Abstract
Strategies for site-specific modulation of genomic sequences in mammalian cells require two components. One must be capable of recognizing and activating a specific target sequence in vivo, driving that site into an exploitable repair pathway. Information is transferred to the site via participation in the pathway by the second component, a donor nucleic acid, resulting in a permanent change in the target sequence. We have developed biologically active triple helix forming oligonucleotides (TFOs) as site-specific gene targeting reagents. These TFOs, linked to DNA reactive compounds (such as a cross-linking agent), activate pathways that can engage informational donors. We have used the combination of a psoralen-TFO and single strand oligonucleotide donors to generate novel cell lines with directed sequence changes at the target site.
Here we describe the synthesis and purification of bioactive psoralen-linked TFOs, their co-introduction into mammalian cells with donor nucleic acids, and the identification of cells with sequence conversion of the target site. We have emphasized details in the synthesis and purification of the oligonucleotides that are essential for preparation of reagents with optimal activity.
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Acknowledgement
This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging.
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Alam, M.R., Thazhathveetil, A.K., Li, H., Seidman, M.M. (2014). Preparation and Application of Triple Helix Forming Oligonucleotides and Single Strand Oligonucleotide Donors for Gene Correction. In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_7
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DOI: https://doi.org/10.1007/978-1-62703-761-7_7
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