Robust, Persistent Transgene Expression in Human ES Cells is Achieved with AAVS1-targeted Integration

¹ Department of Surgery & Cambridge Institute for Medical Research, Department of Pathology, Hinxton, Cambridge, UK.
² Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
³ Department of Surgery & Cambridge Institute for Medical Research, Hinxton, Cambridge, UK.
⁴ Centre for Brain Repair, University of Cambridge, Cambridge UK.
⁵ Department of Pathology, Hinxton, Cambridge, UK. Centre for Brain Repair, University of Cambridge, Cambridge UK.

^* To whom correspondence should be addressed. E-mail: jrs68{at}cam.ac.uk.

	Abstract

Silencing and variegated transgene expression are poorly understood problems that can interfere with gene function studies in human embryonic stem cells (hESCs). We show that transgene expression (EGFP) from random integration sites in hESCs is affected by variegation and silencing, with only half of hESCs expressing the transgene, which is gradually lost after withdrawal of selection and differentiation. We tested the hypothesis that a transgene integrated into the AAV2 target region on chromosome 19, known as the AAVS1 locus, would maintain transgene expression in hESCs. When we used AAV2 technology to target the AAVS1 locus, 4.16% of hESC clones achieved AAVS1-targeted integration. Targeted clones expressed Oct-4, SSEA-3 and Tra-1-60 and differentiated into all three primary germ layers. EGFP expression from the AAVS1 locus showed significantly reduced variegated expression when in selection with 90%±4 of cells expressing EGFP compared to 57%±32 for randomly integrated controls, and reduced tendency to undergo silencing, with 86%±7 hESCs expressing EGFP 25 days after withdrawal of selection, as compared to 39%±31 randomly integrated clones. In addition, QPCR analysis of hESCs also indicated significantly higher levels of EGFP mRNA in AAVS1-targeted clones as compared to randomly integrated clones.