Abstract
Improved biolistic bombardment is observable when using a modification to a double-barrel device. One such modification is a custom attachment that can be retrofit to a standard helium-based gene gun. The custom attachment facilitates DNA and protein delivery. The attachment confines the flow of particles in a barrel similar to the choke on a shotgun barrel.
Claims
1. An attachment for a biolistic particle delivery system that confines the flow of particles to a barrel comprising: an elongated body with a central cavity that defines a major flow path passing through; and a ring located at a top of the cylindrical body, said ring having a diameter greater than a diameter of the cylindrical body; wherein the ring is retrofit to a macrocarrier launch assembly of the biolistic particle delivery system.
2. The attachment of claim 1 wherein the elongated body is a hollow cylinder with a length that is between one and a half times and two times (1.5 to 2) greater than a diameter of the elongated body.
3. The attachment of claim 1 wherein the elongated body tapers from an initial thickness greatest at the location where the ring meets the top of the cylindrical body to a terminal thickness less than the initial thickness.
4. The attachment of claim 1 wherein the major flow path is divided into more than one major flow path by one or more dividers.
5. The attachment of claim 4 wherein the one or more dividers begin at the top of the cylindrical body and extend only to a depth at or within the elongated body.
6. The attachment of claim 5 wherein each of the more than one major flow path is defined by a cavity shape having at least one curved wall and at least one planar wall.
7. The attachment of claim 4 wherein the one or more dividers begin at the top of the cylindrical body and extend to a depth beyond the elongated body, thereby acting as fins to help guide flow even as particles have exited the central cavity.
8. The attachment of claim 1 further comprising minor flow paths located adjacent a periphery of the central cavity, said major flow path separated from the minor flow paths by one or more baffles.
9. The attachment of claim 1 wherein flow of the major flow path is substantially laminar.
10. The attachment of claim 1 wherein the attachment is symmetrical about at least an x-axis and a y-axis and is asymmetrical about a z-axis, said z-axis being defined by an axial axis traversing a longitude of the elongated body.
11. A biolistic particle delivery system comprising: a gas acceleration tube; a source of high-pressure gas operatively connected to a first end of the gas acceleration tube; and the attachment of claim 1 located at a second end of the gas acceleration tube.
12. The biolistic particle delivery system of claim 11 further comprising a screen that allows only the biolistic particles to impact an intended target.
13. The biolistic particle delivery system of claim 11 wherein the biolistic particle delivery system comprises a bombardment chamber, connective tubing attached to a vacuum source, a helium regulator, and a solenoid valve.
14. The biolistic particle delivery system of claim 13 further comprising a rupture disk.
15. The biolistic particle delivery system of claim 11 wherein the biolistic particle delivery system is a helium driven gene gun.
16. A method of delivering biolistic particles comprising: using a high-pressure gas to move biolistic particles through an attachment for a biolistic particle delivery system toward a target having one or more cells selected from the group consisting of: bacterial cells, fungal cells, insect cells, plant cells, animal cells, intracellular organelles, and combinations thereof; wherein the attachment comprises an elongated barrel; and bombarding the targets with coated microcarriers.
17. The method of claim 16 further comprising replacing a stock component with the elongated barrel.
18. The method of claim 17 further comprising reducing a number of outlier particles through use of the stock component by employing said elongated barrel in lieu thereof.
19. The method of claim 17 further comprising reducing a number of cells killed through use of the stock component by employing said elongated barrel in lieu thereof.
20. The method of claim 17 further comprising increasing an efficacy related to penetration of a cell wall.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] Several embodiments in which the present disclosure can be practiced are illustrated and described in detail, wherein like reference characters represent like components throughout the several views. The drawings are presented for exemplary purposes and may not be to scale unless otherwise indicated.
[0064] FIG. 1 shows a biolistic particle delivery system.
[0065] FIGS. 2A-2B shows a microcarrier launch assembly usable with the biolistic particle delivery system of FIG. 1. FIG. 2A shows an assembled view. FIG. 2B shows a disassembled view.
[0066] FIG. 3 shows an elevation view of a target shelf usable with the biolistic particle delivery system of FIG. 1.
[0067] FIG. 4 shows the biolistic bombardment process using the biolistic particle delivery system of FIG. 1.
[0068] FIG. 5A shows a side elevation view components and controls on a helium driven gene gun.
[0069] FIG. 5B shows a side, partial cutaway view of a battery compartment of the helium driven gene gun of FIG. 5A.
[0070] FIG. 6A shows an exploded view of major components used for sample delivery with the helium driven gene gun of FIG. 5A.
[0071] FIG. 6B shows a cross-sectional view of a helium driven gene gun, emphasizing view of a high velocity stream of helium that accelerates gold particles coated with plasmids or RNA to velocities sufficient to penetrate and transform cells, both in vitro and in vivo.
[0072] FIG. 7 shows a schematic view of a helium driven gene gun, including comparison views of a stock component within the helium driven gene gun versus use of the flow guiding barrel(s) described herein.
[0073] FIG. 8A shows a front elevation view of single-flow guiding barrel, according to some aspects of the present disclosure. FIG. 8B shows a rear elevation view thereof. FIG. 8C shows a bottom perspective view thereof. FIG. 8D shows a top perspective view thereof. FIG. 8E shows a side cross section view thereof. FIG. 8F shows a side elevation view thereof. FIG. 8G shows a top cross section view thereof. FIG. 8H shows a top plan view thereof. FIG. 8I shows a top perspective view thereof. FIG. 8J shows a front perspective view thereof. FIG. 8K shows a bottom plan view thereof.
[0074] FIG. 9A shows a front elevation view of double-flow guiding barrel, according to some aspects of the present disclosure. FIG. 9B shows a rear elevation view thereof. FIG. 9C shows a top perspective view thereof. FIG. 9D shows a front perspective view thereof. FIG. 9E shows a side elevation view thereof. FIG. 9F shows a top cross section view thereof. FIG. 9G shows a top plan view thereof. FIG. 9H shows a side cross section view thereof. FIG. 9I shows a bottom plan view thereof. FIG. 9J shows a bottom perspective view thereof. FIG. 9K shows a side perspective view thereof.
[0075] FIG. 10A shows a perspective view of multi-flow guiding barrel, according to some aspects of the present disclosure. FIG. 10B shows a front elevation view thereof. FIG. 10C shows a rear elevation view thereof. FIG. 10D shows a left-side elevation view thereof. FIG. 10E shows a right-side elevation view thereof. FIG. 10F shows a top plan view thereof. FIG. 10G shows a bottom plan view thereof.
[0076] FIG. 11A shows variances in barrel length and diameter for the flow-guiding barrel(s), according to some aspects of the disclosure.
[0077] FIG. 11B illustrates the effect of in barrel length for the stock component included in the OEM gene gun of FIG. 1 as compared to the improved gene gun of FIGS. 7-8 that incorporates a flow-guiding barrel(s).
[0078] FIG. 12 illustrates the effect of variances in barrel diameter for the stock component included in the OEM gene gun of FIG. 1 as compared to the improved gene gun of FIG. 7 that incorporates a flow-guiding barrel(s).
[0079] FIG. 13A charts a demonstration of improvement using the flow guiding barrel and quantifies adjacent thereto a total number of transformed cells for both the flow guiding barrel and the stock component. Onion epidermis cells were bombarded with 24 ng of a plasmid that expresses green fluorescent protein and were imaged after two days. Cells were counted via CellProfiler software, as shown in FIG. 13B (for the stock component) and FIG. 13C (for the flow guiding barrel).
[0080] FIG. 14A charts the number of cells transformed using either the stock component (n=23) with 100% of the DNA and spermidine and the novel barrel extension (n=18) with 10% of the DNA and spermidine. Onion epidermis cells were bombarded with 2.4 ng and 24 ng of a plasmid that expresses green fluorescenct protein and were imaged after two days. Cells were counted via CellProfiler software, as shown in FIG. 14B (for the stock component) and FIG. 14C (for the flow guiding barrel).
[0081] FIG. 15A charts transient protein delivery improvements using a modified barrel. Onion epidermis cells were bombarded with 30 ug of fluorescein isothiocyanate (FITC) labelled bovine serum albumin (BSA) and were imaged after two days. Cells positive with FITC marker were counted via CellProfiler software, as shown in FIG. 15B (for the stock component) and FIG. 15C (for the flow guiding barrel).
[0082] FIGS. 16A-16C show a schematic representation of two plasmids used for the evaluation of CRISPR reagents. FIG. 16A shows CRISPR plasmid pTF6005 that carries a Cas9 expression cassette under the control of maize ubiquitin promoter and Cauliflower Mosaic Virus (CaMV) 35S terminator (T35S); OsPDS gRNA1 is regulated by OsU6 promoter; hygromycin resistance gene (hpt II) is driven by 2CaMV 35S promoter (P35S) and terminated by T35S. RB, T-DNA right border; LB, T-DNA left border; SpR, spectinomycin resistance gene; ColE1 ori, high copy number origin of replication for E. coli; pVS1, origin of replication from plasmid VS1 for Agrobacterium. FIG. 16B shows the reporter plasmid pKL2187 has genes for the red fluorescent protein tdTomato and the green fluorescent protein ZsGreen1. Transcription of the tdTomato gene is driven by a 2 P35S and terminated by an Agrobacterium nopaline synthase terminator (Tnos). The encoded tdTomato protein has an SV40 nuclear localization signal at the N terminus. Transcription of the ZsGreen1 gene is driven by a 2 P35S promoter and terminated by a potato protease inhibitor II terminator (TpinII). The translation start codon is preceded by a TMV Q translational enhancer and is immediately followed by the target sequence of the OsPDS-gRNA1 (SEQ ID NOs: 1 and 2) expressed from pTF6005. The open reading frame for the flexible peptide linker 2 (GGGGS) and ZsGreen1 is out-of-frame by 1-bp with the start codon and is not translated; however, indel mutations at the gRNA target site can bring the ZsGreen1 gene in-frame and restore green fluorescence. AmpR, ampicillin resistance gene. The plasmid pKL2188 is identical to pKL2187 except that the ZsGreen1 gene is in-frame with the start codon. Blue letters, gRNA target sequence; underscored red letter, PAM sequence. FIG. 16C shows the OsPDS target site (SEQ ID NO: 1) as well as one on-target gRNA tested for editing efficiency. The gRNA1 (SEQ ID NO: 3) was driven by OsU6 promoter in the CRISPR plasmid shown in FIG. 16A.
[0083] FIG. 16D charts improved cas9-plasmid delivery and editing efficiency using a modified barrel. Onion epidermis cells were bombarded with 2 ug a CRISPR-Cas9 plasmid construct with OsPDS gRNA1, called pTF6005 as well as a reporter plasmid pKL2197. The onions were incubated at 30 C. in dark for 2 days prior to imaging. These constructs carry a gRNA targeting the OsPDS gRNA1 target site in pKL2187 and are expected to result in GFP expressing cells if cas9 editing occurred. Cells were counted via CellProfiler software, as shown in FIG. 16E (for the stock component) and FIG. 16F (for the flow guiding barrel).
[0084] FIG. 17A charts improved viral-plasmid delivery using Soybean Mosaic Virus expressing green fluorescent protein in Soybean using a modified barrel. Two-week old juvenile soybean plants were bombarded with 2 ug of SMV plasmid expressing green fluorescent protein and then kept in the green house for a remaining two weeks. After two weeks, the soybean inoculated leaves as well as newly grown leaves were imaged for gfp-expression and reverse transcription-polymerase chain reaction (rt-pcr) to confirm the viral infection, as shown in FIG. 17B (stock component) and FIG. 17C (flow guiding barrel).
[0085] FIG. 18A charts improved viral-plasmid delivery using Sugarcane Mosaic Virus expressing green fluorescent protein in Corn using a modified barrel. Two-week old juvenile corn plants were bombarded with 2 ug of SCMV plasmid expressing green fluorescent protein and then kept in the green house for a remaining two weeks. After two weeks, the corn inoculated leaves as well as newly grown leaves were imaged for gfp-expression and reverse transcription-polymerase chain reaction (rt-pcr) to confirm the viral infection, as shown in FIG. 18B (stock component) and FIG. 18C (flow guiding barrel).
[0086] FIG. 19A charts improved penetration depth (i.e., velocity) using a modified barrel. FIG. 19B charts target area/particle distribution using a modified barrel. 1 wt % Agarose gels were bombarded with 30 ug of fluorescein isothiocyanate (FITC) labelled bovine serum albumin (BSA) and were imaged under a fluorescent confocal microscope immediately after to determine particle penetration and distribution. Particles were counted via CellProfiler software, as shown in FIG. 19C (for the stock component) and FIG. 19D (for the flow guiding barrel).
[0087] FIG. 20 aggregates views of various barrels. The first column of FIG. 20 shows various single barrels, including a perspective view of a stock component, a perspective view of a short, flow-guiding single barrel, and a perspective view of a long, flow-guiding single barrel. The second column of FIG. 20 shows various double barrels, including a perspective view of a narrow double barrel, a perspective view of wide double barrel, and a perspective view of a double barrel with a single fin. The third column of FIG. 20 shows various multi-barrels, including a perspective view of a multi-barrel, a perspective view of a short, flow-guiding multi-barrel, and a perspective view of a long, flow-guiding multi-barrel. The fourth column of FIG. 20 shows various multi-flow barrels, including a perspective view of a flow-guiding multi-flow barrel, a perspective view of a flow-guiding multi-flow barrel having a single fin, and a perspective view of a flow-guiding multi-flow barrel having a plurality of fins.
[0088] FIG. 21A compares use of a known barrel versus use of a flow-guiding double-barrel with respect to a total number of transformed cells and performance ratios. FIG. 21B charts the results of testing a double-barrel to the flow guiding double-barrel described herein. The first chart shows an improvement in number of cells transfected using the new design. The second chart measures the ability of the double-barrel to evaluate a consistent performance ratio, i.e. the number of cells transfected via the right barrel compared to the left. A performance ratio of 1 presents the real results, as both sides should be identical. The 3D images show the different barrel designs.
[0089] An artisan of ordinary skill in the art need not view, within isolated figure(s), the near infinite number of distinct permutations of features described in the following detailed description to facilitate an understanding of the present disclosure.
DETAILED DESCRIPTION
[0090] The present disclosure is not to be limited to that described herein. Mechanical, electrical, chemical, procedural, and/or other changes can be made without departing from the spirit and scope of the present disclosure. No features shown or described are essential to permit basic operation of the present disclosure unless otherwise indicated.
[0091] FIG. 7 shows a diagram illustrating the improvements that can be implemented with the helium-based gene gun system 100. The stock component 200S includes the macrocarrier cover lid 202, adjustable nest 204, fixed nest 206 with a retaining spring, stopping screen support ring 208, two spacer rings 210, and five macrocarrier holders 212 described above. The stock component 200S can be swapped out in favor of any one or more of the improved flow guiding barrels described herein 800, 900, 1000. This exchange of components can occur at position 100P within the bombardment chamber 102.
[0092] FIGS. 8A-8K depict a single-flow guiding barrel 800. The barrel 800 comprises a body 802, a retaining ring 804, a single major flow path 806, and optionally, several minor, peripheral flow paths 808 defined by baffles located near the periphery of a central cavity.
[0093] Holes on the barrel 800 wall help release the pressures and bombard taller plants. The materials of the barrel 800 are not limited to hard plastics. For example, the barrel 800 can be made from an inert material constructed to withstand the pressure used within the system.
[0094] The retaining ring 804 acts as a head and includes a larger diameter than that of the barrel 800 so that the barrel 800 does not slide through the holder. The length of the barrel 800 can vary, however in some embodiments is at least four times (4) greater than the thickness of the retaining ring.
[0095] FIGS. 9A-9K depict a double-flow guiding barrel 900. The barrel 900 comprises a body 902, a retaining ring 904, major flow paths 906A & 906B, optionally: several minor, peripheral flow paths 908 defined by minor dividers (e.g., baffles) located near the periphery of the central cavities, and a major divider 910 that separates the major flow paths 906A & 906B. As shown, the major divider can also extend beyond the body 902 and act as a flow guiding fin to deliver particles to a location more proximate the plant tissue.
[0096] Holes on the barrel 900 wall help release the pressures and bombard taller plants. The materials of the barrel 900 are not limited to hard plastics. For example, the barrel 900 can be made from an inert material constructed to withstand the pressure used within the system.
[0097] The retaining ring 904 acts as a head and includes a larger diameter than that of the barrel 900 so that the barrel 900 does not slide through the holder. The length of the barrel 900 can vary, however in some embodiments is at least four times (4) greater than the thickness of the retaining ring.
[0098] FIGS. 10A-10G depict a multi-flow guiding barrel 1000. The barrel 1000 comprises a body 1002, a retaining ring 1004, major flow paths 1006A-1006D, optionally: several minor, peripheral flow paths (not shown) defined by minor dividers (e.g., baffles) located near the periphery of the central cavities, and major dividers 1010 that separates the major flow paths 1006A-1006D. As shown, the major dividers 1010 can also extend beyond the body 1002 and act as a flow guiding fins to deliver particles to a location more proximate the plant tissue.
[0099] Holes on the barrel 1000 wall help release the pressures and bombard taller plants. The materials of the barrel 1000 are not limited to hard plastics. For example, the barrel 1000 can be made from an inert material constructed to withstand the pressure used within the system.
[0100] The retaining ring 1004 acts as a head and includes a larger diameter than that of the barrel 800 so that the barrel 1000 does not slide through the holder. The length of the barrel 1000 can vary, however in some embodiments is at least four times (4) greater than the thickness of the retaining ring.
[0101] Reverse configurations can also exist (see e.g., barrels 2000D-E of FIG. 20), wherein the body of the barrels 800, 900, 1000 can extend well past where the dividers 910, 1010 terminate and therefore leave what looks to be a cutout in its stead.
[0102] Particles are accelerated through a burst of pressurized gas from the top of the gun, through the barrel 800/900/1000, toward plant tissue. As shown in FIGS. 11A-11B, FIG. 12, and barrels 2000A-L of FIG. 20, the length and inner diameter of the barrel 800 have a great effect on the efficacy of the gene gun 800.
[0103] FIG. 20 further depicts a stock component 2000A that comes with the gene gun 800 in the upper left-hand corner. The stock component 2000A is essentially a metal ring that particles travel through after being accelerated by a burst of pressurized gas, and its function is to support a screen that allows only particles to impact the plant tissue. The metal ring is a rudimentary form of a single barrel but does not have the length sufficient to guide flow in order to best transfect cells.
[0104] The remaining barrels 2000B-L show barrels (including some that were shown in FIGS. 8A-8K, 9A-9K, and 10A-10G) that deviate from the OEM design shown in FIG. 2. The barrels retrofit to the biolistic particle delivery system 100 and can replace the stock component entirely.
[0105] In operation, the gene gun 100 accelerates particles via a burst of pressurized gas (e.g., helium, nitrogen, etc.) from the top of the gun. The particles are sent through the barrel 800 and toward plant tissue. The length and inner diameter of the barrel 800 can be critical factors in its improved effectiveness over the stock component.
[0106] The barrels 800, 900, 1000 themselves are an attachment for a helium-based gene gun 100 that confines the flow of particles to a barrel similar to rifling on a traditional firearm. The stock component 200S is a metal ring that particles travel through after being accelerated by a burst of pressurized gas, and its function is to support a screen that allows only particles to impact the plant tissue. The flow guiding barrel attachment has been designed to replace this ring and not only performs its function, but more uniformly directs the airflow to the plant tissue. As the DNA-loaded particles and burst of pressurized gas flow through the device, the confinement of the flow guiding barrel reduces turbulence in the chamber. The major flow path within the cavity of the barrel is therefore substantially laminar. This leads to fewer outlier particles that either kill the cells with their impact or lose too much momentum and are unable to penetrate the cell wall.
[0107] From the foregoing, it can be seen that the present disclosure accomplishes at least all of the stated objectives.
EXAMPLES
[0108] The inventors tested the barrel 800 with one of the most common gene guns used in industry today, the Bio-Rad PDS-1000/He Biolistic Particle Delivery System. The barrel 800 replaced the stock component 200. The flow guiding barrel was produced via 3D printing and the dimensions and material can be customized to other gene gun systems.
[0109] As shown in FIGS. 13A-13C, on average, using the flow guiding barrel leads to at least 23 times (23) more cells modified per shot (153 cells compared to 3324 cells on average). This was tested using onion epidermis cells bombarded with plasmid DNA that expresses a green fluorescent protein (GFP). The critical parameters of DNA and particle amount were kept constant for each bombardment, and plant tissue was sourced from the same onions. The distance between onion tissue and initial particle position was optimized for both conditions to account for the acceleration generated by increased confinement. The improvement in performance due to the flow guiding barrel represents a significant upgrade that has broadly applicable potential.
[0110] As shown in FIGS. 14A-C, on average, using the flow guiding barrel with 10% of the DNA and delivery agent leads to at least 7 times (7) more cells modified per shot (153 cells compared to 1030 cells on average). This was tested using onion epidermis cells bombarded with plasmid DNA that expresses a green fluorescent protein (GFP). The critical parameters of DNA and particle amount were kept constant for each bombardment, and plant tissue was sourced from the same onions. The distance between onion tissue and initial particle position was optimized for both conditions to account for the acceleration generated by increased confinement. The improvement in performance using minimal amounts of DNA due to the flow guiding barrel represents a significant upgrade that can potentially help reduce the number of DNA copies inserted.
[0111] As shown in FIGS. 15A-C, on average, using the flow guiding barrel leads to at least 3 times (3) more cells distributed with protein (259 cells compared to 807 cells on average). This was tested using onion epidermis cells bombarded with FITC labeled BSA to track delivery. The critical parameters of protein and particle amount were kept constant for each bombardment, and plant tissue was sourced from the same onions. The distance between onion tissue and initial particle position was optimized for both conditions to account for the acceleration generated by increased confinement. The improvement in performance and distribution using protein due to the flow guiding barrel represents a significant upgrade that can broadly help improve protein delivery including Cas9, a CRISPR-associated (Cas) endonuclease, or enzyme.
[0112] As shown in FIGS. 16A-F, on average, using the flow guiding barrel leads to at least 3-4 times (3-4) more cells delivered to per shot (82 cells compared to 293 cells on average) as well as 2 times (2) the editing efficiency (23.9% compared to 42.9% on average). The critical parameters of DNA and particle amount were kept constant for each bombardment, and plant tissue was sourced from the same onions. The distance between onion tissue and initial particle position was optimized for both conditions to account for the acceleration generated by increased confinement. The improvement in performance using Cas9 DNA due to the flow guiding barrel represents a significant upgrade that has broadly applicable potential.
[0113] As shown in FIGS. 17A-C, on average, using the flow guiding barrel leads to 100% of the soybean plants being virally infected compared to 66% which is a significant improvement. The critical parameters of DNA and particle amount were kept constant for each bombardment, and plant tissue was sourced from the same onions. The distance between soybean tissue and initial particle position was optimized for both conditions to account for the acceleration generated by increased confinement. The improvement in performance using the soybean mosaic virus due to the flow guiding barrel represents a significant upgrade that can be applied to other plant species.
[0114] As shown in FIGS. 18A-C, on average, using the flow guiding barrel leads to at least 66 times (66) as many plants being infected per experiment (66% compared to 1% infection efficiency). The critical parameters of DNA and particle amount were kept constant for each bombardment, and plant tissue was sourced from the same onions. The distance between corn tissue and initial particle position was optimized for both conditions to account for the acceleration generated by increased confinement. The improvement in performance using the sugarcane mosaic virus due to the flow guiding barrel represents a significant upgrade that can be applied to other plant species.
[0115] As shown in FIGS. 19A-D, on average, using the flow guiding barrel leads to 3 times (3) the penetration depth (22.5 micrometers compared to 7.5 micrometers) and 2 times (2) the particle distribution area (10.89 cm.sup.2 compared to 6.25 cm.sup.2). The critical parameters of pressure, distance, and particle amount were kept constant for each bombardment. The distance between the agarose gel and initial particle position was optimized for both conditions to account for the acceleration generated by increased confinement.
[0116] Barrels 2000D and 2000F were tested to compare use of a known barrel versus use of a flow-guiding double-barrel with respect to a total number of transformed cells and performance ratios. As shown in FIG. 21A, a first chart shows an improvement in number of cells transfected using the new design. As shown in FIG. 21B, a second chart measures the ability of the double-barrel to evaluate a consistent performance ratio, i.e. the number of cells transfected via the right barrel compared to the left. A performance ratio of 1 presents the real results, as both sides should be identical. The 3D images show the different barrel designs.
[0117] According to some examples, an optimal diameter of the barrel preferably ranges from thirteen to twenty-four millimeters (13 to 24 mm); more preferably ranges between eighteen and twenty-two millimeters (18 to 22 mm), and most preferably is approximately twenty millimeters (20 mm) in diameter.
[0118] According to some examples, an optimal length of the barrel preferably ranges from fifteen and eighty-five millimeters (15 to 85 mm), more preferably ranges between thirty and fifty-five millimeters (30 to 55 mm), and most preferably is approximately thirty-five millimeters (35 mm).
[0119] According to some examples, an optimal length of the barrel is preferably between one and five times (1 to 5) greater than the diameter of the barrel, more preferably between one and a quarter times to three times (1.25 to 3) greater than the diameter of the barrel, and most preferably between one and a half times and two times (1.5 to 2) greater than the diameter of the barrel.
[0120] For our device that works universally for all applications, the ideal dimensions are a diameter of 20 mm and a length of 35 mm.
LIST OF REFERENCE CHARACTERS
[0121] The following table of reference characters and descriptors are not exhaustive, nor limiting, and include reasonable equivalents. If possible, elements identified by a reference character below and/or those elements which are near ubiquitous within the art can replace or supplement any element identified by another reference character.
TABLE-US-00001 TABLE 1 List of Reference Characters 100 system 102 bombardment chamber 104 bombardment chamber door 106 rupture disk retaining cap 108 launch assembly shelf 110 vacuum tubing 112 tank 114 helium regulator 116 helium pressure regulator 118 a solenoid valve 120 connective polyether ether ketone (PEEK) tubing 122 on/off power switch 124 fire switch 126 vacuum/vent/hold switch assembly 128 vacuum gauge 130 vacuum/vent rate control valves 132 helium pressure gauge 200 microcarrier launch assembly 200S stock component 202 macrocarrier cover lid 204 an adjustable nest 206 a fixed nest 208 a stopping screen support ring 210 two spacer rings 212 macrocarrier holders 300 plate shelf 400 ballistic process 402 high-pressure helium 404 a rupture disk 406 gas acceleration tube 408 macrocarrier sheet 410 microscopic gold microcarriers 412 target cells 414 stopping screen 416 distance from the rupture disk to the macrocarrier sheet 418 macrocarrier travel distance to the stopping screen 420 distance between the stopping screen and the target cells 500 portable, helium driven gene gun 502 cartridge 504 cylinder lock 506 barrel pin 508 cartridge holder 510 O-ring 512 cylinder advance lever 514 barrel liner 516 safety interlock switch 518 trigger button 520 LED display 522 metal bar 524 battery access cover 526 battery compartment 528 terminals 530 attachment fitting (male) 532 acceleration chamber 534 spacer 600 helium regulator 602 connection to helium tank 604 sleeve 606 female quick connect fitting 608 pressure relief valve 610 high velocity stream of helium 700 helium hose 702 male quick connect fitting 704 female quick connect fitting 100P position at which stock component can be replaced for flow guiding barrels 800, 900, 1000 800 single-flow guiding barrel 802 body 804 retaining ring 806 major flow path 808 peripheral, minor flow paths 900 double-flow guiding barrel 902 body 904 retaining ring 906A first major flow path 906B second major flow path 908 peripheral, minor flow paths 910 major divider 1000 multi-flow guiding barrel 1002 body 1004 retaining ring 1006A first major flow path 1006B second major flow path 1006C third major flow path 1006D fourth major flow path 1010 major dividers 1100A shorter barrel 1100B longer barrel 1100C smaller flow path 1100D larger flow path 2000A stock component 2000B short, flow-guiding single barrel 2000C long, flow-guiding single barrel 2000D narrow double barrel 2000E wide double barrel 2000F double barrel with a single fin 2000G multi-barrel 2000H short, flow-guiding multi-barrel 2000I long, flow-guiding multi-barrel 2000J flow-guiding multi-flow barrel 2000K flow-guiding multi-flow barrel with a single fin 2000L flow-guiding multi-flow barrel with a plurality of fins
Glossary
[0122] Unless defined otherwise, all technical and scientific terms used above have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present disclosure pertain.
[0123] The terms a, an, and the include both singular and plural referents.
[0124] The term or is synonymous with and/or and means any one member or combination of members of a particular list.
[0125] As used herein, the term exemplary refers to an example, an instance, or an illustration, and does not indicate a most preferred embodiment unless otherwise stated.
[0126] The term about as used herein refer to slight variations in numerical quantities with respect to any quantifiable variable. Inadvertent error can occur, for example, through use of typical measuring techniques or equipment or from differences in the manufacture, source, or purity of components.
[0127] The term substantially refers to a great or significant extent. Substantially can thus refer to a plurality, majority, and/or a supermajority of said quantifiable variable, given proper context.
[0128] The term generally encompasses both about and substantially.
[0129] The term configured describes structure capable of performing a task or adopting a particular configuration. The term configured can be used interchangeably with other similar phrases, such as constructed, arranged, adapted, manufactured, and the like.
[0130] Terms characterizing sequential order, a position, and/or an orientation are not limiting and are only referenced according to the views presented.
[0131] The invention is not intended to refer to any single embodiment of the particular invention but encompass all possible embodiments as described in the specification and the claims. The scope of the present disclosure is defined by the appended claims, along with the full scope of equivalents to which such claims are entitled. The scope of the disclosure is further qualified as including any possible modification to any of the aspects and/or embodiments disclosed herein which would result in other embodiments, combinations, subcombinations, or the like that would be obvious to those skilled in the art.
