Abstract
Currently, most genetically engineered rat strains are created by methods that involve random integration of transgenes into the genome. The ability to identify the chromosomal location of the transgene insertion site enables the development of efficient genotyping assays, allows segregation of multiple transgene integration sites to be followed while breeding, and facilitates characterization of possible positional effects on phenotype. Here we describe a method for determining the chromosomal location of transgene insertion that combines restriction endonuclease enzyme digest with subsequent rounds of PCR amplification to produce amplicons representing the chromosomal regions flanking the integrated transgene. This method provides a reliable means for determining the exact location of insertion of transgenes within the genome.
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Tesson L, Cozzi J, Menoret S, Remy S, Usal C, Fraichard A, Anegon I (2005) Transgenic modifications of the rat genome. Transgenic Res 14:531–46
Dellaire G, Chartrand P (1998) Direct evidence that transgene integration is random in murine cells, implying that naturally occurring double-strand breaks may be distributed similarly within the genome. Radiat Res 149:325–9
Liang Z, Breman AM, Grimes BR, Rosen ED (2008) Identifying and genotyping transgene integration loci. Transgenic Res 17:979–83
Ochman H, Gerber AS, Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120:621–3
Rosenthal A (1992) PCR amplification techniques for chromosome walking. Trends Biotechnol 10:44–8
Rosenthal A, Jones DS (1990) Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucleic Acids Res 18:3095–6
Shyamala V, Ames GF (1989) Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR. Gene 84:1–8
Parker JD, Rabinovitch PS, Burmer GC (1991) Targeted gene walking polymerase chain reaction. Nucleic Acids Res 19:3055–60
Yuanxin Y, Chengcai A, Li L, Jiayu G, Guihong T, Zhangliang C (2003) T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends. Nucleic Acids Res 31:e68
Bryda EC, Pearson M, Agca Y, Bauer BA (2006) Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus. Biotechniques 41:715–9
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–10
Kent WJ (2002) BLAT–the BLAST-like alignment tool. Genome Res 12:656–64
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–86
Brownstein MJ, Carpten JD, Smith JR (1996) Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques 20(1004–6):1008–10
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© 2010 Humana Press, a part of Springer Science+Business Media, LLC
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Bryda, E.C., Bauer, B.A. (2010). A Restriction Enzyme-PCR-Based Technique to Determine Transgene Insertion Sites. In: Anegon, I. (eds) Rat Genomics. Methods in Molecular Biology, vol 597. Humana Press. https://doi.org/10.1007/978-1-60327-389-3_20
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DOI: https://doi.org/10.1007/978-1-60327-389-3_20
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Online ISBN: 978-1-60327-389-3
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