Tissue Engineering and the Gene Gun Introduction The gene gun is a high velocity biolistic transfection tool

[Woo2008]. It was created in 1987 by John Sanford at Cornell University as a tool for introducing new genetic material into the cells of plants [McD2003]. Since its inception, it has evolved to apply to other areas. It has even become common enough for some labs and researchers to make handmade versions of the gun [Cox2007]. The uses of the gene gun have expanded into a number of areas, including diolistics, gene therapy, and tissue engineering [OBr2007, McD2003, Wil1991]. Figure 1: The Helios Gene Gun [McD2003]

Figure 2: A Standard Gene Gun [McD2003]

The gene gun is pictured above in handheld form in Figure 1 and standard form in Figure 2. The gene gun employs a method known as bioballistics [OBr2001]. This method uses particle or microprojectile bombardment

techniques [Bio, Wil1991]. This method, while originally applied to plants, is now used for a variety of organisms and organelles: animals, chloroplasts, mitochondria, pollen, fungi, algae, and bacteria [Bio].

The gene gun is one of several methods of gene delivery techniques. Others include microseeding, direct injection, electroporation, use of viruses, and cationic liposomes. The gene gun, like microseeding and electroporation, has the ability to deliver large amounts of DNA. Like electroporation, direct injection of naked DNA, and cationic liposomes, the gene gun is technically simple. [Ble2007] The gene guns disadvantages also overlap with other methods. It has low transfection efficiency, like direct injection, microseeding, electroporation, and cationic liposomes. All of these have at least one of the same advantages as the gene gun. Thus, it is apparent that this is an unavoidable disadvantage that comes with the advantages of the gene gun. The requirement of physical damage to the cell also overlaps with electroporation, which requires damage to the cell membrane, and microseeding, which involves cellular damage. Depending on the desired advantages, this may also be an unavoidable disadvantage. Finally, the lack of specificity is shared with electroporation and cationic liposomes, meaning that this, too, may be an unavoidable disadvantage of the gene gun if certain advantages are priorities for the researcher. [Ble2007] Table 1, below, identifies some of the other methods and their advantages and disadvantages.

Table 1: Overview of Gene Delivery Techniques [Ble2007] Gene Delivery Advantages Technique Gene Gun Can deliver large amounts of DNA, technically simple

Disadvantages

Non-specific, physical damage to cell required for DNA uptake, low transfection efficiency

Direct injection of naked DNA/Plasmid DNA Microseeding

Simple, local delivery, unlimited gene size, non-toxic, most efficient in cardiac and skeletal muscle cells Can deliver large amounts and different types of DNA

Only applicable to tissues accessible by direct injection, very low transfection efficiency, transient gene expression only Low transfection efficiency, cellular damage, limited experience Non-specific, complex equipment, damage to cell membrane required for DNA uptake, low transfection efficiency

Electroporation

Technically simple, can deliver large amounts of DNA

Cationic Liposomes

Technically simple, local delivery, can transfect any cell type, no immunogenicity

Cannot target specific cell types, low transfection efficiency

Retrovirus

Transduces many different cell types, high efficiency of ex vivo transduction, long-term gene expression

Transduces dividing cells only, inefficient transduction in vivo, risk of insertional mutagenesis Immune response, lack of permanent expression, potential wild-type breakthrough, small DNA insert size (8 kb)

Adenovirus

Transfects virtually all cell types, dividing and non-dividing cells, good transfection efficiency in vivo, no integration into host genome

Adeno-associated Virus

Transduces dividing and non-dividing cells, integrates to specific site at chromosome 19, long-term gene expression

Difficult to grow to high titres, risk of insertional mutagenesis, small DNA insert size (4.7 kb), possible immune response and inflammatory reaction Difficult to manipulate due to complex life cycle, risk of wild-type breakthrough

Herpes Simplex Virus 1

Transduces wide variety of cell types, neurotropism, large DNA insert size (30 kb), long term expression feasible

As compared with viral techniques for gene delivery, the gene gun and other non-viral techniques offer notable advantages. Production of large quantities is cheaper when using non-viral methods. The procedure is also simpler and safer, with lower levels of toxicity and immunogenicity. It is ideal for short-term gene expression, but long-term gene expression is possible by cloning cells that have been successfully transfected. [Ble2007]

While the gene gun can deliver genetic material into a multiple organisms and organelles, this paper focuses on the gene guns application to tissue engineering. The gene gun has been used for transfection of multiple types of tissue, including mammalian skin [And2007]. In the following two sections, this paper examines the principles of cellular biology, genetics, engineering, and phsyics that are the foundation of the gene guns functioning. The next section considers the limitations of this tool. The fifth section discusses the various applications of the gene gun to tissue engineering. Finally, this paper outlines a sample protocol for researchers that are utilizing the gene gun.

Basic Science of the Gene Gun The scientific principles that apply once the genetic information is in the cell depend on the goals of introducing the genetic material. This paper focuses on the gene gun as it applies to tissue engineering. Thus, where the foundational scientific principles are different depending on the specific usage of the gene gun and desired outcome, those principles that are relevant to tissue engineering will be the focus in this paper.

The functioning of the gene gun is premised on aspects of cellular biology and genetics. Specifically, the manner in which cells code growth factor proteins and the role that messenger RNA plays in that process are especially

relevant to the gene gun for tissue engineering. Under certain conditions, RNA and DNA can adhere to inert biological particles such as metal atoms. When one of these atoms is coated with the RNA or DNA, it can be introduced into a living cell. [McD2003]

Figure 3, below, shows the how the viral technique operates. The image shows the principles and techniques of genetics and cellular biology upon which the gene gun was based. Picture, instead of the virus, a metal atom and the similarity is apparent. Genetic material is attached to a medium. In the case of the viral technique, it is a virus, and, in the case of the gene gun, it is the metal atom. Instead of introducing into the cell a truncated viral genome with DNA or RNA incorporated, the gene gun uses a metal atom to introduce the genetic materials without the need for a virus. After that, the process within and outside of the cell with respect to production of growth factor proteins is the same. [Goe2006]

Figure 3: Viral Technique for Gene Therapy [Goe2006]

Once inside the cell, the introduced DNA or RNA is incorporated into the nucleus of the cell or is maintained as an episome. Proteins help the gene to be transcribed to messenger RNA. From there, it is translated into growth factor proteins. In this manner, the gene gun is able to facilitate tissue engineering. [Goe2006] The physics and engineering of how it enters the cell is discussed in the following section.

Engineering Behind the Gene Gun The gene gun successfully transports DNA into cells by coating metal microprojectiles and propelling them at a high enough velocity so that these microprojectiles can penetrate the cell wall and cell membrane [OBr2007].

There are different kinds of gene guns and, as mentioned before, some researchers choose to make their own [Cox2007]. One of the more common gene guns is the handheld Helios Gene Gun made by Bio-Rad [McD2003] and figures of how the engineering of the gene gun works use their tool as model [Cox2007].

Figure 4: Basic Diagram of Helium-Powered Gene Gun [Cox2007]

The physical principle of the gene gun that facilitates its design is the acceleration of metal particles using a highvelocity burst of helium. Figure 4, above, gives a basic sketch of the parts of a gene gun. The metal particles that dry on walls of the cartridge (a 1.5 mm tube in the Helios system) are pulled from the surface when the highvelocity helium enters the cartridge. In the acceleration tube, driven by the stream of high-velocity helium, the particles pick up speed. In the expansion tube, the helium loses speed while the particles continue at a high velocity. In the spacer, the particles spread out to take up a larger area than the initial diameter (from 1.5 mm to 12 mm in this case). Differ tube sizes and spacer lengths alter the space of the spread. [Cox2007]

Figure 5, below, provides a more detailed diagram of the engineering of a gene gun. It shows what the mechanisms of the gene gun look like prior to the introduction of high-velocity helium and what happens to the mechanisms once the helium comes in. A plunger releases the high-velocity helium from the gas vent. It goes through the Kapton disc, dislodging the microprojectiles covered in genetic material. After going through the screen, the microprojectiles disperse with wider spacing then when within the gene gun and enter the tissue. [McD2003] This wider spacing is what allows the genetic material to cover a large area than other gene delivery techniques [Ble2007].

There are several variables that alter the effects of the gene gun. Depending on the desired area to be covered and the distance the particles must travel, a different tube and spacer must be used [Cox2007]. Additionally, other factors of the environment impact the effectiveness and the settings that must be used: number of cells used, ability of those cells to regenerate, the fragility of the tissue, type of gun used (helium-powered or gun powder; handheld or standard), and temperature [McD2003].

Figure 5: Detailed Diagram of Handheld Helium-Powered Gene Gun [McD2003]

Limitations The gene gun represents the technical simplicity of particle bombardment. Using traditional gun design the gene gun has created a method propelling large amounts of DNA into a cell rapidly. However, there are limitations to this technology. Though it is able to propel DNA at high velocity it does increase the risk of damage to cells through mass bombardment of particles as well as an increased susceptibility to stray foreign bodies that react badly to the particles. In addition to this, the gene gun method has a relatively low efficiency in successful transfection and in successful long-term transfection. (Goe2006) Due to the high pressure used to increase the velocity of the bullets, cell damage is a regular occurrence which can be a hindrance to any significant progress using the method. The size of the projectiles also needs to be considered when assessing potential damage caused by using this method as projectiles that are too large will increase the damage to cells. The potential for significant cell damage is a serious limitation on the gene gun. For the projectiles to reach their target they need to

be adequate in size and the pressure has to be extremely high. As a result, there is higher chance of damage with every projectile fired. (OBr2007)

Applications The gene gun is a versatile tool that has been used in a variety of different applications. Designed as a particle bombardment or, biolistic instrument the gene gun has been used on a number of subjects. Primarily used on plants, the gene gun has been useful on cells and tissues that prove resistant to other methods of change. Leaves, skin tissue, and seedlings are amongst the targets of the bombardment procedure. Although it was designed ostensibly as a tool to use with plants, the gene gun has also been used on animal subjects as well, specifically in tissue engineering. The biolistic process has successfully transformed animal embryos, cells in culture and animal tissues that are intact. The biolistic process is considered to be a convenient method for transforming tissues and cells as there is no need for particle bombardment prior to performing the procedure. [Bio]

Due to the versatility of the biolistic process, the application has been used on everything from plant cells to human tissue. The design of the gun makes it especially useful in biolistic transfection, propelling DNA and dye into human cells. Specially coated metal projectiles are fired at high velocity so that they can easily penetrate through the membrane and cell walls. A variety of methods to propel the microscopic projectiles have been used including gunpowder and a high pressure gas which is also known as the Helios gene gun. [OBr2007] A popular application for the gene gun is in transduction of naked DNA. This is a safe method of gene delivery and more efficient than some other methods of transduction. The advantage of using this method is that it can deliver large amounts of DNA in a single, technically easy, shot. However, there is susceptibility of physical damage to the cell. For example, foreign materials such as gold particles can flake off into the tissue, effectively causing more harm than good and inhibiting any potential applications for tissue engineering. [Ble2007]

The use of the gene gun has enabled progress to be made in healing wounds, studying tumor biology, and immunization [McD2003]. On a short term basis, the gene gun can prove useful particularly when applied to healing wounds. Wound repair can be incredibly complex, occurring in phases or sections over the course of a period of time. Short-term gene expression is a vital part of the process as well. Unlike other practices where short-term gene expression is unsuitable, in transduction it is the perfect solution for wound repair and transforming damage to the skin tissue. The gene delivery system of biolistic transduction is most suited to this procedure as it is simple technique that requires very little time to complete. [Ble2007]

Another source for tissue engineering is stem cells. Stem cells have the capacity for self-renewal and can be differentiated into multiple cell types. The types of stem cells used in tissue engineering are the embryonic stem cells (ESC) and adult stem cells. ESCs have a variety of applications due to the fact that they are totipotent,

giving them the ability to form all of the cells and tissues of a living organism. Mesenchymal stem cells (MSC) also have a number of applications in gene therapy and have been used in skin and bone tissue repair. [Goe2006] Bone has the potential to spontaneously regenerate though this is limited in a clinical environment when facing larger defects in the bone structure. Though there are several types of cells available to aid the repair of bone, most are limited to short-term repair and therefore are often not used. Thus, the focus for long-term repair has centered on stem cell engineering. MSCs are particularly useful in osteogenic differentiation and formation of bone structure. To administer the MSCs effectively, a transduction is considered a very useful tool and the gene gun enables the bone to be induced for reformation. [Ble2007]

One of the slowest gene therapy procedures is nerve regeneration. The majority of the time the process does not garner the desired results and presently factors that determine the neuroregenerative capability are still being looked into. These capabilities include neurotrophic factors and nerve scaffolds. There has been extensive research into a suitable conduit as an alternative to the current use of the autologous nerve graft. Various choices for guidance channels include synthetic substances such as silicone while biological conduits include arterial and acellular grafts. For nerve regeneration to occur successfully there needs to be cells present that secrete nerve growth factor (NGF). This is essential for all nerve regeneration. Traditional delivery techniques of NGF have suffered serious failings in the control of dosage which has resulted in extremely high levels. The potential for gene therapy to provide more control over growth factors is high and tests on animal subjects have recorded results to suggest that gene therapy enables regeneration at a faster pace. This means that the gene gun may be a relevant alternative to intravenous vaccines when administering NGF. The gene gun method of propulsion is also a safer method and would result in lower morbidity than previous methods considered standard. [Ble2007]

Cell transplantations have been studied in the hopes that they may be able to be utilized as an effective method of distributing insulin for sufferers of Type 1 Diabetes. If a pancreatic islet cell transplant was successful in distribution, it could also mean better control over blood sugars which would effectively cure Type 1 Diabetes. At the moment the problem remains that cell transplants have a short life expectancy when performed. However, research has shown that hepatic cells have had success regenerating the liver and a transplant of hepatocytes could be a better approach for patients suffering with liver based illnesses. Using gene transference combined with tissue engineering this approach could be successful and even offer hope to sufferers of other diseases such as Type 1 Diabetes. At present there is research into the possibilities of using liver stem cells in tissue engineering applications, meaning the gene gun is likely to be implemented in future organ tissue engineering procedures. [Ble2007]

Protocol When shooting tissue and cell samples, it is essential that everything takes place in a sterile environment. This will allow the test subjects to avoid contamination and enable an accurate recording of results. [Woo2008]

To shoot cultured slices have the following ready [Woo2008, McA2004]: Microprojectiles, usually gold or tungsten Cartridge for the microprojectiles (may come pre-loaded) Cultured slices of cells and tissue Gene gun Cartridge holder Barrel liner Forceps Diffusing screen Diffuser Ear protection 2 x 2 nylon mesh Tape

Using the gene gun [Woo2008, McA2004]: 1. Place ear protection over ears. 2. Sterilize the cartridge holder and nylon mesh by exposure to UV light for 30 minutes. 3. To pressurize the guns helium hose and reservoirs, fire a few empty shots at 100 psi. 4. Place microprojectiles into the cartridge holder with the forceps. Skip this step if the cartridge is preloaded. 5. Secure the cartridge holder into the gene gun by locking into place where indicated on the body of the gun. 6. Attach the nylon mesh to the barrel liner with tape. This reduces damage caused by the helium shock wave. This protection becomes more critical when the tissue is more fragile. 7. Add the barrel liner and diffuser screen onto the gene gun using forceps. 8. Turn up the pressure on the gas tank to 180 psi because high pressure is essential in completing a successful transfection. However, a pressure that is too high can cause damage. Some tissue cultures will require pressure as low as 75-110 psi. 9. Position the gene gun at a 90 degree angle and at a distance such that the diffuser screen is about 1 inch from the culture and fire, at closest it should be no less than 0.5 inches away otherwise slice will be damaged. 10. Repeat this last step until all the slices have been transfected successfully. 11. Turn off the gas and carefully remove the barrel and diffuser screen from the gun.

References [Woo2008] Woods, G and Zito, K, Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture, Journal of Visualized Experiments 2008(12): 1-4, 2008. [McD2003] McDonald, M, Gene Guns, http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Gene_gun.html [Cox2007] Cox, G, Optical Imaging Techniques in Cell Biology, CRC Press, Boca Raton, 2007. [OBr2007] OBrien, JA and Lummis, SCR, Diolistics: incorporating fluorescent dyes into biological samples using a gene gun, Trends in Biotechnology 25(11-2): 530-534 [Wil1991] Williams, RS, Johnston, SA, Riedy, M, DeVit, MJ, McElligott, SG, and Sanford, JC, Introduction of foreign genes into the tissues of living mice by DNA-coated microprojectiles, Proceedings of the National Academy of Sciences 88: 2726-2730, 1991. [OBr2001] OBrien, JA, Holt, M, Whiteside, G, Lummis, SC, and Hastings, MH, Modifications to the hand-held Gene Gun: improvements for in vitro biolistic transfection of organotype neuronal tissue, Journal of Neuroscience Methods 112(1): 57-64, 2001. [Bio] Biolistic PDS-1000/HE Particle Delivery System Bulletin 1700, http://www.biorad.com/webmaster/pdfs/Bulletin_1700.pdf [Ble2007] Bleiziffer, O, Eriksson, E, Yao, F, Horch, RE, Kneser, U, Gene transfer strategies in tissue engineering, Journal of Cellular and Molecular Medicine 11(2): 206-223, 2007. [And2007] Andreadis, ST, Gene-Modified Tissue-Engineered Skin: The Next Generation of Skin Substitutes, In K. Lee and D. Kaplan, editors, Tissue Engineering II, 241-274, Springer-Verlag, Berlin, 2007. [Goe2006] Goessler, UR, Riedel, K, Hrmann, K, and Riedel, F, Perspectives of Gene Therapy in Stem Cell Tissue Engineering, Cells Tissues Organs, 183: 169-179, 2006. [McA2004] McAllister, AK, Biolistic Transfection of Cultured Organotype Brain Slices, In WC Heiser, editor, Gene Delivery to Mammalian Cells: Volume 1: Nonviral Gene Transfer Techniques, Humana Press, Totowa, 2004.