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JUN 08, 2017 12:00 PM PDT

Improved CRISPR Cas9 Editing of Pluripotent Stem Cells Utilizing the Latest Technologies from Thermo Fisher Scientific

Speaker
  • Senior Staff Scientist, Cell Biology, Thermo Fisher Scientific
    Biography
      Dr. Newman joined the Primary and Stem Cell Systems Division Life Technologies in 2009 and initially focused on isolation and expansion of human primary cells for development of 3D organotypic models. Currently, Dr. Newman continues to support primary cell efforts and works on development of next generation stem cell culture and differentiation systems, enabling researchers to efficiently culture, expand, cryopreserve, and differentiate their stem cells to various cellular lineages. She received a Ph.D. at the University of Iowa from the lab of Dr. Madeline Shea, focusing on ligand induced allostery of calmodulin and its impact on regulation of the Ryanodine Receptor Type 1. Subsequently, she completed postdoctoral training in the lab of Dr. Ken Prehoda at the University of Oregon, studying the role of intramolecular interactions in regulating cell signaling cascades in the process of assymetric stem cell division.

    Abstract

    The emergence of technology for development of induced pluripotent stem cells (iPSCs) from somatic cells, such as skin and blood cells, has resulted in the ability of researchers to have limitless pool of iPSCs retaining the genetic make-up of the somatic cells from which they were derived.  In conjunction with tools for downstream gene editing, such as clustered regularly interspaced short palindromic-repeat (CRISPR)-Cas9 nuclease systems, iPSCs can be used generate (1) knock-outs to study the impact of genes on cellular processes, or (2) knock-ins to assess the impact of reversal of point mutations on diseased states, or furthermore for generation of reporter cell lines.  Briefly, CRISPR-Cas9 systems provide simple and efficient site-specific targeting that is accomplished by guiding Cas9 nuclease via a variable 20-base guide RNA sequence to the site for formation of a double stranded break. This break can then be repaired via non-homologous end joining (NHEJ) where small insertions or deletions are made in the gene of interest to knock-out its function or via homology-directed repair (HR) in which single nucleotide changes or knock-ins can be accomplished using a donor DNA template for repair.  Together iPSCs and CRISPR-Cas9 systems provide researchers with effective in vitro tools for assessing gene function, disease modeling, and regenerative therapy.  In this webinar, we discuss new technologies available from Thermo Fisher Scientific which support (1) efficient delivery of CRISPR Cas9 to PSCs, (2) improve cell survival following transfection while maintaining normal PSC properties, and (3) improve clonal cell survival following low density seeding of 1, 3, or 5 cells per well of 96-well plate.


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