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Elucidate number of cytosolic siRNA molecules necessary for knockdown

Hedlund, Hampus (2017) MOBY01 20162
Degree Projects in Molecular Biology
Abstract
RNA interference (RNAi) is a biological gene silencing mechanism that functions in every cell, and has the last two decades become a promising approach for genetic therapeutics. With small interfering RNA (siRNA) complementary to the target gene sequence, the siRNA together with the RNA induced silencing complex (RISC), can degrade the coding mRNA, inhibit protein from translation and thus mediate gene downregulation. However, to reach tissues beyond the liver, improved delivery strategies have to be developed. For this development of novel delivery strategies, a better understanding of the various barriers facing siRNA delivery is necessary. As a step to improve this understanding, this project aims to establish a method to elucidate the... (More)
RNA interference (RNAi) is a biological gene silencing mechanism that functions in every cell, and has the last two decades become a promising approach for genetic therapeutics. With small interfering RNA (siRNA) complementary to the target gene sequence, the siRNA together with the RNA induced silencing complex (RISC), can degrade the coding mRNA, inhibit protein from translation and thus mediate gene downregulation. However, to reach tissues beyond the liver, improved delivery strategies have to be developed. For this development of novel delivery strategies, a better understanding of the various barriers facing siRNA delivery is necessary. As a step to improve this understanding, this project aims to establish a method to elucidate the minimum number of intracellular siRNA molecules necessary for knockdown, which is currently unknown. For this purpose, a previosuly developed method to measure the cytosolic delivery of fluorescent siRNA (labeled with Alexa Fluor 647) was adapted for imaging using a regular confocal microscope. A pipeline and analysis of the fluorescence of cultured cells during long-term live cell imaging (12-24h) was developed. Using cells expressing a fluorescent lipid membrane damage sensor (galectin-9-YFP), it was demonstrated that this imaging strategy could detect the vast majority of events resulting in cytosolic delivery of siRNA during transfection lipid mediated delivery. The dose-response relationship of the detected siRNA fluorescence was shown to be linear over three orders of magnitude with respect to siRNA concentration. Finally, substantial efforts to establish stably transfected HeLa clones expressing a destabilized eGFP with a short half-life (~1h) were made. So far, these efforts have resulted in the establishment of a heterogenous pool of stably transfected cells with approx. 5% of the cells having significant expression of eGFP with the expected half-life. Currently, efforts to establish homogenous single-cell derived clones are ongoing. (Less)
Popular Abstract
Today, several diseases, such as cancer, are caused by genes that do not function properly. Genes that of various reasons have become mutated and turned against the human body. What if we could inactivate those harmful genes and cure the resulting diseases? With RNA interference (RNAi), a new gene silencing approach currently researched at the University of Lund, this is believed to be possible.

The silencing or knock-down mechanism works by sending in small interfering RNA (siRNA) molecules into the cells which performs the gene inactivation on a certain gene that has been purposely targeted. The siRNA molecules are taken up by the cells through endosomes, membrane-like bubbles, and some of them burst which results in siRNA leaking... (More)
Today, several diseases, such as cancer, are caused by genes that do not function properly. Genes that of various reasons have become mutated and turned against the human body. What if we could inactivate those harmful genes and cure the resulting diseases? With RNA interference (RNAi), a new gene silencing approach currently researched at the University of Lund, this is believed to be possible.

The silencing or knock-down mechanism works by sending in small interfering RNA (siRNA) molecules into the cells which performs the gene inactivation on a certain gene that has been purposely targeted. The siRNA molecules are taken up by the cells through endosomes, membrane-like bubbles, and some of them burst which results in siRNA leaking into the cells. However, since the RNAi field has only been known for the last two decades, there is much to learn and several obstacles to overcome. One very meaningful question that lacks an answer is to understand how many siRNA molecules that is necessary to completely silence a gene. Research has shown that it could be below 2000 molecules.

To find out, we developed an experimental setup that could measure the presence of siRNA and RNAi in more than 100 cells over time. We then investigated if we could detect all events of siRNA entering the cells via bursted vesicles. We found out that we could indeed detect the majority of releasing siRNA events. To measure knock-down of a target gene, we also established cells which expressed a gene that could easily be monitored for knock-down. The gene-product, a destabilized green fluorescent protein (GFP), has an expected half-life of 1 hour, and we could validate that this applied to our cells as well. Finally, we were able to demonstrate RNAi in a few of these cells.

However, further work lies ahead to obtain cells with a homogenous GFP expression to finally estimate the minimum number of siRNA molecules that is necessary for knock-down. Despite that, we managed to construct a promising experimental foundation to answer that question in the time to come.

Supervisor: Anders Wittrup
Degree Project in Molecular Biology 30 credits, 2017
Lund University, Department of Biology (Less)
Please use this url to cite or link to this publication:
author
Hedlund, Hampus
supervisor
organization
course
MOBY01 20162
year
type
M2 - Bachelor Degree
subject
language
English
id
8906576
date added to LUP
2017-05-10 11:34:46
date last changed
2017-05-10 11:34:46
@misc{8906576,
  abstract     = {RNA interference (RNAi) is a biological gene silencing mechanism that functions in every cell, and has the last two decades become a promising approach for genetic therapeutics. With small interfering RNA (siRNA) complementary to the target gene sequence, the siRNA together with the RNA induced silencing complex (RISC), can degrade the coding mRNA, inhibit protein from translation and thus mediate gene downregulation. However, to reach tissues beyond the liver, improved delivery strategies have to be developed. For this development of novel delivery strategies, a better understanding of the various barriers facing siRNA delivery is necessary. As a step to improve this understanding, this project aims to establish a method to elucidate the minimum number of intracellular siRNA molecules necessary for knockdown, which is currently unknown. For this purpose, a previosuly developed method to measure the cytosolic delivery of fluorescent siRNA (labeled with Alexa Fluor 647) was adapted for imaging using a regular confocal microscope. A pipeline and analysis of the fluorescence of cultured cells during long-term live cell imaging (12-24h) was developed. Using cells expressing a fluorescent lipid membrane damage sensor (galectin-9-YFP), it was demonstrated that this imaging strategy could detect the vast majority of events resulting in cytosolic delivery of siRNA during transfection lipid mediated delivery. The dose-response relationship of the detected siRNA fluorescence was shown to be linear over three orders of magnitude with respect to siRNA concentration. Finally, substantial efforts to establish stably transfected HeLa clones expressing a destabilized eGFP with a short half-life (~1h) were made. So far, these efforts have resulted in the establishment of a heterogenous pool of stably transfected cells with approx. 5% of the cells having significant expression of eGFP with the expected half-life. Currently, efforts to establish homogenous single-cell derived clones are ongoing.},
  author       = {Hedlund, Hampus},
  language     = {eng},
  note         = {Student Paper},
  title        = {Elucidate number of cytosolic siRNA molecules necessary for knockdown},
  year         = {2017},
}