Developing Advanced Tools For In Vivo Genetic Manipulation Ali Yamani1, Phoneopasong Arounlet2, Timothy Phoenix2 1University of Cincinnati College of Medicine, 2University of Cincinnati – James L. Winkle College of Pharmacy Introduction: Pediatric brain tumors are the leading cause of cancer related deaths in children. One obstacle to improving outcomes is a lack of research tools, including mouse models that allow investigation of newly identified genetic alterations and therapeutic targets. To address this, we have made some new preclinical mouse models of pediatric high-grade glioma using in utero electroporation (IUE). This method uses individual gene expression plasmids to exogenously overexpress target genes. However, the co-transfection rate of multiple single gene expression plasmids in IUE is approximately 70-90%. Additionally, overexpression of an exogenous plasmid differs from endogenous gene expression in human cancers. To address the first issue, we developed a “2A” linker plasmid that allows us to co-express up to four different genes from a single promoter. Second, we are testing the CRISPRa (activator) platform, which leverages modified CRISPR-Cas9 to induce targeted endogenous gene expression. The utilization of linker plasmids and CRISPRa will allow us to optimize our mouse models and fidelity to their human cancer counterparts. Methods: When creating the linker plasmid, cDNA sequences were first amplified using PCR. The linker plasmid (pGEM-PTE2A) underwent restriction enzyme linearization. PCR products were ligated with the linearized backbone and transformed into bacteria. DNA was isolated from the bacteria and the cDNA insert was verified by PCR amplification and gel electrophoresis. This process was repeated for the next two insert genes. Sanger sequencing was performed to verify proper gene insertion of the completed linker plasmid. Four gRNAs were selected for CRISPRa to induce endogenous Foxr2 overexpression. gRNAs were cloned then ligated to the PB-gRNA (SAM) backbone. The ligation product was treated with nuclease then transformed into bacteria. Final plasmid products were verified by Sanger sequencing. Foxr2 gRNA and dCas9-SAM plasmids were transfected into neural stem cells and expanded in culture. RNA was isolated from control and Foxr2 gRNA transfected cells and qPCR was performed for Foxr2 expression. Gapdh and Actb were used as control housekeeping genes for qPCR reactions. Results: Sanger sequencing verified the inserts and linker genes were present in the linker plasmid without any notable sequence aberrations. Ongoing studies are being performed to test and validate expression of all three cDNAs from the linker plasmid using in vitro and in vivo methods. In CRISPRa studies, qPCR results showed that Foxr2 gRNA conditions did not result in increased Foxr2 expression. Current work using previously validated and published gRNA sequences to activate Myc expression are being tested in vitro to verify this plasmid system can work in our hands. Conclusion: Usage of the linker gene in IUE will allow multiple genes to be transfected using a single plasmid providing for a more homogenous transfection method. We are currently validating the integrity of the CRISPRa platform. Contact Information: yamaniai@mail.uc.edu Key Words: Linker Plasmid, CRISPRa, Brain Tumor, Cloning, In Utero Electroporation (IUE)