title: Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.
abstract: Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan. junkomasuda@okayama-u.ac.jp.|Mucosal Immunity Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA. junkomasuda@okayama-u.ac.jp.
authors.forename: Junko
authors.identifier: http://orcid.org/0000-0001-5066-3804
authors.initials: J
authors.lastname: Masuda
authors.affiliation: Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, Yokohama, 230-0045, Japan.|Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawamoto
authors.affiliation: Mucosal Immunity Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Warren
authors.initials: W
authors.lastname: Strober
authors.affiliation: Department of Oral Biochemistry, Asahi University School of Dentistry, Hozumi 1851, Gifu, 501-0296, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Takayama
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Akifumi
authors.initials: A
authors.lastname: Mizutani
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Murakami
authors.affiliation: Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, Yokohama, 230-0045, Japan.|Laboratory for Immune Regeneration, RIKEN Center for Integrative Medical Sciences, Yokohama, 230-0045, Japan.
authors.forename: Tomokatsu
authors.initials: T
authors.lastname: Ikawa
authors.affiliation: Mucosal Immunity Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kitani
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Narumi
authors.initials: N
authors.lastname: Maeno
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Shigehiro
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Ayano
authors.initials: A
authors.lastname: Satoh
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Akimasa
authors.initials: A
authors.lastname: Seno
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Vaidyanath
authors.initials: V
authors.lastname: Arun
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Tomonari
authors.initials: T
authors.lastname: Kasai
authors.affiliation: Mucosal Immunity Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Ivan J
authors.initials: IJ
authors.lastname: Fuss
authors.affiliation: Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, Yokohama, 230-0045, Japan.|Division of Cell Regeneration and Transplantation, Advanced Medical Research Center, School of Medicine, Nihon University, Tokyo, 173-8610, Japan.
authors.forename: Yoshimoto
authors.initials: Y
authors.lastname: Katsura
authors.affiliation: Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Seno
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D019556:COS Cells; D002522:Chlorocebus aethiops; D017353:Gene Deletion; D018390:Gene Targeting; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D000071336:Transcription Activator-Like Effectors / Q000378:metabolism*; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D008164:Luminescent Proteins; D012097:Repressor Proteins; D000071336:Transcription Activator-Like Effectors; D000097573:Red Fluorescent Protein; C407909:TCF3 protein, human
keywords: Artificial transcription factor; TALE technology; TCF3 (E2A)