Transgenic rodent models produced by traditional pronuclear microinjection is known to occasion random integration. As such, the often unknown site of integration can have important ramifications, such as potentially disrupting an endogenous gene. Moreover, failing to perform in-depth genetic characterization on your transgenic models will only obscure and likely obstruct proper phenotype interpretation, by promoting erroneous genotype-phenotype correlation.^1-4 Besides, knowledge of the exact genomic position(s) of the integrated transgene is also beneficial for breeding and intercrossing. For example, "when planning crosses between animals that carry a conditional allele and a given Cre allele in case the alleles are on the same chromosome."⁵

Cre/lox: a popular modular switch to control site specific recombination events in mammalian genomes

The Cre/lox system is an incredibly powerful technology widely exploited in the field of mouse genetics. The versatility of the approach allows scientists to have a better grip on the location and timing of gene expression.^6-9 In fact, Cre/lox is often used to generate conditional and cell-type specific KO alleles⁸ but can also be leveraged to activate gene expression. Indeed, delivering a loxP-flanked “stop” sequence (transcriptional termination element) between the promoter and transgene coding sequence will halt gene expression. However, cells with expressed Cre will see Cre recombinase remove the “floxed” sequence to express the transgene.⁶

For this reason, Genentech turned to our TLA-based solutions to map transgene insertion in 7 previously published Cre and CreERT2 transgenic lines. After which, they sought to develop genotyping assays for as many of the Cre transgenic lines as possible. Automated genotyping platforms and robust assays are indeed particularly relevant and attractive for large transgenic animal facilities, where a high volume of genotyping assays is performed.¹⁰

Identifying integration sites at a single nucleotide level and detecting unexpected structural variations

Despite limited sequence data available for the transgenes used, all analyzed mouse lines had the Cre sequence in common. Therefore, the same Cre-specific primer sets were used to perform TLA on all the lines. Not only TLA was able to identify the exact transgene integration sites but our unique analytical QC method was also able to pick up unexpected structural rearrangements accompanying those integration events. Results for each of the transgenic lines have been summarized in Table 1 (below).

^an.a.: Not applicable; nearest gene is >1 Mb away from integration site. Reference genome: mouse genome assembly NCBI37/mm9.
Determination of hemizygous transgene copy number was calculated using quantitative PCR data and was not part of the TLA analysis.

Following the identification of those integration sites, the exact transition sequences were then validated by PCR followed by sequencing, using genomic DNA and primers designed to span the identified junction (i.e. transgene-genome breakpoints). Quantitative PCR genotyping assays were done to distinguish wild-type, hemizygous and homozygous alleles in 4 of the lines. Indeed, since 3 of the 7 lines had genomic duplications surrounding the transgene concatemer, this prevented designing genotyping assays as those will return a positive signal from the WT probe/amplicon.

Importance of a robust genetic analytical QC method for transgenic (animal) models

All in all, this study underscores the importance of thorough genetic characterization of transgenic animal models and therefore, serves as a useful reference for the entire (transgenic) mouse community. This paper demonstrates the unmatched capabilities of our TLA-based approach for the complete targeted sequence of any loci of interest as well as the robust detection of all SNVs and structural variants.

Unrivalled genetic insights of TLA-based solutions over standard technologies

Unlike conventional technologies, TLA can reconstruct a complete or near complete picture of the transgene integration events, even with minimal (prior) knowledge of the region of interest.¹¹ Usually, TLA identifies integration sites via:

• The generated coverage that the integrations will result in (i.e. in the position of the genome where the integrations have occurred, via our whole genome coverage plot)
• The detection of the breakpoint sequences (genomic lesion) that the integrations will result in (i.e. the breakpoints between the transgene and the flanking host genome).

Moreover, TLA can reliably detect SNVs within the transgene sequence and can assess if any (larger) structural variations may have accompanied your genetic engineering.

We invite you to watch our most recent webinar recording, where we presented the relevance of our genetic characterization method in the field of genetically engineered (animal) models.

Webinar

Complete and unbiased genetic characterization for the QC of transgenic animal models

Recorded 15 April 2021

References

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

[2] Mukai,H.Y., Motohashi,H., Ohneda,O., Suzuki,N., Nagano,M. and Yamamoto,M. (2006) Transgene insertion in proximity to the C-myb gene disrupts erythroid-megakaryocytic lineage bifurcation. Mol. Cell. Biol., 26, 7953–7965.

[3] Durkin,M.E., Keck-Waggoner,C.L., Popescu,N.C. and Thorgeirsson,S.S. (2001) Integration of a C-myc transgene results in disruption of the mouse Gtf2ird1 gene, the homologue of the human GTF2IRD1 gene hemizygously deleted in williams–beuren syndrome. Genomics, 73, 20–27.

[4] Vogt,T.F., Jackson-Grusby,L., Wynshaw-Boris,A.J., Chan,D.C. and Leder,P. (1992) The same genomic region is disrupted in two transgene-induced limb deformity alleles. Mamm. Genome, 3, 431–437.

[5] 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.

[6] Kelmenson, P. (2011, September 23). Cre lox breeding for beginners, Part 1. The Jackson Laboratory. https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding

[7] Sauer,B. and Henderson,N. (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. U.S.A., 85, 5166–5170.

[8] Gu,H., Marth,J.D., Orban,P.C., Mossmann,H. and Rajewsky,K. (1994) Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science, 265, 103–106.

[9] Feil,R., Wagner,J., Metzger,D. and Chambon,P. (1997) Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun., 237, 752–757.

[10] Cain-Hom,C., Pabalate,R., Pham,A., Patel,H.N., Wiler,R. and Cox,J.C. (2016) Mammalian genotyping using acoustic droplet ejection for enhanced data reproducibility, superior throughput, and minimized cross-contamination. J. Lab. Autom., 21, 37–48.

[11] de Vree,P.J.P., de Wit,E., Yilmaz,M., van de Heijning,M., Klous,P., Verstegen,M.J.A.M., Wan,Y., Teunissen,H., Krijger,P.H.L., Geeven,G. et al. (2014) Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat. Biotechnol., 32, 1019–1025.