Potential molecular consequences of transgene integration: The R6/2 mouse example

Jessie C Jacobsen,Alex Stortchevoi,Michael E Talkowski,Marcy E Macdonald,Carrie Hanscom,Douglas D Barker,Suzanne J Reid,Serkan Erdin,Colby Chiang,James F Gusella,Ian Blumenthal,Russell G Snell,Renee R Handley,A Jennifer Morton,Carl Ernst

doi:10.1038/srep41120

Abstract

Integration of exogenous DNA into a host genome represents an important route to generate animal and cellular models for exploration into human disease and therapeutic development. In most models, little is known concerning structural integrity of the transgene, precise site of integration, or its impact on the host genome. We previously used whole-genome and targeted sequencing approaches to reconstruct transgene structure and integration sites in models of Huntington’s disease, revealing complex structural rearrangements that can result from transgenesis. Here, we demonstrate in the R6/2 mouse, a widely used Huntington’s disease model, that integration of a rearranged transgene with coincident deletion of 5,444 bp of host genome within the gene Gm12695 has striking molecular consequences. Gm12695, the function of which is unknown, is normally expressed at negligible levels in mouse brain, but transgene integration has resulted in cortical expression of a partial fragment (exons 8–11) 3’ to the transgene integration site in R6/2. This transcript shows significant expression among the extensive network of differentially expressed genes associated with this model, including synaptic transmission, cell signalling and transcription. These data illustrate the value of sequence-level resolution of transgene insertions and transcription analysis to inform phenotypic characterization of transgenic models utilized in therapeutic research.

Highlights

Integration of exogenous DNA into a host genome represents an important route to generate animal and cellular models for exploration into human disease and therapeutic development
There were at least two potential explanations for this increase; 1) the integration of the transgene within the noncoding intronic sequence of Gm12695 profoundly disrupts its normal regulation, resulting in altered expression of this gene in these regions of the brain, or 2) the increased expression of Gm12695 is an indirect consequence of the expression of the R6/2 transgene, as a reaction to the pathogenesis/toxicity caused by expression of the polyglutamine fragment. To distinguish between these options, we examined further mouse lines that are descended from the original R6/2 mouse line but show marked differences in their severity of phenotype due to substantial changes in the length of the CAG trinucleotide repeat
We reasoned that if Gm12695 expression was a consequence of pathogenesis/toxicity induced by the expression of the polyglutamine fragment, its expression would vary across each model harbouring different CAG repeat lengths

Summary

Introduction

Integration of exogenous DNA into a host genome represents an important route to generate animal and cellular models for exploration into human disease and therapeutic development. Using a series of targeted and whole-genome sequencing approaches, we previously revealed significant structural rearrangement of the transgene in R6/2 genomic DNA at nucleotide resolution, including excision/insertion and inversion events that left a single copy of the 5′region and exon 1 CAG expansion region downstream of a 168 bp insertion of bacterial DNA and of intron 1 sequence, along with additional rearrangement of the upstream and intron 1 sequences (Fig. 1, see Chiang et al.[2] for complete details, GenBank: KF990992.1) This structure suggests the R6/2 transgene integration event involved fragments derived from at least three copies of the original injected HTT fragment, as originally speculated by the authors who created the model[3] and suggested by previous analyses[4]. We further these studies by assessing the transcriptional impact of the transgenic integration in the R6/2 mouse

Methods

Results

Conclusion