Nanoparticulate carriers for Mitochondrial Gene Therapy: Optimization of Non-radioactive DNA probe labeling and detection kit analysis of Mitochondrial Transcription by Northern blotting
Nanoparticulate carriers for Mitochondrial Gene Therapy: Optimization of Non-radioactive DNA probe labeling and detection kit analysis of Mitochondrial Transcription by Northern blotting
Student: Carol Kycia
Department: Pharmaceutical Sciences
Advisor:
Abstract
Figure 1: Two Mitochondria isolated from mouse liver by Eyad Katrangi, Ph.D. Candidate; TEM image taken by Bill Fowle.
We are developing nano-particulate carriers for the targeted delivery of DNA to mammalian mitochondria. Our efforts are currently focused on two separate systems: vesicular carriers made from amphiphilic quinolinium compounds that we call DQAsomes and gold nanoparticles surface-modified with mitochondriotropic ligands. However, in order to evaluate the delivery efficiency of these carrier systems, our studies have been limited to the use of indirect methods based on detecting co-localization of labeled cargo DNA with stained intracellular mitochondria. A much stronger proof of delivery would be achieved by the detection of the expression of a gene product from a reporter gene construct designed specifically for expression in the mitochondria.
Similar reporter gene constructs based on the expression of fluorescent proteins like green fluorescent protein (GFP) have been invaluable in the development of novel gene delivery strategies for nuclear gene therapy. Unfortunately no such construct is readily available for mitochondrial expression and is an important reason why despite our recent successes in delivering exogenous DNA into mammalian mitochondria, the delivery of DNA for protein expression in the mitochondrial matrix has remained elusive if not hard to prove. We believe that the development of such a fluorescent reporter gene construct for mitochondrial expression will provide us and the research community in general with a valuable tool for a quick and reliable evaluation of emerging mitochondrial gene delivery strategies.
Long-term Goal:
To develop a mitochondrial reporter gene construct for assessment of mitochondrial transfection efficiency with nanoparticulate mitochondriotropic carriers.
Specific aims:
To design and synthesize specific DNA probes to evaluate in vitro transcription of our mitochondrial expression constructs.
To optimize non-radioactive covalent labeling procedures for the DNA probes.