Preview
Publication Date
Fall 2017
Abstract
Niemann Pick Disease Type C1 (NPC1) is a rare lysosomal storage disorder
that affects 1:150,000 people. The disease is characterized by cholesterol
accumulation within lysosomes, as well as clinical cognitive decline and
neurodegeneration. These symptoms can be attributed to a mutation in the
NPC1 gene that leads to the interruption of the intracellular cholesterol
transport. Using CRISPR Cas9 technology, the ability to make a cellular
model with inducible CRISPR gene regulation can give researchers
considerable insight into the cellular pathology of Niemann Pick Disease
Type C1 as well as the ability to apply targeted drug therapy and potentially
lead to drug discovery. We hypothesize that by using bioinformatics, a
CRISPR cas9 system, and genome editing we will make a functioning
HEK293 NPC1 cell model with inducible CRISPR gene regulation. To build
this system, we 1) used online bioinformatics tools to build primers, 2) Hifi
Assembly of mCherry coding sequence with pUC19 plasmid, 3) utilized
gene editing technology with the purpose of inserting the mCherry (a
fluorescent protein) coding DNA sequence into the NPC1 gene and 4)
inserted the built sequence into HEK 293 cells via CRISPR transfection.
Preliminary results show a successful build of the DNA sequence insert
containing the mCherry coding sequence which has been confirmed by
restriction digest and gel electrophoresis. Once completed the model will be
tested in the hopes that it will follow the cellular mechanisms that lead to the
NPC1 cholesterol transporter dysfunction. With the cellular model
successfully made, and functionality proven, future work will include testing
the cells for targeted drug therapy.
Louis Stokes Alliance for Minority Participation (LSAMP) Scholarship
1
Recognition
LSAMP Travel Award, ABRCMS Travel Award, Carol J. Major Legacy Poster Award