Abstract
Nonviral delivery vectors are highly sought after for gene therapeutic applications and genetic vaccination. Dumbbell-shaped DNA minimal vectors have important advantages as compared with plasmids and minicircle DNA. Here, we describe the rapid, cheap, and efficient production of superior dumbbell vectors at high purity using a process termed 1-2-3 gap–primer PCR. This process represents a 1-tube, 2-enzyme, 3 h procedure that comprises a PCR followed by a ligation. The resulting dumbbells harbor mismatches close to the loop structures, which facilitate nuclear diffusion and result in enhanced gene expression.
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References
Kay MA (2011) State-of-the-art gene-based therapies: the road ahead. Nat Rev Genet 12:316–328
Mok PL, Cheong SK, Leong CF et al (2012) Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells. Cytotechnology 64:203–216
Kaur T, Slavcev RA, Wettig SD (2009) Addressing the challenge: current and future directions in ovarian cancer therapy. Curr Gene Ther 9:434–458
López-Fuertes L, Pérez-Jiménez E, Vila-Coro AJ et al (2002) DNA vaccination with linear minimalistic (MIDGE) vectors confers protection against Leishmania major infection in mice. Vaccine 21:247–257
Schakowski F, Gorschlüter M, Junghans C et al (2001) A novel minimal-size vector (MIDGE) improves transgene expression in colon carcinoma cells and avoids transfection of undesired DNA. Mol Ther 3:793–800
Zanta MA, Belguise-Valladier P, Behr J-P (1999) Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc Natl Acad Sci U S A 96:91–96
Cost GJ (2007) Enzymatic ligation assisted by nucleases: simultaneous ligation and digestion promote the ordered assembly of DNA. Nat Protoc 2:2198–2202
Taki M, Kato Y, Miyagishi M et al (2004) Small-interfering-rna expression in cells based on an efficiently constructed dumbbell-shaped DNA. Angew Chem Int Ed 43:3160–3163
Taki M, Kato Y, Miyagishi M et al (2003) A direct and efficient synthesis method for dumbell-shaped linear DNA using PCR in vitro. Nucleic Acids Symp Ser 3:191–192
Yu H, Jiang X, Hang L et al (2015) Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression. Nucleic Acids Res 43(18):e120
Qiagen (2021) QIAquick spin handbook. https://www.qiagen.com/us/resources/download.aspx?id=95f10677-aa29-453d-a222-0e19f01ebe17&lang=en. Accessed 8 Nov 2021
New England Labs (2020) Tm calculator. https://tmcalculator.neb.com/#!/main. Accessed 8 Nov 2021
Acknowledgments
This work was supported by the National University of Singapore Bridging Grants [NUHSRO/2015/091/Bridging/02 and NUHSRO/2017/068/Bridging/06], the National Medical Research Council of Singapore [New Investigator Grant number NMRC/NIG/1058/2011], and the Ministry of Education of Singapore [Academic Research Fund (AcRF) Tier 1 Faculty Research Committee (FRC) grants number T1-2011Sep-04 and T1-2014Apr-02, the Seed Fund for Basic Science Research number T1-BSRG 2015-05], and the Technology Development Review (TDR) grant number H19H0G1006 of the Agency for Science, Technology and Research (A*STAR) of Singapore all to V.P.
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Loh, P.S., Patzel, V. (2022). Efficient Generation of Superior Dumbbell-Shaped Nonviral DNA Delivery Vectors Using 1-2-3 Gap-Primer PCR. In: Walther, W. (eds) Gene Therapy of Cancer. Methods in Molecular Biology, vol 2521. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2441-8_18
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DOI: https://doi.org/10.1007/978-1-0716-2441-8_18
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