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

Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.

Bibliographic Details
Main Author: Fang, Jennifer, M. Eng. Massachusetts Institute of Technology
Other Authors: Douglas Lauffenburger.
Format: Thesis
Language:eng
Published: Massachusetts Institute of Technology 2007
Subjects:
Online Access:http://hdl.handle.net/1721.1/38244
_version_ 1826213970495668224
author Fang, Jennifer, M. Eng. Massachusetts Institute of Technology
author2 Douglas Lauffenburger.
author_facet Douglas Lauffenburger.
Fang, Jennifer, M. Eng. Massachusetts Institute of Technology
author_sort Fang, Jennifer, M. Eng. Massachusetts Institute of Technology
collection MIT
description Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.
first_indexed 2024-09-23T15:57:44Z
format Thesis
id mit-1721.1/38244
institution Massachusetts Institute of Technology
language eng
last_indexed 2024-09-23T15:57:44Z
publishDate 2007
publisher Massachusetts Institute of Technology
record_format dspace
spelling mit-1721.1/382442019-04-12T15:41:08Z Optimization of organelle fractionation methods for quantitative analysis of gene delivery trafficking kinetics Fang, Jennifer, M. Eng. Massachusetts Institute of Technology Douglas Lauffenburger. Massachusetts Institute of Technology. Biological Engineering Division. Massachusetts Institute of Technology. Biological Engineering Division. Biological Engineering Division. Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006. "September 2006." Includes bibliographical references (p. 141-147). 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. (cont.) The rates with which each vector moves from outside of the cell into the early endosome, to the late endosome, to the cytoplasm and into the nucleus can be quantified. The steps affected by specific modifications to the vector design and the vector properties most important for delivery efficiency can be identified. As vectors are sorted differently in different cell types, this DGE device will allow researchers to gain insight of the cell-specific sorting mechanisms. Ultimately, DGE can aid design of vectors that reach delivery efficiencies comparable to viruses and tailor the vectors to the tissue of interest. by Jennifer Fang. M.Eng. 2007-08-03T18:18:47Z 2007-08-03T18:18:47Z 2006 Thesis http://hdl.handle.net/1721.1/38244 146477581 eng 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. http://dspace.mit.edu/handle/1721.1/7582 147 p. application/pdf Massachusetts Institute of Technology
spellingShingle Biological Engineering Division.
Fang, Jennifer, M. Eng. Massachusetts Institute of Technology
Optimization of organelle fractionation methods for quantitative analysis of gene delivery trafficking kinetics
title Optimization of organelle fractionation methods for quantitative analysis of gene delivery trafficking kinetics
title_full Optimization of organelle fractionation methods for quantitative analysis of gene delivery trafficking kinetics
title_fullStr Optimization of organelle fractionation methods for quantitative analysis of gene delivery trafficking kinetics
title_full_unstemmed Optimization of organelle fractionation methods for quantitative analysis of gene delivery trafficking kinetics
title_short Optimization of organelle fractionation methods for quantitative analysis of gene delivery trafficking kinetics
title_sort optimization of organelle fractionation methods for quantitative analysis of gene delivery trafficking kinetics
topic Biological Engineering Division.
url http://hdl.handle.net/1721.1/38244
work_keys_str_mv AT fangjennifermengmassachusettsinstituteoftechnology optimizationoforganellefractionationmethodsforquantitativeanalysisofgenedeliverytraffickingkinetics