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    • CX-5461 cost The glyceraldehyde phosphate dehydrogenase GAPD

    2018-11-07

    The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene encodes a key component of the glycolytic pathway, a process that turns glucose into ATP in all cells. GAPDH is ubiquitously expressed at consistently high levels in many different cell types, making it an ideal gene upon which to base a gene-expression system (Barber et al., 2005) (GTex Consortium, 2015; Murphy and Polak, 2002) (http://www.gtexportal.org/home/gene/GAPDH). We have generated a series of vectors in which the expression of introduced genes is derived from the GAPDH transcript, ensuring high-level, widespread expression of the transgene. In addition, our vector design links expression of a selectable marker gene to integration dependent trapping the GAPDH promoter, greatly enhancing the probability of obtaining correctly targeted clones.
    Results The structure of this GAPTrap (GT) vector is shown in Figure 1A. Genes of interest are inserted in frame with a T2A sequence (Szymczak and Vignali, 2005; Szymczak et al., 2004) that replaces the GAPDH stop codon. An internal ribosome entry site (Jang et al., 1990) located immediately downstream is used to express selectable marker genes encoding neomycin, hygromycin, or puromycin resistance. In the GT vectors, sequences encoding these CX-5461 cost markers have been optimized for expression in mammalian cells and are designated Meo, Mygro, and Muro. Vectors, Addgene accession numbers and cell lines generated for this study are listed in Table S1. To assist with the generation of targeted clones, we utilized transcription activator-like effector nucleases (TALENs) (Wood et al., 2011; Hockemeyer et al., 2011) or the CRISPR/Cas9 system directed against the point within the GAPDH locus at which the vector is inserted. We found that introduction of a double-stranded break using either TALENs or CRISPRs was essential for the generation of correctly targeted clones. Depending on the vector configuration, targeting efficiencies were often greater than 70%. For example, GT-TdTom and GT-lacZ vectors gave targeting frequencies of 10/12 (83%) and 9/10 (90%) when using TALENs (see Figure S1C). Similarly, CRISPR/Cas9-assisted homologous recombination yielded a targeting efficiency of 80% (12/15) (Figure S1D). To ascertain the frequency at which insertions and deletions (Indels) occurred in the unmodified GAPDH allele of cells containing a GT vector, we sequenced the region corresponding to the point within the GAPDH allele targeted by the GAPDH TALENs. This analysis showed that of 24 GT-reporter lines, 25% had Indels, indicative of non-homologous end-joining (NHEJ) events. Given the relatively low frequency of on-target NHEJ events in the GAPDH locus itself, the presence of off-target cutting events by this pair of TALENs was not assessed. GAPDH functions as a tetramer, and examination of the 3D crystal structure indicated that the C terminus of each GAPDH subunit is located on the exterior surface of the tetramer (Ismail and Park, 2005), suggesting that additional amino acids encoded by the T2A sequence should not interfere with enzymatic activity. However, examination of GAPDH protein using western blot analysis indicated that modified alleles that include an internal ribosome entry site (IRES)-selectable marker cassette were expressed at lower levels than the wild-type GAPDH allele. Thus, although we expect the specific activity of GAPDH-T2A proteins to be the same as that of the wild-type protein, the reduced expression of GAPDH-T2A from the modified allele may explain why cells with two targeted GAPDH alleles could not be isolated (Figure S1E). Using the vectors shown in Figure 1A, we generated a series of PSC lines that expressed EGFP (Cormack et al., 1996), Clover (Lam et al., 2012), mCherry (Merzlyak et al., 2007), mtagBFP2 (Subach et al., 2011), Tandem tomato (tdTomato) (Shaner et al., 2004), luciferase 2 (Luc2) (Promega), a membrane-bound Gaussia princeps luciferase (GLuc) (Santos et al., 2009), and nuclear LacZ (Stanley et al., 2000) (Table S1). Cells expressing the fluorescent markers could be readily visualized by microscopy (Figure 1B).