Project Report

Small-interfering ribonucleic acids (siRNA) are of great interest to many scientists and engineers because siRNAs are used to temporarily silence or "knockdown" gene expression. Knocking down genes has allowed researchers to determine the functions of genes, identify components of biochemical pathways, differentiate stem cells into terminal lineages, and many other applications. Researchers continue to find new applications for siRNA. Thus, the use and delivery of siRNA has become exceptionally vital to researchers in biological, medical, chemical, pharmaceutical, and bioengineering disciplines. To facilitate the delivery of siRNA, many methods have been developed by several prominent companies around the world. Each siRNA delivery method has a variety of advantages and disadvantages regarding different cell types and tissues. Thus, testing and comparing different siRNA delivery methods on different cells is necessary to determine which methods are most optimal for delivering siRNA to specific cells. Furthermore, testing and comparing siRNA delivery methods can allow researchers to identify and develop new ideas for siRNA delivery that utilize the advantages of multiple methods, while minimizing disadvantages. Two siRNA delivery methods of particular interest include the use of Nucleofection, an electroporative method and Atelocollagen, a natural polymer. Electroporation induces the formation of pores in mammalian cells when a voltage is applied across a suspension of cells. The formation of pores allows nucleic acids like siRNA to enter the cell and knockdown gene expression. Alternatively, Atelocollagen is a natural polymer that comes from the skin of cows and is used in a variety of cosmetic products. Atelocollagen is able to form an advantageous complex with siRNA that allows siRNA to enter cells and avoid degradation. Both methods have significant advantages for delivery of siRNA in different settings, but these two methods have never been compared side-by-side. So, the principal investigator (PI) set out to compare these two methods, and determine the advantages and disadvantages of using each method. The PI used a special cell line that was engineered to permanently express an enzyme that could act with an external reagent that made the cells glow. The PI attempted to knockdown the gene that produced the enzyme of interest by delivering specific siRNA targeted against the gene behind enzyme of interest. siRNA was delivered to the cells using both electroporation and Atelocollagen. The PI evaluated the ability of the siRNA to knockdown the gene behind the enzyme of interest and the toxicity effect of each delivery method on the cells. The PI found that both methods were able to knockdown the target gene; however, Atelocollagen seemed to work more effectively than electroporation and Atelocollagen was slightly less toxic to the cells used. Yet, the PI observed that electroporation might be more advantageous to use on cell cultures outside of the body for tissue engineering applications. In addition to the findings of this project, the PI was able to learn new techniques in fluorescent microscopy, and was afforded the opportunity to build a brand new tissue-engineering lab at the host university. The PI formed new collaborative ties with three foreign labs, and was invited to present a special guest lecture at a foreign institution on the research conducted. The PI will continue to work with all three labs on future projects, and is pursuing opportunities to build more collaborative ties with researchers abroad. The PI hopes to find a way to improve the efficiency and efficacy of both siRNA methods in future work with international collaborators.

Agency
National Science Foundation (NSF)
Institute
Office of International and Integrative Activities (IIA)
Application #
1107555
Program Officer
Carter Kimsey
Project Start
Project End
Budget Start
2011-06-01
Budget End
2012-05-31
Support Year
Fiscal Year
2011
Total Cost
$5,700
Indirect Cost
Name
Mellott Adam J
Department
Type
DUNS #
City
Lawrence
State
KS
Country
United States
Zip Code
66049