Investigating the Cell Shape on Gene Transfection Efficiency of Oligonucleotide-assembled Nanocomplexes
Date Issued
2012
Date
2012
Author(s)
Huang, Hui-Wen
Abstract
Cell morphology has a profound effect on a range of cellular events, such as proliferation, differentiation, cytoskeletal organization, or presumably gene expression. Currently, regarding the research of cell shapes, the majority focuses on the changes to gene expression after the shape of the cell has been changed, while only a few focus on the relationship between changing the shape of the cell and the effects it has on gene transfection.
This research uses two different materials, chitosan and TCPS, to culture the Hela cells and successfully change the cell shape. Once the cell shape is changed, two gene transfection vectors were used, the first type being a ExGEN500 and Plasmid DNA complex, and the second type being an oligonucleotide coating on the first type complex. According to the results of this research, the cells cultured on chitosan, both gene transfection vectors are less efficient than the cells cultured on TCPS. On the other hand, from the results of the cell uptake experiment, it is observed that there is no difference between the cell uptake of the complexes with chitosan and the complexes in TCPS. However, from the data of the confocal microscopy, DNA/PEI complexes do not transfect into the cell nucleus when cell culture on chitosan. This may be the reason for the lowered gene transfection efficiency.
In the second part of the research, the gene transfection vectors are first uptaken by cell on TCPS, and then, after the use of trypsin to dramatically change the skeleton of the cells, the cells are readhered to TCPS or suspended above chitosan. From this, it is observed that, under a shortly uptaking time and a shortly culturing period, the gene transfection efficiency of DNA/PEI complex is increased, while under a long uptaking time and long culturing period there is no difference in effect. From this, it can be predicted that changing the form of the cell in the shortly uptaking time period after cell uptake can cause gene transfection proteins to perform earlier.
From the two parts of the experiment, it can be seen that the structure of the cell has a large impact on gene transfection, but if the cell strcture is changed first, and then the cell is subjected to gene transfection, the efficiency of the transfection will be decreased. However, if the cell is first subjected to gene transfection, and the gene transfection complex is subjected to cell uptake for a brief period before the cell structure is changed, the efficiency of the gene transfection will not be decreased to the same extent. Also, the research shows that once the gene transfection particles enter the nucleus of the cell, with regards to the transfection protein, changing the structure of the cell will not cause a large effect on performance. Thus, whether the complexes can enter the cell or not is the key to gene transfection.
Subjects
cell morphology
gene transfection
chitosan
oligonucleotides
polyethyleneimine
Type
thesis
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