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Transgenic mouse model

Unravelling frequent structural variation and insertional mutagenesis in a large-scale transgenic mouse study for The Jackson Laboratory

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Despite the advantages of transgenic models in biomedical research, the traditional microinjection of exogenous/recombinant DNA into zygotic pronuclei is known to drive insertional mutagenesis.1-3 Adding to the mix, transgenes often integrate as a concatemer (i.e. multicopy array).1,2 As such, several factors can influence proper transgene expression. Therefore, thorough genetic characterization of generated transgenic lines is essential for better correlation between genotype-phenotype and the proper selection of founders for experiments and publication.1-3

Until the introduction of our genetic QC solutions, the robust detection of all genetic variation (including structural variants), in and around genes of interest represented a true impediment to genetic research.4 Indeed, the popular FISH assay, although allowing the evaluation of integration sites, is solely capable of mapping transgenes at low resolution and remains largely uninformative when it comes to potential mutagenesis at the integration site. As for inverse PCR, the approach will struggle with the multicopy nature of most transgenes.

To follow-up on Genentech’s publication2, The Jackson Laboratory (JAX) sought to investigate the rate of mutagenesis in a larger sample size that include a variety of transgene types. Here, JAX puts TLA-based solutions to the test by evaluating the capabilities of our genetic QC solutions to also analyze larger constructs such as bacterial artificial chromosomes (BAC), human PAC as well human cosmid vectors.1

QC for the complete genetic characterization of transgene insertion site

In most of the analyzed lines, TLA identified unexpected structural variations accompanying insertion (30/40). Among the observed genomic rearrangements are: 24 deletions (including a deletion that is > 1 Mb), 6 duplications and a complex structural variation that includes simultaneous duplication, deletion, and inversion.1

Interestingly, TLA identified several cases where mapped sequences were fused to E. coli genome (K-12), and not vector sequences. Suggesting that fragments of contaminating E. coli DNA were cointegrating with the transgene. In fact, 10 out of 40 strains were found to have this cointegration, with total composition ranging from 300 bp to > 200 kb.1

Taken together, transgenic alleles were seen to display a high rate of potentially confounding genetic events. Finally, this study clearly demonstrated the unique capabilities of our TLA-based genetic QC solutions to robustly detect all genetic variation (including structural variants) in and around any (trans)genes of interest.1

 

Table 1_JAX paper


"TLA should be considered a “first pass” tool for integration locus discovery"

Together with JAX, we reported here the first large-scale study that sought to evaluate the consequences of random transgene insertions. By leveraging the rapid, efficient and robust capabilities of TLA, transgene integration sites were precisely mapped in 40 widely used transgenic mouse lines.1 All in all, this paper illustrates how transgene insertions could confound some experiments, as transgenic alleles were seen to display a high rate of potentially confounding genetic event. As such, the authors underscored the need to adopt proper QC strategies upon generation of transgenic lines. Finally, this paper underpins the importance of in-depth genetic characterization of transgenic models to guarantee sound genotype-phenotype data interpretation and reproducible experiments in biomedical research.1

In this study, the authors indicate that: “TLA-based discovery of transgenic insertion sites provides a number of practical benefits that should improve quality control for both public repositories and the end user. For example, allele-specific assays can be developed at the integration site to distinguish all genotype classes, allowing for homozygous mating strategies unless precluded by insertional mutagenesis. End users of Cre lines can use knowledge of the genetic locus before attempting to mate to a floxed target allele that is linked to the Cre line, selecting an alternative unlinked line, or scaling their breeding to assure identification of rare recombinants.”1 Finally, The Jackson Laboratory concluded that "TLA should be considered a “first pass” tool for integration locus discovery."1

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References

[1] Goodwin, L. O., Splinter, E., Davis, T. L., Urban, R., He, H., Braun, R. E., Chesler, E. J., Kumar, V., van Min, M., Ndukum, J., Philip, V. M., Reinholdt, L. G., Svenson, K., White, J. K., Sasner, M., Lutz, C., & Murray, S. A. (2019). Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis. Genome research, 29(3), 494–505. https://doi.org/10.1101/gr.233866.117

[2] Cain-Hom C, Splinter E, van Min M, Simonis M, van de Heijning M, Martinez M, Asghari V, Cox JC, Warming S. Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification. Nucleic Acids Res. 2017 May 5;45(8):e62. doi: 10.1093/nar/gkw1329. PMID: 28053125; PMCID: PMC5416772.

[3] Taconic Biosciences, Inc. Transgene Mapping Analysis by Targeted Locus Amplification Technology. https://www.taconic.com/pdfs/Transgene-Mapping-Analysis-A4.pdf

[4] de Vree, P., de Wit, E., Yilmaz, M. et al. Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol 32, 1019–1025 (2014). https://doi.org/10.1038/nbt.2959

 

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