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Reaction parameters of targeted gene repair in mammalian cells

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Abstract

Targeted gene repair uses short DNA oligonucleotides to direct a nucleotide exchange reaction at a designated site in a mammalian chromosome. The widespread use of this technique has been hampered by the inability of workers to achieve robust levels of correction. Here, we present a mammalian cell system in which DLD-1 cells bearing integrated copies of a mutant eGFP gene are repaired by modified single-stranded DNA oligonucleotides. We demonstrate that two independent clonal isolates, which are transcribed at different levels, are corrected at different frequencies. We confirm the evidence of a strand bias observed previously in other systems, wherein an oligonucleotide designed to be complementary to the nontranscribed strand of the target directs a higher level of repair than one targeting the transcribed strand. Higher concentrations of cell oligonucleotides in the electroporation mixture lead to higher levels of correction. When the target cell population is synchronized into S phase then released before electroporation, the correction efficiency is increased within the entire population. This model system could be useful for pharmacogenomic applications of targeted gene repair including the creation of cell lines containing single-base alterations.

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References

  1. Kmiec, E. B. (1999) Targeted gene repair. Gene Ther. 6, 1–3.

    Article  PubMed  CAS  Google Scholar 

  2. Liu, L., Parekh-Olmedo, H., and Kmiec, E. B. (2003) The development and regulation of gene repair. Nat. Rev. Genet. 4, 679–689.

    Article  PubMed  CAS  Google Scholar 

  3. Igoucheva, O., Alexeev, V., Pryce, M., and Yoon, K. (2003) Transcription affects formation and processing of intermediates in oligonucleotide-mediated gene alteration. Nucleic Acids Res. 31, 2659–2670.

    Article  PubMed  CAS  Google Scholar 

  4. Dekker, M., Brouwers, C., and Te, R. H. (2003) Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides. Nucleic Acids Res. 31, E27.

    Google Scholar 

  5. Goncz, K. K., Kunzelmann, K., Xu, Z., and Gruenert, D. C. (1998) Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments. Hum. Mol. Genet. 7, 1913–1919.

    Article  PubMed  CAS  Google Scholar 

  6. Goncz, K. K., Colosimo, A., Dallapiccola, B., Gagne, L., Hong, K., Novelli, G., et al. Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR. Gene Ther. 8, 961–965.

  7. Andersen, M. S., Sorensen, C. B., Bolund, L., and Jensen, T. G. (2002) Mechanisms underlying targeted gene correction using chimeric RNA/DNA and single-stranded DNA oligonucleotides. J. Mol. Med. 80, 770–781.

    Article  PubMed  CAS  Google Scholar 

  8. Rice, M. C., Bruner, M., Czymmek, K., and Kmiec, E. B. (2001) In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae. Mol. Microbiol. 40, 857–868.

    Article  PubMed  CAS  Google Scholar 

  9. Liu, L., Cheng, S., van Brabant, A. J., and Kmiec, E. B. (2002) Rad51p and Rad54p, but not Rad52p, elevate gene repair in Saccharomyces cerevisiae directed by modified single-stranded oligonucleotide vectors. Nucleic Acids Res. 30, 2742–2750.

    Article  PubMed  CAS  Google Scholar 

  10. Rice, M. C., Czymmek, K., and Kmiec, E. B. The potential of nucleic acid repair in functional genomics. Nat. Biotechnol. 19, 321–326.

  11. Brachman, E. E. and Kmiec, E. B. (2003) Targeted gene repair of cyc1 mutations in Saccharomyces cerevisiae directed by modified single-stranded DNA oligonucleotides. Genetics 163, 527–538.

    PubMed  CAS  Google Scholar 

  12. Cole-Strauss, A., Gamper, H., Holloman, W. K., Munoz, M., Cheng, N., and Kmiec, E. B. (1999) Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract. Nucleic Acids Res. 27, 1323–1330.

    Article  PubMed  CAS  Google Scholar 

  13. Kren, B. T., Wong, P. Y., and Steer, C. J. (2003) Short, single-stranded oligonucleotides mediate targeted nucleotide conversion using extracts from isolated liver mitochondria. DNA Repair (Amst) 2, 531–546.

    Article  CAS  Google Scholar 

  14. Gamper, H. B., Parekh, H., Rice, M. C., Bruner, M., Youkey, H., and Kmiec, E. B. (2000) The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts. Nucleic Acids Res. 28, 4332–4339.

    Article  PubMed  CAS  Google Scholar 

  15. Parekh-Olmedo, H., Drury, M., and Kmiec, E. B. (2002) Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids. Chem. Biol. 9, 1073–1084.

    Article  PubMed  CAS  Google Scholar 

  16. Drury, M. D. and Kmiec, E. B. (2003) DNA pairing is an important step in the process of targeted nucleotide exchange. Nucleic Acids Res. 31, 899–910.

    Article  PubMed  CAS  Google Scholar 

  17. Liu, L., Rice, M. C., and Kmiec, E. B. (2001) In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides. Nucleic Acids Res. 29, 4238–4250.

    Article  PubMed  CAS  Google Scholar 

  18. Nickerson, H. D. and Colledge, W. H. (2003) A comparison of gene repair strategies in cell culture using a lacZ reporter system. Gene Ther. 10, 1584–1591.

    Article  PubMed  CAS  Google Scholar 

  19. Liu, L., Rice, M. C., Drury, M., Cheng, S., Gamper, H., and Kmiec, E. B. (2002) Strand bias in targeted gene repair is influenced by transcriptional activity. Mol. Cell Biol. 22, 3852–3863.

    Article  PubMed  CAS  Google Scholar 

  20. van der Steege, G., Schuilenga-Hut, P. H., Buys, C. H., Scheffer, H., Pas, H. H., and Jonkman, M. F. (2001) Persistent failures in gene repair. Nat. Biotechnol. 19, 305–306.

    Article  PubMed  Google Scholar 

  21. Vasquez, K. M., Marburger, K., Intody, Z., and Wilson, J. H. (2001) Manipulating the mammalian genome by homologous recombination. Proc. Natl. Acad. Sci. USA 98, 8403–8410.

    Article  PubMed  CAS  Google Scholar 

  22. Zheng, H. and Wilson, J. H. (1990) Gene targeting in normal and amplified cell lines. Nature 344, 170–173.

    Article  PubMed  CAS  Google Scholar 

  23. Majumdar, A., Puri, N., Cuenoud, B., Natt, F., Martin, P., Khorlin, A., et al. (2003) Cell cycle modulation of gene targeting by a triple helix-forming oligonucleotide. J. Biol. Chem. 278, 11,072–11,077.

    CAS  Google Scholar 

  24. Orren, D. K., Petersen, L. N., and Bohr, V. A. (1995) A UV-responsive G2 checkpoint in rodent cells. Mol. Cell Biol. 15, 3722–3730.

    PubMed  CAS  Google Scholar 

  25. Liu, X. M., Yan, Z., Luo, M., Zak, R., Driskell, R., Li, Z., et al. (2003) Targeted correction of genomic single base-pair mutations using adeno-associated virus under non-selective conditions. Simultaneous Oral Abstract Sessions. Molecular Therapy 7, S159-S160.

    Google Scholar 

  26. Bandyopadhyay, P., Kren, B. T., Ma, X., and Steer, C. J. (1998) Enhanced gene transfer into HuH-7 cells and primary rat hepatocytes using targeted liposomes and polyethylenimine. Biotechniques 25, 282–292.

    PubMed  CAS  Google Scholar 

  27. Thorpe, P., Stevenson, B. J., and Porteous, D. J. (2002) Optimising gene repair strategies in cell culture. Gene Ther. 9, 700–702.

    Article  PubMed  CAS  Google Scholar 

  28. Banks, G. A., Roselli, R. J., Chen, R., and Giorgio, T. D. (2003) A model for the analysis of nonviral gene therapy. Gene Ther. 10, 1766–1775.

    Article  PubMed  CAS  Google Scholar 

  29. Solinger, J. A., Kiianitsa, K., and Heyer, W. D. (2002) Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol. Cell 10, 1175–1188.

    Article  PubMed  CAS  Google Scholar 

  30. Maguire, K. and Kmiec, E. B. (2003) Enhancement of in vivo targeted nucleotide exchange by nonspecific carrier DNA. In: Genetic Recombination Reviews and Protocols (Walman, A. S., ed.), Humana, Totowa, pp. 209–220.

    Google Scholar 

  31. Igoucheva, O., Alexeev, V., and Yoon, K. (2001) Targeted gene correction by small single-stranded oligonucleotides in mammalian cells. Gene Ther. 8, 391–399.

    Article  PubMed  CAS  Google Scholar 

  32. Ellis, H. M., Yu, D., DiTizio, T., and Court, D. L. (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. USA 98, 6742–6746.

    Article  PubMed  CAS  Google Scholar 

  33. Brachman, E. E. and Kmiec, E. B. (2004) DNA replication and transcription direct a DNA strand bias in the process of targeted gene repair in mammalian cells. J. Cell Sci. 117, 3867–3874.

    Article  PubMed  CAS  Google Scholar 

  34. Parekh-Olmedo, H., Engstrom, J., and Kmiec, E. B. (2003) The effect of hydroxyurea and trichostatin A on targeted nucleotide exchange in yeast and mammalian cells. Ann. NY Acad. Sci. 1002, 43–56.

    Article  PubMed  CAS  Google Scholar 

  35. Yoshida, M., Kijima, M., Akita, M., and Beppu, T. (1990) Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J. Biol. Chem. 265, 17,174–17,179.

    CAS  Google Scholar 

  36. Santana, E., Peritz, A. E., Iyer, S., Uitto, J., and Yoon, K. (1998) Different frequency of gene targeting events by the RNA-DNA oligonucleotide among epithelial cells. J. Invest Dermatol. 111, 1172–1177.

    Article  PubMed  CAS  Google Scholar 

  37. Leith, J. T., Heyman, P., DeWyngaert, J. K., Dexter, D. L., Calabresi, P., and Glicksman, A. S. (1983) Thermal survival characteristics of cell subpopulations isolated from a heterogeneous human colon tumor. Cancer Res. 43, 3240–3246.

    PubMed  CAS  Google Scholar 

  38. Leith, J. T., Dexter, D. L., DeWyngaert, J. K., Zeman, E. M., Chu, M. Y., Calabresi, P., and Glicksman, A. S. (1982) Differential responses to x-irradiation of subpopulations of two heterogeneous human carcinomas in vitro. Cancer Res. 42, 2556–2561.

    PubMed  CAS  Google Scholar 

  39. Dexter, D. L., Spremulli, E. N., Fligiel, Z., Barbosa, J. A., Vogel, R., VanVoorhees, A., and Calabresi, P. (1981) Heterogeneity of cancer cells from a single human colon carcinoma. Am. J. Med. 71, 949–956.

    Article  PubMed  CAS  Google Scholar 

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Author notes

  1. Yiling Hu

    Present address: Department of Neuroscience, Dalhousie University, Halifax, Nova Scotia

Authors and Affiliations

  1. Department of Biological Sciences, University of Delaware, 19716, Newark, DE, USA

    Hetal Parekh-Olmedo, Miya Drury, Michael Skogen & Eric B. Kmiec

  2. Department of Chemistry and Biochemistry, University of Delaware, 19716, Newark, DE, USA

    Miya Drury

Authors
  1. Yiling Hu
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  2. Hetal Parekh-Olmedo
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  3. Miya Drury
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  5. Eric B. Kmiec
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Correspondence to Eric B. Kmiec.

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Hu, Y., Parekh-Olmedo, H., Drury, M. et al. Reaction parameters of targeted gene repair in mammalian cells. Mol Biotechnol 29, 197–210 (2005). https://doi.org/10.1385/MB:29:3:197

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