Robust, Persistent Transgene Expression in Human Embryonic Stem Cells Is Achieved with AAVS1-Targeted Integration

Author:

Smith Joseph R.123,Maguire Sean4,Davis Lesley A.23,Alexander Morgan23,Yang Fentang4,Chandran Siddharthan5,ffrench-Constant Charles51,Pedersen Roger A.23

Affiliation:

1. Department of Pathology, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom

2. Cambridge Institute for Medical Research, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom

3. Department of Surgery, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom

4. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom

5. Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom

Abstract

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 (enhanced green fluorescent protein [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 adeno-associated virus type 2 (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, stage-specific embryonic antigen-3 (SSEA3), 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 with 57% ± 32% for randomly integrated controls, and reduced tendency to undergo silencing, with 86% ± 7% hESCs expressing EGFP 25 days after withdrawal of selection compared with 39% ± 31% for randomly integrated clones. In addition, quantitative polymerase chain reaction analysis of hESCs also indicated significantly higher levels of EGFP mRNA in AAVS1-targeted clones as compared with randomly integrated clones. Transgene expression from the AAVS1 locus was shown to be stable during hESC differentiation, with more than 90% of cells expressing EGFP after 15 days of differentiation, as compared with ∼30% for randomly integrated clones. These results demonstrate the utility of transgene integration at the AAVS1 locus in hESCs and its potential clinical application. Disclosure of potential conflicts of interest is found at the end of this article.

Publisher

Oxford University Press (OUP)

Subject

Cell Biology,Developmental Biology,Molecular Medicine

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3