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Published October 23, 2018 | Published + Submitted
Journal Article Open

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes And Neighboring Endogenous Genes

Abstract

Transgenic mouse lines are routinely employed to label and manipulate distinct cell types. The transgene generally comprises cell-type specific regulatory elements linked to a cDNA encoding a reporter or other protein. However, off-target expression seemingly unrelated to the regulatory elements in the transgene is often observed, it is sometimes suspected to reflect influences related to the site of transgene integration in the genome. To test this hypothesis, we used a proximity ligation-based method, Targeted Locus Amplification (TLA), to map the insertion sites of three well-characterized transgenes that appeared to exhibit insertion site-dependent expression in retina. The nearest endogenous genes to transgenes HB9-GFP, Mito-P, and TYW3 are Cdh6, Fat4 and Khdrbs2, respectively. For two lines, we demonstrate that expression reflects that of the closest endogenous gene (Fat4 and Cdh6), even though the distance between transgene and endogenous gene is 550 and 680 kb, respectively. In all three lines, the transgenes decrease 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. These results provide insights into the effects of transgenes and endogenous genes on each other's expression, demonstrate that mapping insertion site is valuable for interpreting results obtained with transgenic lines, and indicate that TLA is a reliable method for integration site discovery.

Additional Information

© 2018 Laboulaye, Duan, Qiao, Whitney and Sanes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Received: 20 July 2018; Accepted: 25 September 2018; Published: 23 October 2018. We thank Karthik Shekhar for statistical help; Max van Min and Judith Bergboer (Cergentis) for assistance in interpreting TLA results and permission to use material from their reports in Figures 3–5; Helen McNeil (Lunenfeld Institute) for providing the Fat4 conditional mutant and Peter Scheiffele. (Biozentrum, Basel) for antibodies to Slm1 and Slm2. Author Contributions: XD, ML, MQ, and IW performed the experiments and analyzed the data. JS conceived the project and analyzed the data. ML and JS wrote the manuscript. All the authors reviewed and edited the manuscript. This work was supported by NIH grants R01 EY022073 and R37 NS029169 to JS and a Klingenstein-Simons Neuroscience Fellowship to XD. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Submitted - 389676.full.pdf

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Created:
September 15, 2023
Modified:
October 23, 2023