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Proc Natl Acad Sci U S A. 2010 Nov 9;107(45):19467-72. doi: 10.1073/pnas.1012677107. Epub 2010 Oct 25.

Generation of induced pluripotent stem cells using site-specific integration with phage integrase.

Author information

1
Departments of Medicine and Laboratory Medicine and Institute for Human Genetics, University of California, San Francisco, CA 94143, USA.

Abstract

To date, a large number of reports have described reprogramming many somatic cell types into induced pluripotent stem (iPS) cells, using different numbers of transcription factors and devising alternate methods of introducing the transcription factor genes or proteins into the somatic cells. Here, we describe a method using bacteriophage ΦC31 integrase to reprogram mouse embryonic fibroblasts and human amniotic fluid cells into iPS cells. These iPS cells showed morphology, surface antigens, gene expression, and epigenetic states similar to ES cells and formed teratomas with three germ layers in nonobese diabetic/severely compromised immunodeficient mice. Importantly, these iPS cells have only a single integration site in each cell line. The locations of integration favor the intergenic regions, and their distances from the adjacent genes extended from several hundred to >1 million bp. The effect of the insertion on the expression of these genes can be studied by RT-PCR. No insertion into microRNA gene loci was detected. Hence, it is possible to select cells in which adjacent gene functions are not affected, or the inserts can be removed if necessary. We conclude that phage integrase-mediated site-specific recombination can produce iPS cells that have undisturbed endogenous gene function and could be safe for future human therapeutic application.

PMID:
20974949
PMCID:
PMC2984165
DOI:
10.1073/pnas.1012677107
[Indexed for MEDLINE]
Free PMC Article

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