1. Gene therapy and viral vectors Lecture 10

2. Physical methods of gene delivery micro-injection electroporation gene gun tattooing laser ultrasound

3. DNA Microinjection Microinjection is the direct-pressure injection of a solution into a cell through a glass capillary. It is an effective and reproducible method for introducing exogenous material into cells in culture. Manipulator and Phase- contrast microscope

4. Methodology Cells: The best cells for microinjecting are large, easily adherent, with a pronounced nucleus, giving them a tall aspect. In theory, any mammalian cell can be injected although some types provide more challenges than others. Contractile cells such as muscle often change shape rapidly in response to being injected (particularly when calcium is present in the medium), and cells that do not lay flat when cultured may need to be held in place with a second holding micropipet.

5. Micropipet: 1. Glass capillary tubing for fabricating micropipet 2. Micropipette puller for preparing the glass micropipettes DNA Any plasmid containing a cytomegalovirus (CMV) promoter-driven reporter gene that may be assayed in individual cells (e.g., green fluorescent protein [GFP] or b- galactosidase) may be used for monitoring the efficiency of microinjection.

6. Microinjection Apparatus: 1. Microscope 2. Micromanipulator 3. Microinjector

7. Applications

8. Animation of microinjection https://www.youtube.com/watch?v=h- Bfc1GPWpE

9. Electroporation Electroporation uses controlled, millisecond electrical pulses to create temporary pores in the cell membrane and allow dramatic cellular uptake of a synthetic DNA immunotherapy previously injected into muscle or skin. The cellular machinery then uses the DNA’s instructions to produce one or more proteins associated with the targeted disease. These foreign protein(s), or antigen(s), mimic the presence of an actual pathogen and induce an immune response to provide future protection against the pathogen or eliminate cells infected with an infectious disease or cancer.

12. Applications Electroporation is the formation of aqueous pores in lipid bilayers by the application of a short (microseconds to milliseconds) high- voltage pulse to overcome the barrier of the cell membrane. This transient, permeabilized state can be used to load cells with a variety of different molecules including ions, drugs, dyes, tracers, antibodies, oligonucleotides, RNA and DNA. Electroporation has proven useful both in vitro, in vivo and in patients, where drug delivery to malignant tumors has been performed. In addition, the data show that electroporation of DNA vaccines in vivo is an effective method to increase cellular uptake of DNA and gene expression in tissue leading to marked improvement in immune responses. Electroporation represents a way of increasing the number of DNA-transfected cells and enhancing the magnitude of gene expression, while reducing intersubject variability and requiring less time to reach a maximal immune response compared to conventional intramuscular injection of the vaccine.

13. Gene gun The gene gun is part of a method called the biolistic (also known as bioballistic) method, and under certain conditions, DNA (or RNA) become “sticky,” adhering to biologically inert particles such as metal atoms (usually tungsten or gold). By accelerating this DNA-particle complex in a partial vacuum and placing the target tissue within the acceleration path, DNA is effectively introduced. Uncoated metal particles could also be shot through a solution containing DNA surrounding the cell thus picking up the genetic material and proceeding into the living cell. A perforated plate stops the shell cartridge but allows the slivers of metal to pass through and into the living cells on the other side. The cells that take up the desired DNA, identified through the use of a marker gene (in plants the use of GUS is most common), are then cultured to replicate the gene and possibly cloned. The biolistic method is most useful for inserting genes into plant cells such as pesticide or herbicide resistance. Different methods have been used to accelerate the particles: these include pneumatic devices; instruments utilizing a mechanical impulse or macroprojectile; centripetal, magnetic or electrostatic forces; spray or vaccination guns; and apparatus based on acceleration by shock wave, such as electric discharge.

14. Gold particlesTungsten particles

15. Applications Another important use of the DNA gun involves the transformation of organelles: chloroplasts, as well as yeast mitochondria. The ability to transform organelles is significant because it enables researchers to engineer organelle-encoded herbicide or pesticide resistances in crop plants and to study photosynthetic processes. DNA delivery with the gene gun also offers new advantages for research in such areas as DNA vaccination/genetic immunization, gene therapy, tumor biology/wound healing, plant virology and many others. The major limitations are the shallow penetration of particles, associated cell damage, the inability to deliver the DNA systemically, the tissue to incorporate the DNA must be able to regenerate, and the equipment itself is very expensive. An objection to this method is that the DNA could be inserted into a working gene in the plant and many of the public worry that this new DNA could then be transferred to wild plants as well and resistance could be conferred to weeds or insects.