Prem K. Premsrirut

4th Year Graduate Student

Department: Cold Spring Harbor Laboratory

Graduate Program: Genetics

Advisor: Dr. Scott Lowe

Abstract:

Title:  Conditional Regulation of Tumor Suppressor Genes in vivo using RNA interference

RNA interference (RNAi) is a powerful method for suppressing gene expression in mammalian systems, not only in vitro but also in vivo, allowing for the generation of sequence specific knockdown in mice. We have shown that RNAi can cause sufficient knockdown to recapitulate the phenotypes of knockout mice, particularly in cancer models, and unlike traditional knockout mice, RNAi has the powerful advantage of reversibility, since the endogenous gene remains intact. Our first generation reversible RNAi mouse model provided the proof of principle that RNAi technology can be used to spatially, temporally and reversibly regulate gene expression of probably any endogenous gene; however, it was generated by classical transgenesis using standard pronuclear injection, a method which lacks control over the integration site and copy number of the transgene. This method demands that numerous mice be screened in order to identify founders with robust expression of short hairpin RNA (shRNA), and that various tissues be tested due to the variability of expression in each founder. In order to circumvent these drawbacks, we are now using the FLP/FRT recombinase system targeting the ColA1 locus in previously engineered embryonic stem (ES) cells, a method developed in the Jaenisch lab. This approach places a tet-regulated microRNA (miRNA) immediately downstream of the endogenous ColA1 gene in ES cells, which are subsequently used to generate ES cell-derived mice by tetraploid embryo complementation. With this system, we are able to rapidly generate numerous reversible gene knockdown mouse models providing us with powerful tools to, in principle, dissect the function of nearly any endogenous gene in vivo. For the cancer field, reversible knockdown translates to the ability to study the roles of tumor suppressor genes on tumor maintenance and determine whether their sustained inactivation is required for tumor survival and progression, results which could have enormous implications for cancer therapy.

Publications:
(MSTP-supported publications indicated with an *)

Dickins, R.A., McJunkin, K., Hernando, E., Premsrirut, P.K., Krizhanovsky, V., Burgess, D.J., Kim, S.Y., Cordon-Cardo, C., Zender, L., Hannon, G.J. and Lowe, S.W. (2007). Tissue-specific and reversible RNAi in transgenic mice. Nature Genetics. 39:914-921.