title: Chromatin insulator elements block transgene silencing in engineered human embryonic stem cell lines at a defined chromosome 13 locus.
abstract: Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5' and 3' of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken β actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs.
authors.affiliation: Primary and Stem Cell Systems, Life Technologies Corporation, Carlsbad, California, USA.
authors.forename: Chad C
authors.initials: CC
authors.lastname: Macarthur
authors.forename: Haipeng
authors.initials: H
authors.lastname: Xue
authors.forename: Dennis
authors.initials: D
authors.lastname: Van Hoof
authors.forename: Pauline T
authors.initials: PT
authors.lastname: Lieu
authors.forename: Miroslav
authors.initials: M
authors.lastname: Dudas
authors.forename: Andrew
authors.initials: A
authors.lastname: Fontes
authors.forename: Andrzej
authors.initials: A
authors.lastname: Swistowski
authors.forename: Thomas
authors.initials: T
authors.lastname: Touboul
authors.forename: Rina
authors.initials: R
authors.lastname: Seerke
authors.forename: Louise C
authors.initials: LC
authors.lastname: Laurent
authors.forename: Jeanne F
authors.initials: JF
authors.lastname: Loring
authors.forename: Michael S
authors.initials: MS
authors.lastname: German
authors.forename: Xianmin
authors.initials: X
authors.lastname: Zeng
authors.forename: Mahendra S
authors.initials: MS
authors.lastname: Rao
authors.forename: Uma
authors.initials: U
authors.lastname: Lakshmipathy
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
mesh_terms: D002454:Cell Differentiation; D002461:Cell Line, Transformed; D019070:Cell Lineage; D002843:Chromatin / Q000235:genetics*; D002882:Chromosomes, Human, Pair 13; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D038101:Insulator Elements / Q000235:genetics*; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D019076:Transgenes
chemical_list: D002843:Chromatin; D020648:Peptide Elongation Factor 1; D049452:Green Fluorescent Proteins; D019426:Integrases