1. safe as ultrasound waves appear to have no adverse effects
2. BIOLOGICAL MECHANISM
2.1. TRANSDUCTION
2.1.1. viral RNA transcribed by reverse transcriptase to produce double stranded DNA
2.1.2. 2 types
2.1.2.1. adenoviral transduct
2.1.2.1.1. virus enters host cell via endocytosis
2.1.2.1.2. adenoviral DNA remains episomal (it does not integrate into host genome)
2.1.2.2. lentiviral transduct
2.1.2.2.1. permanently integrates into host cell's genome
2.1.2.2.2. DNA then enters nucleus
2.1.2.2.3. transgene integrated into host genome via lentiviral integrase enzymes
2.1.2.2.4. TAKE INTO ACCOUNT OF: EFFECTS CAUSED BY GENOMIC INTEGRATION
2.1.3. delivery using viral vector
2.1.4. transgene
2.1.4.1. added to complete viral genome OR
2.1.4.2. used to replace one or more viral genes
2.1.4.3. delivered as part of recombinant viral genome
2.1.4.4. purpose: to exploit virus's natural ability to infect & transfer nucleic acid and animal cells.
2.2. BACTOFECTION (BACTERIA- MEDIATED GENE DELIVERY)
2.2.1. delivery using bacterial gene
2.2.1.1. f(x): to deliver therapeutic genes into target cells)
2.2.1.1.1. bacterial vectors that contain expression plasmid with therapeutic genes interact with target cells (intestinal cell)
2.2.2. relies on bacteria (also invades animal cells)
2.2.3. transgene
2.2.3.1. delivered not as part of bacteria genome BUT on plasmid carried by the bacterium
2.2.3.1.1. dependents on the formation of a fine DNA/calcium phosphate co-precipitate
2.2.4. how does it happen?
2.2.4.1. bacteria penetrates into cell
2.2.4.2. plasmids with therapeutic genes are released into cytoplasm upon bacterial vector lysis
2.2.4.3. released plasmid transported into nucleus
2.2.4.4. therapeutic genes expressed by host cell machinery
3. NON-BIOLOGICAL MECHANISM
3.1. CHEMICAL TRANSFECTION
3.1.1. Cells are persuaded to take up DNA from their surroundings when DNA presented as a synthetic complex
3.1.1.1. a complex will overall positive charge
3.1.1.1.1. allow it to interact with negatively charged cell membrane and promote endocytosis
3.1.1.2. a lipophilic complex
3.1.1.2.1. fuses with the membrane
3.1.2. involves formation of a complex that facilitates interactions with plasma membrane of cell via electrostatic attraction or fusion of lipophilic vesicles
3.1.3. Calcium phosphate method
3.1.3.1. taken up by endocytosis
3.1.3.1.1. suitable for cell growing in monolayer/suspension but not clump
3.1.3.2. precipitate must coat the cells
3.1.3.3. low proportion of cells that took up DNA (1-2%)
3.1.3.4. inexpensive and simple method
3.1.3.5. transfection efficiency
3.1.3.5.1. depends on cell constitution
3.1.3.5.2. pH
3.1.3.5.3. quality
3.1.3.5.4. amount of used nuclotides
3.1.3.6. toxic
3.1.4. Polyplexes method
3.1.4.1. alternative chemical transfection
3.1.4.1.1. to solve the problem where some cell lines are affected by co-precipitate due to its toxicity and hence, difficult to transfect in calcium phosphate method
3.1.4.2. using polycationic compounds that form soluble complexes (polyplexes) through spontaneous electrostatic interactions with DNA
3.1.4.3. The earliest of this method utilized DEAE-dextran (diethylaminoethyl dextran), a soluble polycationic carbohydrate
3.1.4.3.1. initially, it's devised to introduce viral RNA and DNA into cells but later use as a method for plasmid DNA transfer
3.1.5. Lyposomes and lypoplexes method
3.1.5.1. alternative chemical transfection
3.1.5.1.1. package DNA inside a fusogenic phospholipid vesicle which interact with target cell membrane and facilitates DNA uptake
3.1.5.2. First approach- used bacterial protoplast containing plasmids to transfer DNA into mammalian cells
3.1.5.2.1. bacterial cells were transformed with a plasmid vector and then treated with chloramphenicol
3.1.5.3. advantages
3.1.5.3.1. very efficient
3.1.5.3.2. gentle
3.1.5.3.3. ability to transform cells in live animals
3.1.5.4. disadvantage
3.1.5.4.1. labor-intensive
3.1.5.5. example
3.1.5.5.1. yeast cells with cell wall removed (spheroplasts) has been used to introduced YAC DNA into mouse ES cells
3.1.5.5.2. liposome-based procedure, liposome is a artificial phospholipid vesicle
3.1.5.5.3. a breakthrough- cationic/neutral lipid mixtures can spontaneously form stable complexes with DNA lipoplexes that interact productively with cell membrane
3.1.5.6. transfection efficiency improved
3.1.5.6.1. combine liposomes and lipoplexes with viral proteins
3.2. PHYSICAL TRANSFECTION (direct transfer by physical force)
3.2.1. Microinjection
3.2.1.1. technique that guaranteed to generate successful
3.2.1.2. introduces DNA into large cells such as oocytes, also can inject directly into tissues such as skin and muscle
3.2.1.3. eg: single cell manipulation and transgenic animals
3.2.1.4. Precise but time-consuming and expensive
3.2.2. Ultrasound
3.2.2.1. involves the exposure of cells to a rapidly oscillating probe
3.2.2.2. Process: cavitation (formation and collapse of bubbles in the liquid, including the cell membrane)
3.2.2.2.1. Particle bombardment
3.2.2.3. transient appearance allows DNA to cross the membrane into the cytoplasm
3.2.2.4. Suitable for many cell types such as mouse fibroblasts
3.2.3. Electroporation
3.2.3.1. low-frequency ultrasound allows efficient delivery of nucleic acids into mammalian cells
3.2.3.2. generation of transient, nanometer sized pores by electricity, pores allow DNA to enter
3.2.3.3. parameter: intensity and duration of electric pulse
3.2.3.4. simple to carry out with high reproducible but high input cost and require large amount of cells
4. why are mammalian cells vastly used in gene delivery?
4.1. mammalian cells allow the production of recombinant human proteins with authentic PTMs
4.1.1. authentic PTMs, not carried by
4.1.1.1. bacteria
4.1.1.2. yeast
4.1.1.3. plants
5. gene transfer to animal cells
5.1. delivery mechanism
5.1.1. physical transfection
5.1.2. chemical- mediated transfection
5.1.3. transduction
5.1.4. bactofection