The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.

Breast cancer is the most commonly occurring cancer among women in the United States. As with all other cancer types, breast cancer formation is the result of alterations in tumor suppressor genes, which regulate diverse cellular activities to prevent tumor development, and oncogenes, which promote tumor formation. In this study, we further characterized two candidate breast cancer tumor suppressor genes, MTUS2 and LHX8, and their role in breast cancer development. Soft agar assays showed that the knockdown of MTUS2 and LHX8 in MCF10AT cells led to increased colony formation compared to non-silencing control. Furthermore, through q-RT-PCR analysis, we found that the relative mRNA expression of MTUS2 and LHX8 is down-regulated in a panel of breast cancer cell lines compared to the pre-malignant breast epithelial cell line MCF10AT. Finally, when we ectopically expressed MTUS2 or LHX8 in MCF7 cells it resulted in slower proliferation of the cells compared to an empty vector control. Taken together, these results provide further information concerning the roles MTUS2 and LHX8 play in breast tumorigenesis. This study implicates two breast cancer tumor suppressor genes, which can provide a unique tool for identifying novel pathways involved in the formation of breast cancer. Citation Format: Diane Fru, Victoria Ruhl, Michael R. Green. Characterizing candidate breast cancer tumor suppressors: MTUS2 and LHX8. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B29. Note: This abstract was withdrawn after the Proceedings was printed and was not presented at the conference.