By Doug Macron
Researchers from Cold Spring Harbor Laboratory this month published data demonstrating that a newly developed transgenic RNAi approach can temporarily and reversibly inhibit the expression of a gene necessary for survival in an adult mouse.
The ability to suppress and then re-activate essential genes "will not only help in pinpointing timeframes during which [these] genes are important during development, but will also be tremendously useful in cancer or other disease-related research," Scott Lowe, a CSHL researcher and one of the paper's senior authors, said in a statement. "We could, for example, first allow tumors to grow in mice, then shut off a gene that we think might be a good therapeutic target to test whether it saves the animals.”
"This ability to toggle gene activity at any stage is a major advance over other gene knockdown techniques that are not reversible," CSHL President and study co-author Bruce Stillman added. "This approach in animal models is the closest genetic equivalent to treating human patients with a single dose of a small molecule-based therapy targeted at a particular gene. So this system will be invaluable for testing novel therapeutic targets and evaluating their efficacy and side effects."
While techniques to inactivate specific genes at specific times exist for simple model organisms, such tools are not available for mice, according to the paper, which appeared in the Proceedings of the National Academy of Sciences.
Researchers do have some options, such as conditional deletion alleles “in which the gene of interest is flanked by loxP recombination sites [and] enable acute gene inactivation upon expression of Cre recombinase,” according to the paper. But such approaches are costly and time-consuming, and Cre-mediated gene excision is incomplete and has varying efficiency.
“Most importantly, conditional gene deletion is not reversible, so these models cannot be used to determine the effect of transient gene inactivation, during windows of development or in the adult,” they noted.
To address this issue, the investigators turned to shRNAs, which, in a transgenic setting, can “recapitulate knockout mouse phenotypes when constitutively or inducibly expressed, thus providing an alternative to Cre-mediated gene deletion.”
They added that shRNAs under the control of the reversible tetracycline-responsive element can inducibly and reversibly knock down specific genes, while “shRNA transgenesis is faster than generation of conventional and conditional knockouts because it obviates the need for site-specific homologous recombination.
In addition, “RNAi suppresses gene function in trans, [therefore] only a single transgenic allele is necessary, thus reducing animal husbandry,” the CSHL team wrote.
Previously, the researchers, in collaboration with CSHL's Greg Hannon, developed a rapid and scalable method of generating inducible shRNA transgenic mice, and examined its utility in inhibiting a variety of genes. As described in a paper in Cell earlier this month, they generated eight tet-regulated shRNA transgenic lines targeting luciferases, the transcription factor Oct4, and various tumor suppressors. These were able to potently silence their target genes in a broad range of tissues in vivo, according to that paper.
In this month's PNAS paper, Lowe and colleagues sought to test whether the system could be used against genes necessary for survival by targeting those involved in DNA synthesis.
They conducted an shRNA screen in colorectal cancer cells to identify genes that are involved in this process and are sensitive to RNAi inhibition, and honed in on Rpa3, a subunit of a protein with well-characterized roles in DNA replication and repair. Then, the team generated transgenic mice with tetracycline-responsive element-driven, Rpa3-targeting shRNAs, whose expression triggered reversible cell cycle arrest.
In adult mice, shRNA expression led to rapid atrophy of the intestinal epithelium, which in turn caused rapid weight loss and death within 8 to 11 days of shRNA induction, according to the PNAS paper. Withdrawal of tetracycline led to a complete reversal of villus atrophy and weight loss.
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“Hence, our system shows that transient inactivation of the replication gene Rpa3 causes a reversible inhibition of DNA synthesis but is not invariably lethal, thus providing a system to study this essential process at different developmental stages and, through the use of tissue-specific transactivators, in particular cell types,” the researchers wrote.
“More broadly, transient inactivation of essential genes in the whole organism is an exciting prospect for mouse genetics,” the added. “On a practical level, our platform can be modified through simple cloning to target any gene, leading to transgenic mice within [roughly four months], up to three times faster than standard gene targeting approaches.”
At the same time, the ability to toggle gene expression at any development state helps bring “mouse genetics forward to meet that of other model organisms, which have long used temperature-sensitive mutations to this end,” the team wrote. “Adding a new layer of versatility, the sequence-specific nature of RNAi allows the rational targeting of any gene of interest inducibly and reversibly.”
The approach is not without its limitations, the authors noted.
“Many applications of inducible shRNA transgenics require knockdown throughout the tissue of interest,” they stated. But in the experiments detailed in PNAS, mice treated with tetracycline sometimes regained weight and survived after an initial weight loss, which suggests that there were a “small number” of non-shRNA-expressing cells that can compensate for arrested cells where the shRNA expression is maintained.
“Moving forward, more transactivator strains that drive high levels of truly ubiquitous expression from the [tetracycline-responsive element] must be developed,” they wrote.
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