The application of Molecular Biochemistry for transfection studies
in eukaryotic systems is well documented. Of the numerous methods
employed for the introduction of foreign DNA into eukaryotic cells,
the use of low density lipoproteins (LDL) as carriers of DNA into
cells has not been reported.
LDL was isolated, characterized with respect to its protein and
lipid components, and then variously modified in an attempt to
enhance its affinity for DNA. It was found that both unmodified
and modified LDL could interact with DNA, at physiological pH.
The carbodiimide modified LDL (ECDI - LDL) showed the greatest
affinity for DNA.
LDL and ECDI - LDL were used to study LDL receptor binding in
skin fibroblasts. This was followed by a study of receptor
binding activities of both unmodified LDL and ECDI - LDL complexed
to DNA (pBR322). Although the extent of binding of ECDI - LDL
and ECDI - LDL - DNA complexes to plasma membranes was greater,
the internalization and degradation of both modified and unmodified
LDL complexes were equivalent. This additional binding was
attributed to non - receptor - specific affinity of the carbodiimide
modified complexes for the plasma membrane.
The transfection of foreign DNA into eukaryotic cells in culture
was monitored by assaying for the expression of the cloning vector,
pSV2cat, complexed to LDL or ECDI - LDL and introduced into the
cells by LDL receptor - mediated endocytosis. Of the cell lines
in which the expression of the pSV2cat recombinant DNA was monitored,
the human lung fibroblasts showed the greatest activity of the
expressed chloramphenicol acetyl transferase enzyme. Although
transfection efficiency was lower than that of the calcium
phosphate - DNA coprecipitation procedure, the LDL receptor -
mediated transfection of eukaryotic cells was carried out under
physiological conditions and may be applicable in vivo. / Thesis (Ph.D)-University of Durban-Westville, 1987.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9700 |
| Date | 09 October 2013 |
| Creators | Khan, Zainub. |
| Contributors | Hawtrey, A. O., Ariatti, Mario. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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