hESC engineering by integrase-mediated chromosomal targeting.
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.
Liu
Ying
Y
Invitrogen Corporation, Carlsbad, CA, USA.
Lakshmipathy
Uma
U
Ozgenc
Ali
A
Thyagarajan
Bhaskar
B
Lieu
Pauline
P
Fontes
Andrew
A
Xue
Haipeng
H
Scheyhing
Kelly
K
MacArthur
Chad
C
Chesnut
Jonathan D
JD
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
D017931:DNA Primers; D019426:Integrases