Stable and reproducible transgene expression independent of proliferative or differentiated state using BAC TG-EMBED

Pankaj Chaturvedi, Binhui Zhao, David L. Zimmerman, Andrew S Belmont

Research output: Contribution to journalArticle

Abstract

Reproducible and stable transgene expression is an important goal in both basic research and biotechnology, with each application demanding a range of transgene expression. Problems in achieving stable transgene expression include multi-copy transgene silencing, chromosome-position effects, and loss of expression during long-term culture, induced cell quiescence, and/or cell differentiation. Previously, we described the “BAC TG-EMBED” method for copy-number dependent, chromosome position-independent expression of embedded transgenes within a BAC containing ~170 kb of the mouse Dhfr locus. Here we demonstrate wider applicability of the method by identifying a BAC and promoter combination that drives reproducible, copy-number dependent, position-independent transgene expression even after induced quiescence and/or cell differentiation into multiple cell types. Using a GAPDH BAC containing ~200 kb of the human GAPDH gene locus and a 1.2 kb human UBC promoter, we achieved stable GFP-ZeoR reporter expression in mouse NIH 3T3 cells after low-serum-induced cell cycle arrest or differentiation into adipocytes. More notably, GFP-ZeoR expression remained stable and copy-number dependent even after differentiation of mouse ESCs into several distinct lineages. These results highlight the potential use of BAC TG-EMBED as an expression platform for high-level but stable, long-term expression of transgene independent of cell proliferative or differentiated state.

Original languageEnglish (US)
Pages (from-to)376-391
Number of pages16
JournalGene therapy
Volume25
Issue number5
DOIs
StatePublished - Aug 1 2018

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Transgenes
Cell Differentiation
Chromosomes
NIH 3T3 Cells
Biotechnology
Cell Cycle Checkpoints
Adipocytes
Cell Culture Techniques
Serum
Research
Genes

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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Stable and reproducible transgene expression independent of proliferative or differentiated state using BAC TG-EMBED. / Chaturvedi, Pankaj; Zhao, Binhui; Zimmerman, David L.; Belmont, Andrew S.

In: Gene therapy, Vol. 25, No. 5, 01.08.2018, p. 376-391.

Research output: Contribution to journalArticle

Chaturvedi, Pankaj ; Zhao, Binhui ; Zimmerman, David L. ; Belmont, Andrew S. / Stable and reproducible transgene expression independent of proliferative or differentiated state using BAC TG-EMBED. In: Gene therapy. 2018 ; Vol. 25, No. 5. pp. 376-391.
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