title: hESC engineering by integrase-mediated chromosomal targeting.
abstract: Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency allowing for long-term expression of transgenes. In this chapter, we describe a retargeting system where phiC31 integrase is used to deliver a chromosomal target for a second integrase, R4. The engineered hESC line can be adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture and upon differentiation. The system described here represents a method to rapidly assemble complex plasmid-based assay systems, controllably insert them into the hESC genome, and have them actively express in pluripotent as well as in differentiated lineages there from.
authors.affiliation: Invitrogen Corporation, Carlsbad, CA, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Uma
authors.initials: U
authors.lastname: Lakshmipathy
authors.forename: Ali
authors.initials: A
authors.lastname: Ozgenc
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Thyagarajan
authors.forename: Pauline
authors.initials: P
authors.lastname: Lieu
authors.forename: Andrew
authors.initials: A
authors.lastname: Fontes
authors.forename: Haipeng
authors.initials: H
authors.lastname: Xue
authors.forename: Kelly
authors.initials: K
authors.lastname: Scheyhing
authors.forename: Chad
authors.initials: C
authors.lastname: MacArthur
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
mesh_terms: D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques; D002460:Cell Line; D002877:Chromosomes, Human / Q000235:genetics*; D018920:Coculture Techniques; D015925:Cryopreservation; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
chemical_list: D017931:DNA Primers; D019426:Integrases