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Effect of Various Concentrations of Helper Lipids in DNA Transfection Efficiency . There are many illnesses caused by defective or mutated genes that are passed down from generation to generation. At present no effective cures for such diseases have been found. The best solution on a long term basis is to introduce functional genes in place of the defective ones. This process of replacing, adding or correcting defective genes by introducing a working gene is known as gene therapy. At the moment gene therapy is accomplished using various vectors (carriers) to carry the specific gene of interest into the cell. Certain viruses are used for this purpose. Though viruses are the most efficient carriers available at present, they, however, have certain disadvantages. One of these is the attack by the immune system of the host resulting in damage to the introduced gene. Other disadvantages include, the limited size of the gene that can be delivered, and the unexpected response of the viral gene as well. The other method is non-viral delivery which includes chemical as well as physical means of delivering the gene. Cationic liposomes provide a promising vector for use in gene therapy. There are many advantages to using liposomes. First of all, it does not limit the size of DNA that can be carried into the cell. Also, it is not prone to attack by the immune system, lipid toxicity is relatively low, and it can be produced rather easily. But as of now it is much lower in efficiency compared to the viral method possibly because the optimum conditions of DNA delivery and stability have not yet been fully worked out. The purpose of this lab's research is to find out optimal condition for delivery so as to increase the efficiency of the cationic liposome in delivering the gene into the cell. Introduction of genes into cells is achieved by a method called transfection. The cationic lipid currently used in the lab is, dioleoyl trimethylammonium propane (DOTAP). There are two main reasons for using DOTAP, which has a positively charged hydrophilic head. One is that, the positive charge of the liposome helps condense the negatively charged DNA and thus holds it in the structure. The second reason is that the negative charge of the plasma membrane and the positively charged liposome creates an electrostatic attraction that ensures the intake of DNA by the cell. DOTAP is combined with two neutral helper lipids dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC). The helper lipids being neutral in charge help in stabilizing DOTAP and keep it from fast degradation. Two important differences can be seen in the structures of DOPE and DOPC. DOPE has a much smaller head compared to DOPC and this causes a difference in the structure of the complex as well as the efficiency. From work done earlier in the lab the structures of the complexes have been studied through x-ray diffraction. There is the hexagonal structure where the liposomes form tubes that arrange itself into the hexagonal lattice, with the DNA forming a rod in the middle (Fig 2). The other structure is the lamellar which consists of the DNA sandwiched in between lipid bilayers (Fig 2). DOPC maintains a lamellar structure all the time. On the other hand, increasing the ratio of DOPE in the system causes a structural transformation from lamellar to hexagonal (Fig 2). Cationic lipid/DNA (CL-DNA) complexes contain the supercoiled DNA plasmid that carries the Luciferase gene (LUC, structure shown in Fig 3). This gene was obtained from the N.American firefly. If the complex is taken up by the cells, the gene transcribed and the luciferase protein produced, then the protein, when mixed with a certain reagent produces a reaction that causes it to give off light. This intensity or amount of light can be measured using the luminometer where, based on the light given off, the amount of protein produced can be assayed. The efficiency of the transfection can therefore be found. CL-DNA complexes are incubated with mouse cells for transfection. The steps involved in the transfection process is given in Fig 4. Data on transfection efficiency has been obtained from studies done earlier on the CL/DNA system (Fig 5). The blue lines indicate the DOTAP/DOPC system while the red lines indicate the DOTAP/DOPE system. Initially, the curves for both DOPC and DOPE are the same indicating that they have the same transfection efficiency. At this region DOPE and DOPC are both in the lamellar phase which accounts for them both having the same efficiency. At increasing amounts of Helper Lipid in the system, the curves start moving apart. While the curve for DOPC starts to decrease the efficiency curve for the DOPE system remains high. This indicates a functional change between the two systems. At higher ratios the DOPE exists in the purely hexagonal phase while the DOPC remains in the lamellar phase. This summer my project involved experiments to find out whether the transfection efficiency can be further increased by having both DOPE and DOPC in the complex in the right amounts. For this purpose DOPE was added to DOPC in increasing ratios. Thus starting with just DOPC in the system, increasing amounts of DOPE were added and the results studied to determine the effect it had on the transfection efficiency. Return to the RISE 2000 project list |
