GENE LIST AND STATUS

Our first major project will seek to understand the organization and morphologies of the major intracellular structures and regulatory complexes in undifferentiated stem cells and cardiomyocytes derived from them. To do this, we will use genome-edited cell lines, tagged with fluorescent , and light microscopy to determine the numbers, locations, and dynamic changes of these intracellular structures and regulatory complexes.

This year, we hope to generate ~20 different FP-tagged isogenic hiPSC clonal lines representing 15-17 intracellular structures for live cell imaging. We plan to generate at least two clonal lines for each structure when possible. The table below shows the editing and cell line generation status to date. When completed, we plan to make these lines available to the research community.

AICS# Structure Gene FP Status AICS0005 Adhesions Paxillin PXN EGFP Available* AICS0012 Alpha- TUBA1B mEGFP Available* AICS0031 Alpha-tubulin TUBA1B mTagRFP-T Screening & QC* AICS0011 Mitochondria TOM20 TOMM20 mEGFP Available* AICS0013 Nucleus LaminB1 LMNB1 mEGFP Available* AICS0029 LaminB1 LMNB1 tdTomato Screening & QC* AICS0017 Cell-cell junctions Desmoplakin DSP mEGFP Available* AICS0016 Beta-actin ACTB mEGFP Completed* AICS0010 ER Sec61-beta SEC61B mEGFP Completed * AICS0014 Nucleolus Fibrillarin FBL mEGFP Completed * AICS0024 Myosin IIB MYH10 mEGFP Completed * AICS0032 Centrosome Centrin CETN2 mTagRFP-T Screening & QC* AICS0020 Intermediate VIM mEGFP Screening & QC* filaments AICS0023 Tight junctions ZO-1 TJP1 mEGFP Completed * AICS0030 Autophagosomes LC3 MAP1LC3B mEGFP Screening & QC* AICS0036 Cytoplasm GFP AAVS1-CAG-GFP mEGFP Screening & QC* AICS0025 Golgi Beta-galactoside alpha- ST6GAL1 mEGFP Edited pool * 2,6-sialyltransferase 1 AICS0022 Lysosome LAMP1 LAMP1 mEGFP Edited pool* AICS0033 Peroxisome PMP34 SLC25A17 mEGFP Design phase AICS0040 Endosome Rab5A RAB5A mEGFP Design phase AICS0044 Plasma membrane CAAX-GFP AAVS1-CAG-GFP mEGFP Design phase

CRISPR/Cas9 is used to introduce a green or red fluorescent protein at the endogenous locus of the listed above and the resulting gene-edited cells are enriched by flow cytometry (Pre-clonal edited pool). Subsequent clones generated from these edited pools are genotyped and subjected to both molecular and cellular QC (Screening & QC) followed by scale up and final QC including karyotype and sterility testing. Clones that have completed all QC steps are subjected to whole exome sequencing and RNA-Seq analysis. *Indicates confirmation of correct cellular localization by imaging.

February 2017 v.1.0 cellscience.alleninstitute.org Gene List and Status page 1 of 1