The integration site of the APP transgene in the J20 mouse model of Alzheimer's disease

Abstract

Background: Transgenic animal models are a widely used and powerful tool to investigate human disease and develop therapeutic interventions. Making a transgenic mouse involves random integration of exogenous DNA into the host genome that can have the effect of disrupting endogenous gene expression. The J20 mouse model of Alzheimer's disease (AD) is a transgenic overexpresser of human APP with familial AD mutations and has been extensively utilised in preclinical studies and our aim was to determine the genomic location of the J20 transgene insertion. Methods: We used a combination of breeding strategy and Targeted Locus Amplification with deep sequencing to identify the insertion site of the J20 transgene array. To assess RNA and protein expression of Zbtb20, we used qRT-PCR and Western Blotting. Results: We demonstrate that the J20 transgene construct has inserted within the genetic locus of endogenous mouse gene Zbtb20 on chromosome 16 in an array , disrupting expression of mRNA from this gene in adult hippocampal tissue. Preliminary data suggests that ZBTB20 protein levels remain unchanged in this tissue, however further study is necessary. We note that the endogenous mouse App gene also lies on chromosome 16, although 42 Mb from the Zbtb20 locus. Conclusions: These data will be useful for future studies utilising this popular model of AD, particularly those investigating gene interactions between the J20 APP transgene and other genes present on Mmu16 in the mouse.

Keywords: APP; Alzheimer’s disease; Amyloid Precursor Protein; J20; Transgenic; Zbtb20; mouse model.

Figure 1.. Breeding scheme and observed ratios of offspring in a two-generation cross between Chmp2b ^-/- and J20 ^+/- animals.

( A) Two-generation breeding scheme to generate Chmp2b ^-/-; J20 ^+/- animals. ( B) Observed ratios of offspring genotype differed significantly from the expected ratio χ2(3, N = 90.21) = 0.58, p < 0.0001.

Figure 2.. Insertion site of the J20 APP transgene (Tg) on mouse chromosome 16.

( A) Representation of Mmu16 showing insertion site of the J20 Tg array. Also shown are the positions of endogenous mouse App and Chmp2b. ( B) TLA applied to the APPSwInd transgene in the J20 mouse. Read mapping to the mouse (mm10) genome assembly shows the exact Tg insertion site (red arrows) with associated genomic deletion (yellow bar). Gene annotation is shown below labelling a small portion of intron 1 of the Zbtb20 gene. ( C) TLA reads mapped to the APP Tg sequence. Complete coverage was achieved, showing that head to tail concatemerization of the Tg has occurred at position 12088 ablating the ampicillin cassette of the plasmid construct. Coloured lines represent SNPs found in the Tg sequence compared to the published sequence. A linearised map of the plasmid construct is shown below.

Figure 3.. Expression of Zbtb20 mRNA and protein in 3 month old wildtype and J20 ^+/- hippocampus.

( A) Zbtb20 mRNA expression is significantly reduced in J20 hippocampus compared to wildtype (WT), detected by quantitative RT-PCR using primers spanning exons 8–9 (Mann Whitney U = 1, n = 5 wildtype/6 J20. P = 0.009). ( B) ZBTB20 protein levels are unchanged in J20 hippocampus compared to wildtype animals (Mann-Whitney U = 10, n = 5 wildtype/4 J20, P = 1). Sample sizes were too low to test for normality so non-parametric testing was conducted. Error bars show standard error of the mean; uncropped blots can be viewed at http://doi.org/10.17605/OSF.IO/4UGZF.