title: Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives.
abstract: The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Fabian
authors.initials: F
authors.lastname: Oceguera-Yanez
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Shin-Il
authors.initials: SI
authors.lastname: Kim
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Ghee Wan
authors.initials: GW
authors.lastname: Tan
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; iPS Portal Inc., Kyoto 602-0841, Japan.
authors.forename: Long
authors.initials: L
authors.lastname: Xiang
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hatani
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kondo
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ikeya
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Yoshida
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Haruhisa
authors.initials: H
authors.lastname: Inoue
authors.affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Hakubi Center for Advanced Research, Kyoto University, Kyoto 606-8501, Japan. Electronic address: woltjen@cira.kyoto-u.ac.jp.
authors.forename: Knut
authors.initials: K
authors.lastname: Woltjen
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D002460:Cell Line; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics*; D003001:Cloning, Molecular; D000229:Dependovirus; D017930:Genes, Reporter; D005818:Genetic Engineering; D056426:Genetic Loci; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D019219:Parvovirinae / Q000235:genetics; D011401:Promoter Regions, Genetic; D014162:Transfection; D019076:Transgenes
chemical_list: D049452:Green Fluorescent Proteins
keywords: AAVS1; CRISPR/Cas9; Gene targeting; Human induced pluripotent stem cell (iPSC); Nuclease; TALEN