 369　(19 83)] containing a 281-bp fragment containing a polyadenylation position containing It is. The remaining two samples are untreated controls.

All treated samples were subjected to one impact treatment using a particle gun. Immediately after treatment, all samples were transferred to corn suspension medium and incubated in the dark at 28°C for 3 Hold for 6 hours t:4 GUS cytochemical evaluation was performed on all samples 36 hours after treatment. Individuals who are positive for GUS in both the launch and DuPont protocols Overall, the difference between the method of the present invention and the DuPont protocol By adjusting the ratio, the DuPont protocol without nitric acid pretreatment The gene expression for each treatment on each day was compared and the results are summarized in Table 2. Ru.

Table 2 Item 28 7 Present invention 207 258 292 1. 8:1.07 DuPont 84 146 180 3rd day 7 Present invention 148 211 301 2. 9:1.07 DuPont 4 73 100 4th day 7 Present invention 228 380 485 3. 5:1.07 DuPont 8 110 194 Day 5 7 Present invention 62 217 389 2. 4:1゜07 DuPont 0 91 137 The significant data in Table 2 are those relating to the invention versus Duporet ratio. similar Although it is a tungsten particle, it uses tungsten particles that are not pretreated with nitric acid. The present invention produces a much higher percentage of cells turning blue than the standard DuPont method. The ζmi GtJS gene (pDP460) was successfully developed to prevent discoloration. It is taken into cells and expressed there.

Example 3 In this example, we used beads pretreated with nitric acid as described in Example 2. Tongues that were not pretreated according to standard DuPont protocols It is compared to stainless steel beads. The purpose of this example is to successfully incorporate cells into cells. Not only that, but after uptake, the DNA is integrated into the plant DNA and foreign DNA is The purpose is to show whether accompanying cell division occurs. In other words, DNA becomes a cell (This allows foreign DNA to be incorporated into plant DNA.) The aim is to show that it is possible to successfully grow plants that In this example, Standard nitric acid pretreated tungsten vinyl with cotyledons of Xanthitobacco The DNA expressed using the Plasmid pDP456 contains two different types of transcription units (PTUs) It is something. The first PTU has sites -421--90 replicated in tandem, CaM An enhanced promoter spanning nucleotides −421 to +2 of V35S [R, C, Gardner et al, Nucleic Ac1ds Res, 9.2 8.71 (1981)], p spanning the 5° leading sequence of tobacco mosaic virus. 79-bp Hindll-5al I from JIllol [D, R, G a1lie, D, E, 5leat, J, W, WattsS P, C, Turne r,T.

PJL A, Wil5onS Nucleic Ac1dsRes, 15.　 3257 (1987)], 1 from pRAJ275 spanning the GUS decoding sequence. 870-bp fragment [R, Jeff Ferson, S, Burgess.

D, HirshS Proc, Natl, Acad, Sci, U, S, A, , 83, 8447 (1986) Ko, and pUc18 [C, Yani sch-Perron S J.

V　i　　i　r　a, J, M　e　s　s　i　n　gSG　e　n　e　 33.103 (1985)], Agrobacterium, tumefac iens Navarin Synthase Gene [VL.

Bevan, W, K Barnes S M, D, Chilton, Sc 1en ce II, 369 (1983) containing polyadenylation positions from 281 -bp fragment. The second PTtJ is NPT instead of GUS II decoding sequence [R, T, Frayley etal, Proc, Natl, A cad, sci, u.

S, A, 80.4803 (1983)]. It is the same. In this example, four in vitro grown Xanti cotyledons were mesetal, is Plant Mo1. Biol, 14:261-268 ( Shock treated as described in (1990). Two independent experiments in which cotyledons were bombarded for each microparticle/DNA protocol and go nine Ultrafine particle/DNA DuPont ratio using the standard DuPont protocol previously described. The process of the present invention was carried out in accordance with the above-mentioned example. All DNA samples were subjected to one impact treatment. After particle gun treatment, DNA treatment was performed. All samples were cultured in the prescribed culture described in the specifications of Tomes et al. Move to the ground Record the number of colonies recovered for each treatment and use NPTI as shown in Table 3. Verify that pH 1456 was properly incorporated into each sample, shown as I+. Table 3 shows that the protocol or duplication of the present invention Samples from Pon Inc. protocol DNA protocol NPTI I25, CL 456 Present invention 10 52, C1456 Present invention 10 52, C2456 Present invention - 20, C1456 Present invention 10 26, C1456 Honsenju 26, C2456 present invention - 26, C3456 Present invention 10 26, C4456 Present invention 10 55, C1456 Present invention 10 50, C2456 Present invention 10, (J456 DuPont + 12, C1456 DuPont + 14, C1456 DuPont + 14, C2456 DuPont + 2, C1456 DuPont + 41, C1456 DuPont - 43, C1456 DuPont + 34, C1456 DuPont + 80 of the cotyledons recovered from all samples treated with the method protocol of the invention. 9% of the colonies were stably transformed.Also, the DuPont DNA protocol Only 6% of cotyledons recovered from all treated samples were stably transformed. In other words, the method of the present invention showed a higher number of transformations. Example 4 When DuPont's protocol is compared to the protocol of the present invention, nitric acid treatment Example 4 was carried out to confirm whether it brought about improvement in transformation. In the example, genotype 2-122-4 (W23X3) embryogenic maize suspension station was used as the genetic material, the DNA was pDP460, and by particle gun treatment the DNA was pDP460. The method of the present invention is carried out using nitric acid treatment and the DuPont DNA/particle mixing process. Compared to DuPont's protocol, which uses only nitrate and nitric acid, nine In this example, we compared 40 samples, which were treated with pDP460 DNA. , 20 samples were tested in each experiment, and none of the treated samples were treated with a particle gun. Standard protocol for mixing DNA/particles as described in Example 2 with one bombardment treatment. The DuPont method described in Example 2 was then carried out, followed by particle gun treatment. Transfer all samples to corn callus medium and keep at 28°C in the dark for treatment. After 36 hours, all samples were subjected to GUS cytochemical evaluation. Based on the number of visibly blue-stained cells present in the sample, two Intermediate gene expression was compared between the treatments and the data are summarized in Table 4 below.

Table 4 Individual GUS details Protocol N Minimum Average Maximum Coefficient of Variation DuPont Original 20 113 326.2 491 25.19% Invention Standard 20 203 430.9 728 29.61% You can understand from Table 4 Between DuPont's protocol and the protocol of the present invention (ma, statistics) statistically significant difference (values are calculated with 95% confidence using analysis of variance) These results show that, in fact, the nitric acid pretreatment ζ Mariki λ (Ko Taka 0 Intermediate Gene September 28, 1994 PCT/US93100817 3. Patent applicant Nationality United States August 18, 1993 1. Shock treatment of living cells with biological material-coated particles accelerated by a particle gun. A method for transporting biological substances into living cells by transporting the particles into biological cells. 111- Before coating with a chemical substance, the particles are pretreated with a strong inorganic acid and then washed. A method for transporting biological substances into living cells characterized by 2. Claim 1 in which the strong acid is selected from the group consisting of nitric acid, sulfuric acid, hydrochloric acid, and phosphoric acid. Gum 3 according to Claim 2, Strong acidity j Gum 4 according to Claim 2, which is nitric acid The method according to claim 3, wherein the concentration of is 0.1M to 1.0M. 5. The method according to claim 4, wherein the concentration of nitric acid is 0.1M to 0.5M. 6. Is the particle made of tungsten, palladium, platinum, and gold, or an alloy thereof? The beads according to claim 1, which are beads selected from the group consisting of 7. The method according to claim 6, wherein the beads are tungsten beads. -8. The diameter of the piece is about 0.5 microns - about 3.0 microns. Gum according to item 6-9, the gum according to claim 7, in which the particles are stirred during pretreatment. How to place nine 10. The method according to claim 9, wherein the stirring is performed by sonic crushing treatment. 11. The method according to claim 9, wherein the stirring time is about 5 minutes to about 60 minutes. 12. The gum according to claim 11, wherein the stirring time is about 10 minutes to about 20 minutes. 13. The method according to claim 1, wherein the biological substance is DNA. 14. The method according to claim 1, wherein the biological substance is RNA. 15. In an improved method for successfully transporting DNA into living cells, cleaning Pre-treat the tungsten microparticle beads with an effective amount of nitric acid, and Wash the pretreated tungsten beads and incubate the tungsten beads with DNA. The DNA-coated tungsten beads are then accelerated and delivered to target living cells. The above method consists of 16. The method according to claim 15, characterized in that the beads are used during the pretreatment. 17. The method according to claim 16, wherein the molar concentration of nitric acid is O6IM~0.5M. Also 18. Continuation of claim front page where the beads are agitated by sonication (72) Inventor: Riichi Bangs Carmel, Sioux Springs, Indiana, USA 46032 coat 6

Claims (18)

[Claims] 1. Bombarding living cells with biological material-coated particles accelerated by a particle gun A method for transporting biological substances into living cells by transporting the particles into biological cells. The particles are pretreated with a strong inorganic acid before being coated with a chemical substance. A method of transporting biological substances into living cells. 2. Claim 1, wherein the strong acid is selected from the group consisting of nitric acid, sulfuric acid, hydrochloric acid, and phosphoric acid. The method described in section. 3. 3. The method according to claim 2, wherein the strong acid is nitric acid. 4. 4. The method according to claim 3, wherein the nitric acid concentration is 0.1M to 1.0M. 5. 5. The method according to claim 4, wherein the nitric acid concentration is 0.1M to 0.5M. 6. The particles are made of tungsten, palladium, platinum, and gold, or alloys thereof. The method according to claim 1, wherein the beads are selected from the group consisting of: 7. 7. The method of claim 6, wherein the beads are tungsten beads. 8. Claim 6, wherein the beads have a diameter of about 0.5 microns to about 3.0 microns. The method described in section. 9. 8. The method according to claim 7, wherein the particles are agitated during pre-treatment. 10. 10. The method according to claim 9, wherein the stirring is performed by sonication. 11. The method of claim 9, wherein the stirring time is about 5 minutes to about 60 minutes. 12. 12. The method of claim 11, wherein the stirring time is about 10 minutes to about 20 minutes. 13. 2. The method according to claim 1, wherein said biological material is DNA. 14. 2. The method according to claim 1, wherein said biological material is DNA. 15. In an improved method for successfully transporting DNA into living cells, small amounts of Pre-treat the tungsten microparticle beads with an effective amount of nitric acid, and Wash the pretreated tungsten beads and incubate the tungsten beads with DNA. The DNA-coated tungsten beads are then accelerated and delivered to target living cells. The above method consists of: 16. The method according to claim 15, wherein the beads are continuously stirred during the pretreatment. Law. 17. The method according to claim 16, wherein the nitric acid molar concentration is 0.1M to 0.5M. Law. 18. 18. The method of claim 17, wherein the beads are agitated by sonication.