RNA interference: improved method of reversible gene shutdown
LifeSciencesToday based on the materials of Cold Spring Harbor Laboratory:
CSHL team perfects non-lethal way of switching off essential genes in miceOne way to study the function of a gene is to turn it off and observe what effect the loss of its activity has on the body.
However, if a gene is important for survival, permanently disabling it will kill the organism before its function is determined. Cold Spring Harbor Laboratory (CSHL) scientists have overcome this obstacle with the help of RNA interference technology (RNAi). They learned to temporarily turn off an important gene in adult mice, and then turn it on again before this change kills the animals.
In the article Reversible suppression of an essential gene in adult mice using transgenic RNA interference, published in the journal Proceedings of the National Academy of Sciences, researchers describe the possibilities of this approach using the example of switching off a gene necessary for DNA replication. The gene remained disabled until the mice began to lose weight and were on the verge of death. Turning on the gene successfully reversed these symptoms and saved the animals.
The ability to regulate gene activity in this way in animals "will not only help in identifying the period during which vital genes are important at the developmental stage, but will also be extremely useful in cancer and other diseases research," says CSHL Professor Scott Lowe, a researcher at the Howard Hughes Medical Institute (HHMI). (Scott Lowe). "Using mice, we could, for example, first let the tumors grow, and then turn off the gene. We think it could be a good therapeutic target to test whether it will save the animals."
To suppress the activity of genes using RNA interference, scientists use molecules of short hairpin-shaped RNAs (short hairpin-shaped RNAs, shRNAs) that bind to the corresponding RNA regions of the target gene and cause its destruction. This makes it possible to suppress the synthesis of the protein encoded by this gene. Having refined and optimized over the past few years the process of using such shRNAs to suppress gene activity in mice, Low and his colleagues came up with a technology platform recently published in the journal Cell, which allowed them to create genetically modified, or transgenic, mice within 4 months, which is three times faster than using standard approaches (A Rapid and Scalable System for Studying Gene Function in Mice Using Conditional RNA Interference).
The purpose of this study was to test whether the new optimized system is reliable enough to ensure effective targeting of critical genes. To find a fully suitable target gene for research, scientists first screened the RNAi "library", or a large collection of short hairpin RNAs. They had to select only those molecules that block proliferation by suppressing a particular gene. Screening revealed shRNAs against one part – subunit 3 – replication protein A (RPA3), a protein complex discovered in the laboratory of co-director of the study of CSHL President Professor Bruce Stillman in the 1980s. Replication protein A promotes DNA replication by stabilizing the unwinding of its chains and recruiting other necessary proteins.
Scientists have linked shRNAs molecules, the target of which is RPA3, with a fluorescent protein. These molecules were then used to create transgenic mice. In such mice, receiving a certain drug with food, for example, tetracycline, provokes RNA interference and the shutdown of the RPA3 gene. Fluorescent tags made it easy to track tissues in which the RPA3 target gene was turned off.
When scientists induced PRA3 suppression in adult mice, the animals' intestinal tissue rapidly atrophied, causing them to lose weight and die within 8-11 days. But if the drug was removed when the mice were almost on the verge of death, tissue atrophy and weight loss were completely canceled, and the animals survived.
Unlike the intestinal tissue cells of normal mice (above)
mouse cells with the short hairpin RNA gene of the PRA3 protein complex turned off
they do not proliferate, which leads to tissue atrophy (below).
Photo: Scott Lowe
Lowe, Stillman and their colleagues plan to continue studying such mice to understand the mechanisms of tissue preservation and regeneration. However, they are very happy about the possibility and wider use of the approach they have developed.
"The ability to switch the activity of genes at any stage is a big step forward compared to other methods of switching off genes that are not reversible," says Professor Stillman. "This approach in animal models is the closest genetic equivalent of treating patients with a single dose of a small molecule–based drug that targets a specific gene. The importance of this system for testing new therapeutic targets and evaluating their effectiveness and side effects cannot be overestimated."
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