Polycistronic Lentiviral Vector for “Hit and Run” Reprogramming of Adult Skin Fibroblasts to Induced Pluripotent Stem Cells (original) (raw)

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Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham

, Birmingham, Alabama,

USA

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Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham

, Birmingham, Alabama,

USA

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,

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham

, Birmingham, Alabama,

USA

Search for other works by this author on:

,

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham

, Birmingham, Alabama,

USA

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,

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham

, Birmingham, Alabama,

USA

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,

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham

, Birmingham, Alabama,

USA

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,

Department of Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham

, Birmingham, Alabama,

USA

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Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham

, Birmingham, Alabama,

USA

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Received:

19 December 2008

Accepted:

31 January 2009

Published:

12 February 2009

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Chia-Wei Chang, Yi-Shin Lai, Kevin M. Pawlik, Kaimao Liu, Chiao-Wang Sun, Chao Li, Trenton R. Schoeb, Tim M. Townes, Polycistronic Lentiviral Vector for “Hit and Run” Reprogramming of Adult Skin Fibroblasts to Induced Pluripotent Stem Cells, Stem Cells, Volume 27, Issue 5, May 2009, Pages 1042–1049, https://doi.org/10.1002/stem.39
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Abstract

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3′ long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic “hit and run” vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.

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

Copyright © 2009 AlphaMed Press

This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open\_access/funder\_policies/chorus/standard\_publication\_model)

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