Patent Application
20120295345
Microinjection of foreign materials into a biological structure such as a living cell can be problematic. Various transfection techniques include the microinjection of foreign genetic material such as DNA into the nucleus of a cell to facilitate the expression of foreign DNA. For example, when a fertilized oocyte (egg) is transfected, cells arising from that oocyte will carry the foreign genetic material. Thus in one application, organisms can be produced that exhibit additional, enhanced, or repressed genetic traits. In some cases, researchers have used microinjections to create strains of mice that carry a foreign genetic construct causing macrophages to auto-fluoresce and undergo cell death when exposed to a certain drugs. Such transgenic mice have since played roles in investigations of macrophage activity during immune responses and macrophage activity during tumor growth.
Prior art microinjectors function in a similar manner to macro-scale syringes: a pressure differential forces a liquid through a needle and into the cell. In some cases a glass needle that has been fire drawn from a capillary tube can be used to pierce the cellular and nuclear membranes of an oocyte. Precise pumps then cause the expulsion of minute amounts of genetic material from the needle and into the cell. Researchers have produced fine microinjection needles made from silicon nitride and silica glass that are smaller than fire drawn capillaries. These finer needles generally also employ macro-scale pumps similar to those used in traditional microinjectors.
The present disclosure provides systems, devices, and methods for protecting a biological material during delivery into a biological structure. In one aspect, for example, a device for protecting and delivering a preselected biological material into a biological structure can include a lance operable to maintain a charge capable of associating a biological material thereto and at least one protective region formed on or in the lance, where the protective region protects the biological material during delivery into a biological structure.
In another aspect, a method of protecting a biological material during delivery into a biological structure can include bringing into proximity outside of the biological structure a lance having at least one protective region and a preselected biological material, charging the lance with a polarity and a charge sufficient to electrically associate at least a portion of the biological material with the at least one protective region, and inserting the lance into the biological structure while the biological material is protected from damage and/or dislodgement by the at least one protective region. The method can also include discharging the lance to release at least a portion of the biological material from the at least one protective region of the lance and withdrawing the lance from the biological structure.
In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set forth below.
The singular forms “a,” “an,” and, “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” can include reference to one or more of such supports, and reference to “an oocyte” can include reference to one or more of such oocytes.
As used herein, the term “biological material” can refer to any material that has a biological use and can be delivered into a biological such as a cell or a cell organelle. As such, “biological material” can refer to materials that may or may not have a biological origin. Thus, such material can include natural and synthetic materials, as well as chemical compounds, dyes, and the like.
As used herein, the term “charged biological material” may be used to refer to any biological material that is capable of being attracted to or associated with an electrically charged structure. Accordingly, charged biological material may be used to refer to those molecules having a net charge, as well as those molecules that have a net neutral charge but possess a charge distribution that allows attraction to the structure.
As used herein, the term “peptide” may be used to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. A peptide of the present invention is not limited by length, and thus “peptide” can include polypeptides and proteins.
As used herein, the term “uncharged” when used in reference to a lance may be used to refer to the relative level of charge in the lance as compared to a charged biological material. In other words, a lance may be considered to be “uncharged” as long as the amount of charge on the needle structure is insufficient to associate a useable portion of a charged biological material. Naturally what is a useable portion may vary depending on the intended use of the biological material, and it should be understood that one of ordinary skill in the art would be aware of what a useable portion is given such an intended use. Additionally it should be noted that a lance with no measurable charge would be considered “uncharged” according to the present definition.
As used herein, the term “sample” when used in reference to a sample of a biological material may be used to refer to a portion of biological material that has been purposefully attracted to or associated with the lance. For example, a sample of a biological material such as DNA that is described as being associated with a lance would include DNA that has been purposefully attracted thereto, but would not include DNA that is attracted thereto through the mere exposure of the lance to the environment. One example of DNA that would not be considered to be a “sample” includes airborne DNA fragments that may associate with the lance following exposure to the air.
As used herein, “associate” is used to describe biological material that is in electrostatic contact with a structure due to attraction of opposite charges. For example, DNA that has been attracted to a structure by a positive charge is said to be associated or electrically associated with the structure.
As used herein, the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is “substantially” enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a composition that is “substantially free of” particles would either completely lack particles, or so nearly completely lack particles that the effect would be the same as if it completely lacked particles. In other words, a composition that is “substantially free of” an ingredient or element may still actually contain such item as long as there is no measurable effect thereof.
As used herein, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “a little above” or “a little below” the endpoint without affecting the desired result.
As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 1 to about 5” should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually.
This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
The present disclosure provides methods, devices, and associated systems for protecting a biological material during delivery into a cell. Also provided are methods, devices, and associated systems for maximizing the amount of biological material that can be delivered into a cell. Various conventional techniques have been used to introduce a biological material into a cell, and in some cases the biological material can become damaged or disassociated from the device during delivery. This can be problematic for techniques utilizing devices whereby the biological material is associated with an external surface of the delivery instrument, such as a lance.
Generally, the present methods and systems utilize the electrical association and dissociation of a biological material to a lance or other delivery device as a mechanism for delivering the biological material into a biological structure, such as a cell, cellular organelle, or any other structure of a biological origin. Because the biological material can be loaded onto the lance and subsequently released via changes in the charge state of the lance, internal microinjection channels are not required for the delivery of the biological material. As such, a lance can be smaller in size and can be formed in configurations that may not be possible with prior delivery devices. These delivery devices can have an outer shape and cross-section that is significantly smaller than traditional injection pipettes. Such smaller outer shapes may be less disruptive to cellular structures, and thus may allow delivery of the biological with less cellular damage.
Once a biological material has been electrically associated with a tip portion of the lance, the lance can be inserted through an outer portion of the biological structure. With a tip portion of the lance located within the biological structure, the lance can be discharged to release at least a portion of the biological material. Once the biological material has been delivered, the lance can be withdrawn.
In one aspect,
As is shown in
It should be noted, that various materials begin to decompose (e.g. by electrolysis) at voltages above a certain threshold voltage referred to as the decomposition voltage. The decomposition voltage can be different for different materials. In some cases, such decomposition can generate oxygen and hydrogen at the positively charged lance and the negatively charged return electrode, respectively. These electrolysis products can cause damage to the lance and negatively affect the cell being injected. As such, in one aspect the voltage that can be used to charge the lance can be at or below the decomposition voltage. In one specific aspect, the lance is charged with a voltage from about 1 V below the decomposition voltage to about the decomposition voltage. In another aspect, the lance is charged with a voltage from about 2 V below the decomposition voltage to about the decomposition voltage.
Once the zygote 104 is restrained, the lance 102 can be oriented into a position relative to the zygote into which the biological material will be introduced, as is shown in
As is shown in
As is shown in the upper right inset of
Following release of the biological material, the lance 102 can be withdrawn from the zygote 104 as is shown in
When a lance having biological material associated with its surface is inserted through a biological structure such as, for example, a cell membrane, the biological material can become damaged and/or displaced or otherwise disassociated from the lance due to the movement of the biological structure relative to the surface of the lance. Such disruptive effects can decrease the quantity of, and possibly the quality of, biological material that is actually delivered into the biological structure. In addition to external biological structures such as cell walls or cell membranes, internal biological structures can cause similar disruptive effects, thus possibly limiting the biological material arriving at a target location within the biological structure.
Accordingly, methods, systems, and devices for protecting biological material during delivery into a biological structure are provided. In one aspect, for example, a method can include bringing into proximity outside of a biological structure a lance having at least one protective region and a preselected biological material. The lance is charged with a polarity and a charge sufficient to electrically associate at least a portion of the biological material at least partially within the at least one protective region. The lance is then inserted into the biological structure while the biological material is protected from damage and/or displacement by the at least one protective region. Following insertion into the biological structure, the lance is discharged to release at least a portion of the biological material from the at least one protective region of the lance. Releasing the biological material from the protective region thus delivers the biological material into a location within the cell. The lance can then be withdrawn from the biological structure.
It should be noted that in some aspects the protective region may provide sufficient protection that the biological material may remain in the protective region during lance insertion into the cell. For example, in one aspect the lance can be charged to associate the biological material within the protective region. The lance can be discharged and inserted into the cell, while the biological material remains associated with the protective region. Once in the cell, the lance can be oppositely charged to disassociate the biological material from the protective region.
In another aspect, a device for protecting and delivering a preselected biological material into a biological structure is provided including a lance that is operable to maintain a charge capable of associating a biological material thereto, and at least one protective region formed on or in the lance. The protective region(s) is operable to protect the biological material during delivery into a biological structure.
Turning to
Various sizes and configurations of protective regions are contemplated, and any such size and/or configuration capable of providing protection to biological material during delivery is considered to be within the present scope. In one aspect, for example, the protective regions can be of a size sufficient to protect a biological material from damage and/or disassociation from the lance. As such, the size of the protective region can vary depending on the biological material being associated therewith. For example, small molecules can be accommodated in smaller protective regions as compared to large macromolecules such as DNA. That being said, in one aspect protective regions can be from about 5 nm to about 1 micron in size. In another aspect, protective regions can be from about 10 nm to about 500 nm in size. In yet another aspect, protective regions can be from about 50 nm to about 250 nm in size. In another aspect, the protective regions can sufficiently large to protect a biological material from damage and/or disassociation from the lance and sufficiently small to minimize snagging on membranous structures such as the cellular membrane. It should be noted that, when referring to a size of the protective regions, measurements can be the approximate size of openings or depressions in the lance surface, the approximate size of the protective region within the lance body, the approximate surface area of the protected region, or the like.
It should be noted that it is not necessarily the size of a protective region that may cause damage to cellular structures during delivery, but rather the size with respect to the location and configuration on the lance. For example, a 1 μm protrusion from the side of lance may cause significantly more damage to the cell than a 2 μm hollow region extending into the lance with little or no protrusion. Furthermore, the size of a protrusion from a protective region that results in undesirable damage to a biological structure can vary depending on the cell. Such protrusions can be larger in big cells such as zygotes as compared to small cells such as neurons. That being said, in one aspect the portion of a protective region that causes disruption to a biological structure can be less than or equal to about 50 nm. In another aspect, the portion of a protective region that causes disruption to a cellular structure can be less than or equal to about 10 nm. In yet another aspect, the portion of a protective region that causes disruption to a cellular structure can be less than or equal to about 1 nm.
One exemplary technique for creating a lance having an outer surface covered with protective regions is to fabricate the lance from a polycrystalline material through a micromachining process. Randomly oriented crystallographic grains of the polycrystalline material can cause surface depressions at the grain boundaries between the crystals. These surface depressions can be of a size sufficient to function as protective regions for the biological material. An SEM image of one example of a polycrystalline silicon lance is shown in
In other aspects, a lance can contain larger protective regions for protecting biological material during delivery. In one aspect, as is shown in
In addition to protective regions extending partially through the body of the lance, in some aspects a protective region can extend entirely through the body of the lance. As is shown in
In another aspect, as is shown in
As has been described, the size and configuration of the protective region can vary depending on the design of the lance, the lance material, and the nature of the delivery procedure, including various user preferences. For example, in one aspect a protective region can be up to a size or volume that maintains sufficient material in the lance to provide adequate cross sectional strength during delivery of the biological material. In another aspect, multiple protective regions can be up to a collective size or volume that maintains sufficient material in the lance to provide adequate cross sectional strength during delivery of the biological material.
In addition to the micromachining of the polycrystalline lance example described above, various techniques are contemplated for forming protective regions in the lance surface, and any such technique is considered to be within the present scope. For example, such techniques can include MEMS fabrication, etch processing, micromachining, laser ablation, physical abrasion, and the like, including combinations thereof. Additionally, materials having a structure that generates protective regions during manufacture of the lance, such as porous materials, can also be used.
Additionally, the protective regions can provide additional benefits to the lance. For example, one issue that may arise as a lance decreases in size relates to the surface area that is available for association with a biological material. A smaller lance tip has a smaller surface area for holding a charge as compared to a larger lance tip. This smaller surface area can limit the amount of biological material that can be associated therewith. By introducing protective regions into the lance, the surface area of the lance tip is increased, thereby increasing the amount of biological material that can be associated with the charged lance tip. Accordingly, in one aspect protective regions can be provided in a lance to increase the surface area of the lance.
As has been described, a charge is introduced into and held by the lance in order to electrically associate the biological material to the lance. Various lance materials are contemplated for use in constructing the lance, and any material that can be formed into a lance structure and is capable of carrying a charge is considered to be within the present scope. Non-limiting examples of lance materials can include a metal or metal alloy, a conductive glass, a polymeric material, a semiconductor material, carbon nanotube, and the like, including combinations thereof. In one aspect, a lance can be a carbon nanotube filled with a material such as carbon, silicon, and the like. Non-limiting examples of metals can include indium, gold, platinum, silver, copper, palladium, tungsten, aluminum, titanium, and the like, including alloys and combinations thereof. Polymeric materials that can be used to construct the needle structure can include any conductive polymer, non-limiting examples of which include polypyrrole doped with dodecyl benzene sulfonate ions, SU-8 polymer with embedded metallic particles, and the like, including combinations thereof.
Non-limiting examples of useful semiconductor materials can include germanium, gallium arsenide, and silicon, including various forms of silicon such as amorphous silicon, monocrystalline silicon, polycrystalline silicon, and the like, including combinations thereof. Indium-tin oxide is a material that is also contemplated for use as a lance material. Additionally, in some aspects the lance can be a conductive material that is coated on a second material, where the second material provides the physical structure of the lance. Examples can include metal-coated glass or metal-coated quartz lances. The lance can also include a hollow, non-conductive material, such as a glass, where the hollow material is filled with a conductive material. Depending on the design, the lance can be manufactured using various techniques such as wire pulling, chemical etching, MEMs processing, and the like.
Any size and/or shape of lance capable of delivering biological material into a cell is considered to be within the present scope. The size and shape of the lance can also vary depending on the cell receiving the biological material. The effective diameter of the lance, for example, can be sized to maximize survivability of the cell. It should be noted that the term “diameter” is used loosely, as in some cases the cross section of the lance may not be circular. Limits on the minimum diameter of the lance can, in some cases, be a factor of the material from which the lance is made and the manufacturing process used. In one aspect, for example, the lance can have a tip diameter of from about 5 nm to about 3 microns. In another aspect, the lance can have a tip diameter of from about 10 nm to about 2 microns. In another aspect, the lance can have a tip diameter of from about 30 nm to about 1 micron. In a further aspect, the lance can have a tip diameter that is less than or equal to 1 micron. As such, in many cases the tip diameter of the lance can be smaller than the resolving power of current optical microscopes, which is approximately 1 micron. As is noted above, lance tips are contemplated that can have cross sections that are not circular. In such cases, it is intended that the circumference of a circle defined by the tip diameters disclosed above would be substantially the same as an outer circumferential measurement of a non-circular lance tip.
The length of the lance can be variable depending on the design and desired attachment of the lance to the lance manipulation system. Also, the portion of the lance that is contacting and/or passing through a portion of the cell can vary in length depending on the lance design and the depth of the area into which the biological material is to be delivered. For example, delivering biological material to an area located near the surface of a cell can be accomplished using a shorter lance as compared to delivery to an area located deep within a cell. This would not preclude, however, the use of longer lances for delivery into areas near the cellular surface. For example, a relatively long lance may be used to deliver biological material in an application where only a small portion (e.g., only the tip) of the lance penetrates a cell. It should be noted that the lance length can be tailored to the delivery situation and to the preference of the individual performing the delivery.
Thus the length of the lance can be any length useful for a given delivery operation. For example, in some aspects, the lance can be up to many centimeters in length. In other aspects, the lance can be from a millimeter to a centimeter in length. In another aspect, the lance can be from a micron to a millimeter in length. In one specific aspect, the lance can be from about 2 microns to about 500 microns in length. In another specific aspect, the lance can be from about 2 microns to about 200 microns in length. In yet another specific aspect, the lance can be from about 10 microns to about 75 microns in length. In a further specific aspect, the lance can be from about 40 microns to about 60 microns in length.
Additionally, the shape of the lance, at least through the portion of the lance contacting the cell, can vary depending on the design of the lance and the depth to which the biological material is to be injected into the cell. A high lance taper, for example, may be more disruptive to cellular membranes and internal cellular structures than a low taper. In one aspect, for example, the lance can have a taper of from about 1% to about 10%. In another aspect, the taper can be from about 2% to about 6%. In yet another aspect, the taper can be about 3%. The taper of the lance can also be described in terms of the size of the disruption in the cell membrane following insertion. In one aspect, for example, the approximate diameter of the disrupted area of the cell membrane following lance insertion is from about 0.5 nanometers to about 8 microns. In another aspect, the approximate diameter of the disrupted area of the cell membrane following lance insertion is from about 2 micron to about 5 microns.
The overall shape and size of the lance can also be designed to take into account various factors, including those involved with the delivery procedure, as well as the materials utilized to make the lance. For example, in one aspect a lance can be designed having sufficient cross sectional strength to allow biological material delivery, while at the same time minimizing the damage done to the biological structure from the lance's cross sectional area. As another example, the lance can be designed to have a cross sectional area sufficient to minimize damage to the biological structure, while at the same having sufficient surface area to which biological material can be associated.
Different materials can also affect the design of the size and shape of the lance. Some materials may not hold a charge sufficient to associate the biological material to the lance tip at smaller sizes. In such cases, larger size lances can be used to facilitate a higher charge capacity. It may be difficult to form particular sizes and shapes of the lance from certain materials. In such cases, the lance size and shape can be designed to the properties of the desired material. For example, a material such as gold may not be capable of supporting the lance tip at very small diameters due to inadequate strength at smaller sizes, or it may not be possible or feasible to create a very small diameter tip with gold. If the use of a gold lance is desired, the lance size and shape can thus be designed with the properties of gold in mind.
In another aspect, lances in accordance with the present disclosure can be constructed so as to facilitate the electrical association of biological material within the protective region or protective regions. In some aspects, biological material can be associated within the protective region while minimizing the association of biological material elsewhere on or in the lance. As is shown in
Biological material can be delivered into a variety of biological structures, including various cell types. Both prokaryotic and eukaryotic cells are contemplated that can receive biological material, including cells derived from, without limitation, mammals, plants, insects, fish, birds, yeast, fungus, and the like. Additionally, cells can include somatic cells or germ line cells such as, for example, oocytes and zygotes. The enhanced survivability of cells with the present techniques can allow the use of cells and cell types that have previously been difficult to microinject due to their delicate nature. Additionally, various types of biological materials are contemplated for delivery into a biological structure, and any type of biological material that can be electrostatically delivered is considered to be within the present scope. Non-limiting examples of such biological materials can include DNA, cDNA, RNA, siRNA, tRNA, mRNA, microRNA, peptides, synthetic compounds, polymers, dyes, chemical compounds, organic molecules, inorganic molecules, and the like, including combinations thereof. In one aspect, the biological material can include DNA, cDNA, RNA, siRNA, tRNA, mRNA, microRNA, and combinations thereof. In another aspect, the biological material can include DNA and/or cDNA.
A charging system used to charge the lance can include any system capable of electrically charging, maintaining the charge, and subsequently discharging the lance. Non-limiting examples can include batteries, DC power supplies, photovoltaic cells, static electricity generators, capacitors, and the like. The charging system can include a switch for activation and deactivation, and in some aspects can also include a polarity switch to reverse polarity of the charge on the lance. In one aspect the system may additionally include multiple charging systems, one system for charging the lance with a charge, and another charging system for charging the lance with an opposite polarity charge. In one example scenario, an initially uncharged lance is brought into contact with a sample of a biological material. The biological material can be in water, saline, or any other liquid capable of maintaining biological material. A charge opposite in polarity to the biological material is applied to the lance, thus associating a portion of the biological material with the lance. The lance can then be moved into the biological structure, and lance can be discharged, thus releasing the biological material.
The lance can be manipulated by any mechanism capable of aligning and moving the lance. Such a lance manipulation system can include any system or device capable of orienting and moving a lance. Non-limiting examples of lance manipulation systems include mechanical systems, magnetic systems, piezoelectric systems, electrostatic systems, thermo-mechanical systems, pneumatic systems, hydraulic systems, and the like. In one aspect, the lance manipulation system can be one or more micromanipulators. The lance may also be moved manually by a user. For example, a user may push the lance along a track from first location to a second location.
In one aspect, the lance can be moved by the lance manipulation system in a reciprocal motion along an elongate axis of the lance. In other words, the lance can move forward into a biological structure and backward out of the biological structure along the same path. By moving along the elongate axis of the lance, the minimum cross sectional area of the lance is driven through biological structure such as a cell membrane and/or an organelle of interest. This minimal cross sectional exposure can limit the cellular disruption, thus potentially increasing the success of the biological material delivery procedure.
For cellular delivery, the cell can be manipulated and or held in position by a variety of mechanisms. It should be noted that any technique, device, or system for manipulating and/or holding a cell in position is considered to be within the present scope. In one aspect, for example, the cell can be held in position by a suction pipette. A slight suction at the end of such a pipette can hold a cell for sufficient time to accomplish a biological material delivery procedure into the cell. Additionally, supporting arms or other physically restraining structures can be used to hold the cell in position during the delivery procedure. Various configurations for support structures would be readily apparent to one of ordinary skill in the art once in possession of the present disclosure, and such configurations are considered to be within the present scope.
Further exemplary details regarding lances, charging systems, lance manipulation systems, and cellular restraining systems can be found in U.S. patent application Ser. Nos. 12/668,369, filed Sep. 2, 2010; 12/816,183; filed Jun. 15, 2010; 61/380,612, filed Sep. 7, 2010; and 61/479,777, filed on Apr. 27, 2011, each of which is incorporated herein by reference.
It should be noted that the design of a system for delivering biological material into a biological structure can vary due to the interdependencies of various system parameters. Combinations of features can thus influence other features, both in terms of system design and in terms of system use. Features can thus be mixed and matched to create a delivery system for a given purpose or desirable performance. For example, the materials and configuration chosen for the lance may have properties allowing a greater or lesser charge capacity, thus influencing the voltage, current, and electrical timing of the charging and discharging. A smaller tip diameter can more effectively enter a biological structure with potentially less damage, but may have a smaller surface area for the association of biological material. The association capacity of the lance for biological material can thus be increased, for example, by utilizing lance materials capable of holding a higher relative charge, or by utilizing a non-circular shape for the lance tip that increases surface area while minimizing the penetration damage of the lance. Thus, if a particular feature is desired for a lance, other features can be varied to accommodate such a design. As such, it should be understood that the various details described herein should not be seen as limiting, particularly those involving dimensions or values. It is contemplated that a wide variety of design choices are possible, and each are considered to be within the present scope.
It is to be understood that the above-described compositions and modes of application are only illustrative of preferred embodiments of the present invention. Numerous modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention and the appended claims are intended to cover such modifications and arrangements. Thus, while the present invention has been described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiments of the invention, it will be apparent to those of ordinary skill in the art that numerous modifications, including, but not limited to, variations in size, materials, shape, form, function and manner of operation, assembly and use may be made without departing from the principles and concepts set forth herein.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/487,535, filed on May 18, 2011, which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61487535 | May 2011 | US |
