A Transient Transgenic RNAi Strategy for Rapid Characterization of Gene Function during Embryonic Development

RNA interference (RNAi) is a powerful strategy for studying the phenotypic consequences of reduced gene expression levels in model systems. To develop a method for the rapid characterization of the developmental consequences of gene dysregulation, we tested the use of RNAi for “transient transgenic” knockdown of mRNA in mouse embryos. These methods included lentiviral infection as well as transposition using the Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems. This approach can be useful for phenotypic validation of putative mutant loci, as we demonstrate by confirming that knockdown of Prdm16 phenocopies the ENU-induced cleft palate (CP) mutant, csp1. This strategy is attractive as an alternative to gene targeting in embryonic stem cells, as it is simple and yields phenotypic information in a matter of weeks. Of the three methodologies tested, the PB transposon system produced high numbers of transgenic embryos with the expected phenotype, demonstrating its utility as a screening method

I. An Overview of the Library Support Process Used at the Joint Genome Institute Production Genomics Facility

I. AN OVERVIEW OF THE LIBRARY SUPPORT PROCESS USED AT THE JOINT GENOME INSTITUTE PRODUCTION GENOMICS FACILITY Cathe Adam, Karli Ikeda, Sandy Huynh, Jayadevi Krishnakumar, Jim Fey, Diane Bauer, Sanna Anwar, Mansi Chovatia, Nicole Shapiro, Miranda Harmon-SmithLibrary support is the first step in production sequencing at the Joint Genome Institute s (JGI) Production Genomics Facility (PGF). The goal of library support is to generate 384 well glycerol stock plates of each library for a given project. These plates are then passed on to the Sequencing Prep group for Rolling Circle Amplification. Transformation stocks of the 3, 8, and 40kb libraries for a given project are received into library support from cloning technology. The stocks are stored at -80oC until plated. Libraries are plated according to priority. The colonies produced from a plating event are then picked using robotic colony pickers and are inoculated into 384-well destination plates containing LB/glycerol + antibiotic. The destination plates are grown overnight and visualized on a Spectramax Optical Density reader to identify wells without growth. Wells containing no growth are aspirated and replaced with colony growth from a "fix source" plate generated from the same library. The destination plates are then sealed and stored at -80oC until sent to the Sequencing Prep area for Rolling Circle Amplification (RCA). This poster will elaborate on these steps in the library support process and detail further the project tracking and management, plating and picking procedures and the instruments that are utilized throughout these steps

I. An Overview of the Library Support Process Used at the Joint Genome Institute Production Genomics Facility

I. AN OVERVIEW OF THE LIBRARY SUPPORT PROCESS USED AT THE JOINT GENOME INSTITUTE PRODUCTION GENOMICS FACILITY Cathe Adam, Karli Ikeda, Sandy Huynh, Jayadevi Krishnakumar, Jim Fey, Diane Bauer, Sanna Anwar, Mansi Chovatia, Nicole Shapiro, Miranda Harmon-SmithLibrary support is the first step in production sequencing at the Joint Genome Institute s (JGI) Production Genomics Facility (PGF). The goal of library support is to generate 384 well glycerol stock plates of each library for a given project. These plates are then passed on to the Sequencing Prep group for Rolling Circle Amplification. Transformation stocks of the 3, 8, and 40kb libraries for a given project are received into library support from cloning technology. The stocks are stored at -80oC until plated. Libraries are plated according to priority. The colonies produced from a plating event are then picked using robotic colony pickers and are inoculated into 384-well destination plates containing LB/glycerol + antibiotic. The destination plates are grown overnight and visualized on a Spectramax Optical Density reader to identify wells without growth. Wells containing no growth are aspirated and replaced with colony growth from a "fix source" plate generated from the same library. The destination plates are then sealed and stored at -80oC until sent to the Sequencing Prep area for Rolling Circle Amplification (RCA). This poster will elaborate on these steps in the library support process and detail further the project tracking and management, plating and picking procedures and the instruments that are utilized throughout these steps