A platform for rapid generation of single and multiplexed reporters in human iPSC lines

Scientific Reports
Ying PeiXianmin Zeng

Abstract

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript, we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore, we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages, further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays.

References

Oct 27, 2007·Stem Cells·Bhaskar ThyagarajanJonathan D Chesnut
Nov 26, 2009·Science·Matthew J Moscou, Adam J Bogdanove
Dec 17, 2009·Toxicology·Kinga Vojnits, Susanne Bremer
Jul 28, 2010·Biochemical Society Transactions·Delphine LaustriatMarc Peschanski
Mar 10, 2011·Nature Reviews. Genetics·Hao ZhuGeorge Q Daley
Aug 11, 2011·Current Gene Therapy·Alessandra Recchia, Fulvio Mavilio
Dec 2, 2011·Nature Reviews. Cancer·Michel SadelainFrederic D Bushman
Mar 28, 2012·Cell Transplantation·Pai-Jiun HoB Linju Yen
Oct 4, 2012·Expert Review of Cardiovascular Therapy·Xiaoming SunReginald Liew
Oct 19, 2012·Journal of Cardiovascular Translational Research·Daniel SinneckerKarl-Ludwig Laugwitz
Jan 5, 2013·Science·Prashant MaliGeorge M Church
Jan 18, 2014·Nature Reviews. Genetics·Viviane Tabar, Lorenz Studer
Mar 25, 2014·Pharmacology & Therapeutics·Daniel SinneckerAlessandra Moretti
Jun 6, 2014·Science Translational Medicine·Elena MatsaJoseph C Wu

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Citations

Jan 31, 2016·Molecular and Cellular Neurosciences·Stephen J HaggartyRoy H Perlis
Dec 26, 2015·Lancet Neurology·Joshua G HunsbergerP Murali Doraiswamy
Mar 5, 2016·Cell Stem Cell·Robert PassierChristine Mummery
Sep 29, 2015·Stem Cell Reports·Behnam Ahmadian BaghbaderaniMahendra S Rao
May 11, 2016·Chemical Reviews·Gretchen MeinkeFrank Buchholz
Jan 27, 2017·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Kohei HommaMakoto Kaneda
Dec 12, 2018·Expert Opinion on Drug Metabolism & Toxicology·Ágota ApátiBalázs Sarkadi
Mar 16, 2019·Annals of the New York Academy of Sciences·M Catarina Silva, Stephen J Haggarty
Jul 1, 2017·Orphanet Journal of Rare Diseases·Francis AguisandaWei Zheng
Jan 10, 2018·Frontiers in Cell and Developmental Biology·David M GonzalezKristen J Brennand
Dec 4, 2020·Nucleic Acids Research·Amer EliasMikhail Kolot
Oct 30, 2021·Frontiers in Genome Editing·Amy McTagueManju A Kurian

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Methods Mentioned

BETA
PCR
transfection
PCRs
Assay

Software Mentioned

FACSDiva

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