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Integration of exogenous DNA into mouse embryonic stem cell chromosomes shows preference into genes and frequent modification at junctions

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Abstract

Chromosomal integration of exogenous DNA in mammalian cells allows stable gene expression for a variety of biological applications. Although it is presumably mediated by DNA repair machinery, little is known regarding site preferences and other characteristics. We isolated and analyzed 256 chromosomal-plasmid DNA integration junctions from 158 plasmid integrants after electroporation in mouse embryonic stem (ES) cells. The frequency of integrations in transcription units (40%) showed a slight but significant increase over the frequency estimated by computer simulation of random events (30%), suggesting preferential integration into genes. Microarray analysis revealed preference into genes, which are expressed in mouse ES cells. In contrast, bias toward integrations around transcriptional start sites, CpG islands and repeat elements was not observed. Furthermore, all host chromosome sequences as well as the majority of plasmids (96%) at the integration junctions were modified by deletions and/or insertions of additional nucleotides. Detailed analyses revealed frequent stem loop/hairpin formation mediated by weak homologies near plasmid ends before integration. Our study sheds light on a natural fate of exogenous DNA, which preferentially integrates into transcriptionally active chromosomal sites and by an imprecise end-joining pathway, associated with highly frequent modification of the end sequences.

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Abbreviations

ES cells:

Embryonic stem cells

DSBs:

Double-strand breaks

HR:

Homologous recombination

NHEJ:

Non-homologous end-joining

SCID-X1:

X-linked severe combined immune deficiency

MLV:

Moloney murine leukemia virus

AAV:

Adeno-associated virus

rDNA:

Ribosomal RNA gene

AdVs:

Adenoviral vectors

PGK:

Phosphoglycerate kinase

Ad5:

Human adenovirus type 5

LINE:

Long interspersed nucleotide element

SINE:

Short interspersed nucleotide element

LTR:

Long terminal repeats

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Acknowledgments

We thank Dr. Toru Shimada (University of Tokyo, Tokyo, Japan) and Naoko Omuro (University of Tokyo, Tokyo, Japan) for high-quality sequencing, and Dr. Masami Muramatsu (Saitama Medical University, Saitama, Japan), Dr. Hiroyuki Nakai (University of Pittsburgh School of Medicine, Pittsburgh, PA) and the members of Mitani lab for helpful discussions. This work was supported in part by the Ministry of Education, Science, Sports, and Culture, in particular by a Ministry Grant to Research Center for Genomic Medicine, Saitama Medical University.

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Authors and Affiliations

  1. Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, 350-1241, Japan

    Keiichiro Suzuki, Fumi Ohbayashi & Kohnosuke Mitani

  2. Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, 350-1241, Japan

    Itoshi Nikaido & Yasushi Okazaki

  3. Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, 350-1241, Japan

    Akihiko Okuda

  4. Network Information Center, Teikyo University, Itabashi, Tokyo, 173-8605, Japan

    Haruyoshi Takaki

Authors
  1. Keiichiro Suzuki
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  2. Fumi Ohbayashi
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  3. Itoshi Nikaido
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  4. Akihiko Okuda
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  5. Haruyoshi Takaki
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  6. Yasushi Okazaki
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  7. Kohnosuke Mitani
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Correspondence to Kohnosuke Mitani.

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Responsible Editor: Yoichi Matsuda.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

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Suzuki, K., Ohbayashi, F., Nikaido, I. et al. Integration of exogenous DNA into mouse embryonic stem cell chromosomes shows preference into genes and frequent modification at junctions. Chromosome Res 18, 191–201 (2010). https://doi.org/10.1007/s10577-010-9111-5

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  • DOI: https://doi.org/10.1007/s10577-010-9111-5

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