Generation of platform human embryonic stem cell lines that allow efficient targeting at a predetermined genomic location.
Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency, allowing for long-term expression of transgenes. In the present work, we describe a retargeting system where we used phiC31 integrase to target a plasmid to a pseudo-attP site in the cellular genome. The integration site was mapped and the chromosomal location evaluated for potential to be transcriptionally active in differentiated cells. The target plasmid, thus inserted, carried a wild-type R4 attB site that acts as a target for further integration of expression constructs. We engineered 2 hESC lines, BG01V and H9, to contain the target and showed that genetic elements such as promoter-reporter pairs can be inserted at the target efficiently and specifically. The retargeting construct has been adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture, upon random differentiation, and directed induction into neural lineages. 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 undifferentiated as well as in differentiated cells.
Primary and Stem Cell Systems, Life Technologies Corporation, Carlsbad, California 92008, USA.
Ying
Y
Liu
Bhaskar
B
Thyagarajan
Uma
U
Lakshmipathy
Haipeng
H
Xue
Pauline
P
Lieu
Andrew
A
Fontes
Chad C
CC
MacArthur
Kelly
K
Scheyhing
Mahendra S
MS
Rao
Jonathan D
JD
Chesnut
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D019070:Cell Lineage; D049109:Cell Proliferation; D002882:Chromosomes, Human, Pair 13 / Q000235:genetics; D002999:Clone Cells; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D020868:Gene Silencing; D056426:Genetic Loci / Q000235:genetics; D015894:Genome, Human / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D015203:Reproducibility of Results
D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D049452:Green Fluorescent Proteins