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First published online October 25, 2007
Stem Cells Vol. 26 No. 1 January 2008, pp. 119 -126
doi:10.1634/stemcells.2007-0283; www.StemCells.com
© 2008 AlphaMed Press

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TECHNOLOGY DEVELOPMENT

Creation of Engineered Human Embryonic Stem Cell Lines Using phiC31 Integrase

Bhaskar Thyagarajana, Ying Liua, Soojung Shina, Uma Lakshmipathya, Kelly Scheyhinga, Haipeng Xuea, Catharina Ellerströmb, Raimund Strehlb, Johan Hyllnerb, Mahendra S. Raoa, Jonathan D. Chesnuta

aInvitrogen Corporation, Carlsbad, California, USA;
bCellartis AB, Göteborg, Sweden

Key Words. Human embryonic stem cells • phiC31 integrase • Site-specific integration

Correspondence: Bhaskar Thyagarajan, B.V.Sc., Ph.D., Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008, USA. Telephone: 760-268-7460; Fax: 760-602-6691; e-mail: bhaskar.thyagarajan{at}invitrogen.com

Received April 17, 2007; accepted for publication October 9, 2007.
First published online in STEM CELLS EXPRESS   October 25, 2007.



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 EF1{alpha} 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 EF1{alpha} 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.

Disclosure of potential conflicts of interest is found at the end of this article.




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