Abstract 4224: A quick and easy way of assembling TALE/TALEN modules for genetic modifications in mammalian cells .

Abstract

Abstract The transcription activator-like effectors (TALEs) are a family of virulence factors produced by plant bacterial pathogens, and they are able to regulate expression of specific host genes by binding to the corresponding promoter regions in a sequence-specific manner. The transcription activator-like effector nuclease (TALEN) is made by fusing a nuclease to the DNA binding modules of TALE, which would allow for generation of DNA deletions, knockouts or knockins. Since their discovery, genes from different organisms have been successfully targeted using designer TALEs through assembling sequence specific modules, which is a critical step to make TALE/TALEN vectors. However, current methods of assembling DNA binding modules, such as the golden-gate ligation, are still time consuming and labor intensive. Moreover, they are also technically challenging. In the present study, we adopted a single step approach to assemble TALE modules. For proof of principle, we used p53 binding site (Tgacttgcatgtacatgcc) derived from the lncRNA-RoR promoter for proof of principle. First, we designed four gBlock DNA oligos which carried 30 bp overlapping between two adjacent oligos. Then, we linked them together by PCR and subsequently directly cloned into an expression vector which carries two flanking TALE sequences. To determine the function of this construct, we generated a reporter vector carrying this p53 binding site and mCherry and showed that this newly made construct was able to activate the mCherry reporter. Experiments are underway to test if the p53 specific TALE is able to activate the endogenous lncRNA-RoR. Citation Format: Juan Zhang, Yin-Yuan Mo. A quick and easy way of assembling TALE/TALEN modules for genetic modifications in mammalian cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4224. doi:10.1158/1538-7445.AM2013-4224