In genetic research, transgenic mice are often generated for 3 reasons¹:

1. To scrutinize regulatory sequences that regulate the location and timing of gene expressions
2. To investigate the roles of a gene product (by expressing it), or a protein that obstructs it
3. To express reporters in specific cell types

However, off-target expression (unrelated to the regulatory elements in the transgene) is often detected. To evaluate whether those unpredictable expression patterns are influenced by the site of (transgene) insertion, Harvard researchers turned to our proprietary TLA-based QC solutions.² By leveraging our unique genetic QC method, they sought to identify the exact integration sites of 3 well-characterized transgenes (i.e. HB9-GFP, Mito-P, and TYW3) that appeared to exhibit insertion site-dependent expression in retina.¹

A reliable method for integration site discovery

In all 3 analyzed transgenic lines, TLA was able to precisely map the genomic position of transgene integration sites. More specifically, our TLA-based data revealed insertion of:

• HB9-GFP transgene near the Cdh6 locus on mouse chromosome 15
• Mito-P transgene near Fat4 on chromosome 3
• TYW3 transgene near Khdrbs2 on chromosome 1

Despite a 550 and 680 kb distance separating the transgene and endogenous gene in 2 of the analyzed lines, the closest endogenous gene (i.e. Fat4 and Cdh6, respectively) appeared to still exert strong influence over (transgene) expression.

In all cases, the transgenes decreased the expression of the neighboring endogenous genes. In each case, the affected endogenous gene was expressed in at least some of the cell types that the transgenic line has been used to mark and study.

Taken together, this study provides new insights into insertion site-dependent transgene expression. The authors conclude by underlining the fact that these results further substantiate the usefulness of insertion site identification for "gene discovery as well as assessing effects of transgene insertions that would otherwise go undetected".¹

Mapping transgene integration sites should become standard practice in transgenic studies

Thanks to improved and more reliable methods to identify insertion site^2-5, there is growing evidence that interactions between transgenes and endogenous genes are frequent.^6-14 For this reason, authors suggest making transgene mapping a more common practice to better understand and interpret transgene expression patterns.

In conclusion, this study clearly underscores the value of mapping transgene integration sites, and the authors even elaborate on the threefold benefits:

1. Upon identifying neighboring genes, their expression can be assayed to determine whether a transgenic line is hypomorph
2. If the endogenous gene is expressed in cells marked by the transgene, it becomes a candidate effector of that cell’s development or function
3. Once the insertion site has been identified, it is possible to develop reliable genotyping strategies (i.e. design line-specific primers) to determine zygosity without having recourse to relative fluorescent RT-PCR intensities^3,5

Webinar

Click below to watch the recording of our latest webinar to learn more about our genetic QC solutions for genetically engineered (animal) models.

References

[1] Laboulaye, M. A., Duan, X., Qiao, M., Whitney, I. E., & Sanes, J. R. (2018). Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes. Frontiers in molecular neuroscience, 11, 385. https://doi.org/10.3389/fnmol.2018.00385

[2] 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

[3] 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.

[4] 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

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

[6] Cohen-Tannoudji, M., Babinet, C., and Wassef, M. (1994). Early determination of a mouse somatosensory cortex marker. Nature 368, 460–463. doi: 10.1038/368460a0

[7] Young, P., Qiu, L., Wang, D., Zhao, S., Gross, J., and Feng, G. (2008). Single-neuron labeling with inducible cre-mediated knockout in transgenic mice. Nat. Neurosci. 11, 721–728. doi: 10.1038/nn.2118

[8] Huberman, A. D., Wei, W., Elstrott, J., Stafford, B. K., Feller, M. B., and Barres, B. A. (2009). Genetic identification of an On-Off direction-selective retinal ganglion cell subtype reveals a layer-specific subcortical map of posterior motion. Neuron 62, 327–334. doi: 10.1016/j.neuron.2009.04.014

[9] Haverkamp, S., Inta, D., Monyer, H., and Wässle, H. (2009). Expression analysis of green fluorescent protein in retinal neurons of four transgenic mouse lines. Neuroscience 160, 126–139. doi: 10.1016/j.neuroscience.2009.01.081

[10] Trenholm, S., Johnson, K., Li, X., Smith, R. G., and Awatramani, G. B. (2011). Parallel mechanisms encode direction in the retina. Neuron 71, 683–694.doi: 10.1016/j.neuron.2011.06.020

[11] Kay, J. N., Voinescu, P. E., Chu, M. W., and Sanes, J. R. (2011b). Neurod6 expression defines novel retinal amacrine cell subtypes and regulates their fate. Nat. Neurosci. 14, 965–972. doi: 10.1038/nn.2859

[12] Dhande, O. S., Estevez, M. E., Quattrochi, L. E., El-Danaf, R. N., Nguyen, P. L., Berson, D. M., et al. (2013). Genetic dissection of retinal inputs to brainstem nuclei controlling image stabilization. J. Neurosci. 33, 17797–17813. doi: 10.1523/JNEUROSCI.2778-13.2013

[13] Krishnaswamy, A., Yamagata, M., Duan, X., Hong, Y. K., and Sanes, J. R. (2015). Sidekick 2 directs formation of a retinal pathway that detects differential motion. Nature 524, 466–470. doi: 10.1038/nature14682

[14] Peng, Y. R., Tran, N. M., Krishnaswamy, A., Kostadinov, D., Martersteck, E. M., and Sanes, J. R. (2017). Satb1 regulates contactin 5 to pattern dendrites of a mammalian retinal ganglion cell. Neuron 95, 869–883. doi: 10.1016/j.neuron.2017.07.019