Optimization of organelle fractionation methods for quantitative analysis of gene delivery trafficking kinetics

Nonviral vector research and development has been stunted by a lack of knowledge and understanding of how vectors are trafficked within the cell. Research currently involves mass screenings of different combinations of vector components without a true understanding of how each component interacts with the target cell. Few tools are currently available for scientists to quantitatively examine these vector-to-cell interactions or determine the rate limiting steps within the gene delivery pathway. Thus, researchers cannot fully optimize the vector design to reach maximal delivery efficiency. This project seeks to address this issue by modifying a density gradient electrophoresis (DGE) device originally developed on Mel Juso cells to segregate primary rat hepatocyte lysate into nuclear, early endosomal, late endosomal/lysosomal, and cytoplasmic fractions. We found that according to the Horseradish Peroxidase assay, late endosomes and lysosomes consistently localize to fractions 11-13 and early endosomes in fractions 18 to 21. There was minimal labeling in fractions 14 through 17 demonstrating that separation of the organelles was achieved. With this higher resolution fractionation, movement through the endosomal pathway can be studied in greater detail.

Department dc:contributor.department

Massachusetts Institute of Technology. Biological Engineering Division.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2006

Author dc:creator

• Fang, Jennifer, M. Eng. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• Douglas Lauffenburger.

• Biological Engineering Division.

Statement dc:rights

• MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/38244

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/38244