Abstract
Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.
The original chapter was corrected: The affiliation of Dr. Andreas Kolb is University of Aberdeen, Foresterhill and not University of Foresterhill as stated originally in the EPUB version.
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References
Coates CJ, Kaminski JM, Summers JB et al (2005) Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools. Trends Biotechnol 23:407–419
Turan S, Zehe C, Kuehle J et al (2013) Recombinase-mediated cassette exchange (RMCE)—a rapidly-expanding toolbox for targeted genomic modifications. Gene 515:1–27
Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096–1258096
Kolb AF, Coates CJ, Kaminski JM et al (2005) Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction. Trends Biotechnol 23:399–406
Proudfoot C, McPherson AL, Kolb AF et al (2011) Zinc finger recombinases with adaptable DNA sequence specificity. PLoS One 6:e19537
Ousterout DG, Gersbach CA (2016) The development of TALE nucleases for biotechnology. Methods Mol Biol 1338:27–42
Kolb AF (2001) Selection-marker-free modification of the murine β-casein gene using a lox2722 site. Anal Biochem 290:260–271
Gaj T, Sirk SJ, Barbas CF (2014) Expanding the scope of site-specific recombinases for genetic and metabolic engineering. Biotechnol Bioeng 111:1
Loonstra A, Vooijs M, Beverloo HB et al (2001) Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. PNAS 98:9209–9214
Malla S, Dafhnis-Calas F, Brookfield JFY et al (2005) Rearranging the centromere of the human Y chromosome with phiC31 integrase. Nucleic Acids Res 33:6101–6113
Sorrell DA, Robinson CJ, Smith J-A et al (2010) Recombinase mediated cassette exchange into genomic targets using an adenovirus vector. Nucleic Acids Res 38:e123–e123
Lassnig C, Kolb AF, Strobl B et al (2005) Studying human pathogens in animal models: fine tuning the humanized mouse. Transgenic Res 14:803–806
Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71
Shmerling D, Danzer CP, Mao X et al (2005) Strong and ubiquitous expression of transgenes targeted into the beta-actin locus by Cre/lox cassette replacement. Genesis 42:229–235
Robinson C, Kolb AF (2009) Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion. Exp Cell Res 315:508–522
Buchholz F, Angrand PO, Stewart AF (1998) Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16:657–662
Buchholz F, Stewart AF (2001) Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat Biotechnol 19:1047–1052
Voziyanov Y, Konieczka JH, Stewart AF et al (2003) Stepwise manipulation of DNA specificity in Flp recombinase: progressively adapting Flp to individual and combinatorial mutations in its target site. J Mol Biol 326:65–76
Magin TM, McWhir J, Melton DW (1992) A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. Nucleic Acids Res 20:3795–3796
Price J, Turner D, Cepko C (1987) Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. PNAS 84:156–160
Graham FL, Smiley J, Russell WC et al (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36:59–72
Pfeifer A, Ikawa M, Dayn Y et al (2002) Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. PNAS 99:2140–2145
Mao Y, Yan R, Li A et al (2015) Lentiviral vectors mediate long-term and high efficiency transgene expression in HEK 293T cells. Int J Med Sci 12:407–415
Acknowledgments
This work was supported by the BBSRC (Gene Technologies underpinning Health Care #12599; CASE studentship BB/J012343/1), the Genomia Seed Fund, and the Hannah Development Fund.
The authors declare no conflicts of interest.
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Kolb, A.F. et al. (2017). Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors. In: Eroshenko, N. (eds) Site-Specific Recombinases. Methods in Molecular Biology, vol 1642. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7169-5_9
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DOI: https://doi.org/10.1007/978-1-4939-7169-5_9
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Publisher Name: Humana Press, New York, NY
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