title: Creation of engineered human embryonic stem cell lines using phiC31 integrase.
abstract: It has previously been shown that the phage-derived phiC31 integrase can efficiently target native pseudo-attachment sites in the genome of various species in cultured cells, as well as in vivo. To demonstrate its utility in human embryonic stem cells (hESC), we have created hESC-derived clones containing expression constructs. Variant human embryonic stem cell lines BG01v and SA002 were used to derive lines expressing a green fluorescent protein (GFP) marker under control of either the human Oct4 promoter or the EF1alpha promoter. Stable clones were selected by antibiotic resistance and further characterized. The frequency of integration suggested candidate hot spots in the genome, which were mapped using a plasmid rescue strategy. The pseudo-attP profile in hESC differed from those reported earlier in differentiated cells. Clones derived using this method retained the ability to differentiate into all three germ layers, and fidelity of expression of GFP was verified in differentiation assays. GFP expression driven by the Oct4 promoter recapitulated endogenous Oct4 expression, whereas persistent stable expression of GFP expression driven by the EF1alpha promoter was seen. Our results demonstrate the utility of phiC31 integrase to target pseudo-attP sites in hESC and show that integrase-mediated site-specific integration can efficiently create stably expressing engineered human embryonic stem cell clones.
authors.affiliation: Invitrogen Corporation, Carlsbad, California 92008, USA. bhaskar.thyagarajan@invitrogen.com
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Thyagarajan
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Soojung
authors.initials: S
authors.lastname: Shin
authors.forename: Uma
authors.initials: U
authors.lastname: Lakshmipathy
authors.forename: Kelly
authors.initials: K
authors.lastname: Scheyhing
authors.forename: Haipeng
authors.initials: H
authors.lastname: Xue
authors.forename: Catharina
authors.initials: C
authors.lastname: Ellerström
authors.forename: Raimund
authors.initials: R
authors.lastname: Strehl
authors.forename: Johan
authors.initials: J
authors.lastname: Hyllner
authors.forename: Mahendra S
authors.initials: MS
authors.lastname: Rao
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
mesh_terms: D001287:Attachment Sites, Microbiological / Q000235:genetics; D001435:Bacteriophages; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D003001:Cloning, Molecular; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000502:physiology; D011401:Promoter Regions, Genetic; D014162:Transfection
chemical_list: D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D020648:Peptide Elongation Factor 1; D049452:Green Fluorescent Proteins; D019426:Integrases