A simple and highly efficient method to identify the integration site of a transgene in the animal genome

Uemura, Shun; Nagaoka, Tadahiro; Yokoyama, Minesuke; Igarashi, Michihiro; Kishi, Masashi

doi:10.1016/j.neures.2013.11.007

Cited by 10 publications

(8 citation statements)

References 24 publications

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“…The most commonly used method for identification of transgene insertion sites is based on inverse PCR (iPCR) (10,11). This method relies on knowledge of appropriate restriction sites located in the transgene and on sequence information to design appropriate primers for the iPCR.…”

Section: Introductionmentioning

confidence: 99%

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

Cain-Hom¹,

Splinter

Min

et al. 2017

Nucleic Acids Res

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Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events.

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Section: Introductionmentioning

confidence: 99%

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

Cain-Hom¹,

Splinter

Min

et al. 2017

Nucleic Acids Res

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“…Ai14 ( Rosa26-CAG-loxp-stop-loxp-tdTomato ) (Guenthner et al 2013), TetO-Cre (Uemura et al 2014), TetO-H2BGFP (Chakkalakal et al 2012) and Albumin-Cre (Cui et al 2016) mice were obtained from The Jackson Laboratory. All mice were housed in an SPF environment.…”

Section: Methodsmentioning

confidence: 99%

Generation of H11-albumin-rtTA Transgenic Mice: A Tool for Inducible Gene Expression in the Liver

Meng

Chen

et al. 2019

G3 Genes|Genomes|Genetics

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The modification of the mouse genome by site-specific gene insertion of transgenes and other genetic elements allows the study of gene function in different developmental stages and in the pathogenesis of diseases. Here, we generated a “genomic safe harbor” Hipp11 ( H11 ) locus-specific knock-in transgenic mouse line in which the albumin promoter is used to drive the expression of the reverse tetracycline transactivator (rtTA) in the liver. The newly generated H11-albumin-rtTA transgenic mice were bred with tetracycline-operator-Histone-2B-green fluorescent protein (TetO-H2BGFP ) mice to assess inducibility and tissue-specificity. Expression of the H2BGFP fusion protein was observed exclusively upon doxycycline (Dox) induction in the liver of H11-albumin-rtTA/TetO-H2BGFP double transgenic mice. To further analyze the ability of the Dox-inducible H11-albumin-rtTA mice to implement conditional DNA recombination, H11-albumin-rtTA transgenic mice were crossed with TetO-Cre and Ai14 mice to generate H11-albumin-rtTA/TetO-Cre/Ai14 triple transgenic mice. We successfully confirmed that the Cre-mediated recombination efficiency was as strong in Dox-induced H11-albumin-rtTA /TetO-Cre/Ai14 mice as in the control albumin-Cre/A14 mice. Finally, to characterize the expression-inducing effects of Dox in H11-albumin-rtTA/TetO-H2BGFP mice in detail, we examined GFP expression in embryos at different developmental stages and found that newly conceived H11-albumin-rtTA/TetO-H2BGFP embryos of Dox-treated pregnant female mice were expressing reporter GFP by E16.5. Our study demonstrates that these new H11-albumin-rtTA transgenic mice are a powerful and efficient tool for the temporally and spatially conditional manipulation of gene expression in the liver, and illustrates how genetic crosses with these new mice enable the generation of complex multi-locus transgenic animals for mechanistic studies.

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“…Most are forms of PCR, including inverse PCR (iPCR), ligation-mediated PCR, and linear amplification PCR (LAM-PCR). 9 , 10 , 11 , 12 , 13 , 14 These methods each involve using restriction enzymes to digest the input DNA, which is then ligated to terminal adapter sequences. Therefore, these methods are limited to detecting integration events proximal to the chosen restriction enzyme(s).…”

Section: Introductionmentioning

confidence: 99%

Rapid, accurate mapping of transgene integration in viable rhesus macaque embryos using enhanced-specificity tagmentation-assisted PCR

Ryu

Chan

Wettengel

et al. 2022

Molecular Therapy - Methods & Clinical Development

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A simple and highly efficient method to identify the integration site of a transgene in the animal genome

Cited by 10 publications

References 24 publications

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

Generation of H11-albumin-rtTA Transgenic Mice: A Tool for Inducible Gene Expression in the Liver

Rapid, accurate mapping of transgene integration in viable rhesus macaque embryos using enhanced-specificity tagmentation-assisted PCR

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