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Helen's Project Page - RISE summer 2000 |
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Intern: Helen Chang, Biochemistry/Cell Biology, UC, San Diego
Mentor: Heather Miles
Faculty Supervisor: Cyrus Safinya
Department: Materials |
Using flourescence microscopy to understand the behavior of cationic
lipid/DNA complexes for gene delivery.
Gene therapy enables the successful transfer of foreign DNA into a cell to
correct defective or add missing genes. This phenomenon is currently studied
vicariously in science and is being examined with multiple parameters. There are
many factors that contribute to the successful transfer of foreign DNA into
mammalian cells, but presently the methods of viral gene delivery is far more
prevalent than its counterpart, nonviral gene delivery. There are clear
differences between the two methods of gene delivery systems, but the most
significant difference is that viral gene delivery is currently more efficient.
Since nonviral gene delivery can take in large pieces of foreign DNA into a
cell and does not trigger the human immune system immediately, being able to increase the efficiency of nonviral gene delivery will be a remarkable breakthrough in this vast field.
The phrase, ænonviral gene delivery,Æ refers to the fact that nonviral vectors, or nonviral carriers, are used to aid the transfer of foreign DNA fragments into a cell. Just in the past few years, the development of synthetic nonviral vectors has become a popular study with the main intention of trying to increase the efficiency of nonviral gene delivery. Dr. Cyrus SafinyaÆs group at the University of California, Santa Barbara is primarily concentrated on the use of Cationic Lipids (CL) as a nonviral vector in gene delivery. In using fluorescence microscopy (inverted and confocal microscopy), my first objective for my summer project was to understand the methods of introducing foreign DNA into a cell, or in other words, the process of transfection. My second main objective was to understand the methods and techniques used to master the confocal and inverted microscopes in order to visualize the structures and images of the individual cationic liposomes and DNA, or the complexes themselves, from the multiple transfection experiments that were performed.
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Figure 1 shows a simple schematic of gene therapy, where we take cationic lipids, used as the nonviral vector, to introduce the foreign DNA into a cell. Once the CL/DNA complex has passed through the cellular membrane, we hope that the DNA is broken free from the complex and released in the cell. The DNA will then pass through the nuclear membrane into the nucleus, which will eventually be transcribed into RNA and then expressed into protein.
My part of the project was primarily focused on the process of transfection alone (points 1-2). Although I briefly learned the techniques to study the expression of DNA during the summer, I focused on studying the images taken from transfection. Another intern in the research group focused on how much protein was expressed from foreign DNA (points 3-4).
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