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

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.


Changed t-tests to non-parametric tests (Mann-Whitney U) in
for qPCR and western blot experiments as normal distribution of data could not be tested or assumed due to sample sizes being too low.
3. Changed text in results and discussion to agree with reviewers that protein data is only preliminary and future studies should test for ZBTB20 protein changes in multiple tissue at multiple timepoints with negative control samples.

Introduction
The Tg(PDGFB-APPSwInd)20Lms (MGI:3057148, here referred to as 'J20') mouse model is a transgenic animal that overexpresses mutant human APP protein (amyloid precursor protein), and is widely used as a model of amyloid deposition and pathogenesis in the study of Alzheimer's disease (AD). J20 mice recapitulate many AD-like phenotypes, including synaptic loss, amyloid plaque deposition and cognitive impairment (Hong et al., 2016;Mucke et al., 2000;Palop & Mucke, 2016).
The model was developed by Mucke and colleagues using the PDGF-APPSw,Ind transgene construct described previously (Games et al., 1995;Rockenstein et al., 1995), which includes a human APP mini-gene, carrying the familial AD-linked 717 V-F (Indiana) mutation (Murrell et al., 1991) and 670/671 KM-NL (Swedish) double mutation (Mullan et al., 1992). The transgene construct was designed so that the APP mini-gene included genomic sequence for APP introns 6-8, allowing expression of hAPP695, hAPP751 and hAPP770 isoforms. The PDGF-APPSw,Ind transgene expression is driven in neurons throughout the brain by the human platelet-derived growth factor β chain (PDGFβ) promoter (Harris et al., 2010;Sasahara et al., 1991). The J20 mouse is an important model: currently, 125 articles have been catalogued in the Mouse Genome Database bibliography (Blake et al., 2017), which report genotypic and/or phenotypic data from this mouse. This strain has been used for several classical genetic studies to determine the interaction of genes of interest with the APP transgene including a seminal report of the importance of Tau to Aß-associated neuronal dysfunction (Roberson et al., 2007). Moreover, this model has been used to elucidate the role of Aß in synaptic dysfunction (Palop & Mucke, 2016;Palop et al., 2007;Sanchez et al., 2012).
Transgenic mice are conventionally generated by direct injection of linear foreign DNA into the pronucleus of fertilised zygotes. Once inside the cell, these linear fragments undergo circularisation and concatemer formation before integrating into the host genome as a tandem array (Bishop & Smith, 1989). In principle, transgenes insert randomly into the host genome; however, ∼45% of integration sites lie within host gene regions (∼13.2 exonic, 31.6% intronic), potentially as a result of increased accessibility of transcriptionally active DNA (Yan et al., 2013). Integration of a transgene array into coding sequences can induce new mutations (for example, haploinsufficiency) (Haruyama et al., 2009), and so it is important to know the site of integration for a transgene array in a mouse model.
A recent study suggested an association of a heterozygous deletion of the CHMP2B gene (charged multivesicular body protein 2B) with early-onset Alzheimer's disease (Hooli et al., 2014). Interestingly, mutations in CHMP2B are a rare genetic cause of Frontotemporal dementia (Skibinski et al., 2005). We have previously reported generation of a Chmp2b knockout mouse (Ghazi-Noori et al., 2012). To determine if Chmp2b deletion modulates APP/Aß biology in vivo, we attempted to cross our Chmp2b knockout with the J20 mouse, to study potential double mutant progeny. The Chmp2b locus lies on mouse chromosome 16.
Here, we present the outcome of these genetic cross experiments and the resulting mapping of the J20 transgene array integration site by Targeted Locus Amplification (TLA) with deep sequencing. We discuss how integration of the transgene affects expression of the flanking loci.

Animal welfare
Mice were housed in controlled conditions in accordance with guidelines from the UK Medical Research Council in Responsibility in Use of Animals for Medical Research (1993). Two female J20 positive animals were killed at 6 months of age to provide splenic material for the TLA study. Furthermore, 3 month hippocampal tissue was collected from J20 animals: N=5, 3 male, 2 female. C57BL/6J controls: N=5, 3 male, 2 female. All used for qRT-PCR and western blotting. All experiments were conducted under license from the UK Home Office and with Local Ethical Review approval. Tg(PDGFB-APPSwInd)20Lms/ 2Mmjax animals (J20) were obtained from The Jackson laboratory (stock no. 034836) and maintained on a C57BL/6J background in our animal facility. Chmp2b knockout animals were already available in our animal facility (Ghazi-Noori et al., 2012). Mice had access to a mouse house with bedding material and wood chips. All animals had continual access to water and RM1 (Special Diet Services) (stock animals) or RM3 (Special Diet Services) (breeding animals) chow ad libitum. Mice were housed in individually ventilated cages in a specific pathogen free facility with a 12 hour light/dark cycle.
Genotyping of J20 and Chmp2b knockout mice DNA was extracted from tail tip or ear biopsy by the HOTSHOT method (Truett et al., 2000).
Extraction of spleen cells from J20 animals Mice were sacrificed by rising concentration of CO 2 and confirmed by dislocation of the neck and the spleen dissected and kept on ice. Splenocytes were then dissociated through a 40µm mesh filter and the cells collected by centrifugation at 4°C at 500 × g for 5 minutes. The supernatant was discarded and the pellet re-suspended in 1 ml red blood cell lysis buffer (4.13g NH 4 Cl, 0.5g KHCO 3 , 193.5µl 0.5M EDTA dissolved in 500ml H 2 O) for three minutes at room temperature to lyse splenic erythrocytes. To terminate the lysis reaction, 0.5ml phosphate buffered saline (PBS) was added and the splenocytes were collected again by centrifugation at 500 × g for 5 minutes. The supernatant was discarded and the pellet re-suspended in 0.5ml PBS before a final centrifugation step for 2 minutes. The supernatant was discarded and the pellet was re-suspended in 1ml freezing buffer (PBS with 10% fetal calf serum and 10% dimethyl sulphoxide). Samples were stored at -80°C before preparation for TLA processing.

Targeted Locus Amplification
Processing of samples for TLA was performed by Cergentis B.V. (Utrecht, The Netherlands), as previously described (de Vree et al., 2014). A primer pair targeted to the APP transgene sequence was used to perform the TLA. Sequences of the PCR primers are (5' to 3'): 1917_APP_F GAAACTCATCT-TCACTGGCA; 1698_APP_R GGGTAGACTTCTTGGCAATA. PCR products were purified and library prepped using the Illumina NexteraXT protocol and sequenced on an Illumina Miniseq sequencer.
Sequence alignment and analysis of TLA TLA reads were mapped using BWA-SW, which is a Smith-Waterman alignment tool. This allows for partial mapping, which is optimally suited for identifying break-spanning reads. The mouse Mm10 genome assembly version was used for mapping. Visualisation and interpretation of the data were performed using the Integrative Genomics Viewer (IGV) from the Broad Institute (Robinson et al., 2011).
RNA extraction and quantitative reverse transcription PCR RNA was extracted from whole hippocampus from J20 animals and age and litter matched controls. Total RNA was extracted using the Qiagen miRNeasy kit and reverse transcribed using the Applied Biosystems High-Capacity RNA-to-cDNA™ Kit.
Quantitative RT-PCR was carried out on the Zbtb20 gene transcript using a predesigned PrimeTime ® probe-based qPCR assay (assay ID: Mm.PT.58.41805451, Integrated DNA Technologies [IDT]) targeted to exons 8-9 (RefSeq transcript NM_181058) with a FAM probe. TaqMan reactions were run with Taqman Universal Master Mix 2 on a 7500 Fast machine (Applied Biosystems) using standard cycling conditions. Transcript levels were normalised against Applied Biosystems mouse Actb (Assay ID: 4352933E) and Integrated DNA Technologies B2m (assay ID: Mm.PT.39a.22214835) endogenous controls in independent experiments and the results averaged geometrically. Both controls contained VIC probes.
Tissue preparation and western blotting for ZBTB20 For analysis of ZBTB20 in hippocampus, J20 and age/sexmatched wildtype littermate controls were dissected under icecold PBS before homogenisation in radioimmunoprecipitation buffer (150mM sodium chloride, 50mM Tris, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate) with Protease Inhibitor Cocktail Set 1 (Merck). Total protein concentration was determined using Bradford assay (Bio-Rad). Samples from individual animals were run separately and not pooled.
Equal amounts of hippocampal brain proteins were denatured in LDS sample buffer (ThermoFisher) and β-Mercaptoethanol for 10 minutes at 100°C, prior to separation by SDS polyacrylamide gel electrophoresis in 4-12% pre-cast gels (ThermoFisher). Separated proteins were transferred to 0.2µm nitrocellulose membrane and blocked in 5% milk/phosphate buffered saline (with 0.05% Tween 20, PBST) for one hour at room temperature. The membrane was then cut horizontally at the 49KDa band (SeeBlue Plus II protein ladder, Invitrogen) and the lower half was incubated in mouse monoclonal antibody to β-Actin (A5441, Sigma-Aldrich) diluted 1:200,000 in 1% bovine serum albumin (BSA)/PBST overnight at 4°C. The upper half was incubated overnight with rabbit polyclonal primary antibody against ZBTB20 (23987-1-AP, ProteinTech) diluted 1:1000 in 1% BSA/PBST. After washing with PBST the upper and lower membranes were incubated with HRP-conjugated secondary rabbit and mouse antibodies, respectively, diluted in 1% BSA/ PBST for 1 hour at room temperature. SuperSignal™ West Pico Chemiluminescent Substrate and X-ray film was used to visualise bands, Image J 1.49c software (NIH) was used to analyse band intensity. Graphpad prism 5 (Graphpad Software, Inc.) was used to plot graphs and SPSS 25 (IBMCorp) was used to perform statistical analyses.
The second stage cross resulted in 76 progeny, but the observed ratio of genotypes significantly differed from the expected ratio ( Figure 1b). Strikingly no Chmp2b -/-;TgAPP J20/progeny were produced. This suggests that either Chmp2b -/-;TgAPP J20 may not be viable or the TgAPP allele might be on the same chromosome as Chmp2b -mouse chromosome 16 (Mmu16), preventing typical Mendelian segregation.

Targeted locus Amplification analysis of J20 transgene insertion
To determine the cause of the absence of Chmp2b -/-;TgAPP J20 offspring, we mapped the site of the J20 TgAPP transgene insertion, and so sequenced flanking regions around the insertion site by TLA. Sequence analysis (Figure 2a) showed that the TgAPP insertion site lies on Mmu16. Targeted reads mapped the integration breakpoints to genomic co-ordinates Mmu16: 43,127,050 (3' end of the transgene array) and Mmu16: 43,127,512 (5' end of the transgene array). In addition, sequencing around the insertion site revealed a 41.17kb deletion in the mouse genome between chr16:43,085,979 and chr16:43,127,149 ( Figure 2b). Thus the lack of Chmp2b -/-;TgAPP J20 offspring is the result of the insertion of TgAPP J20 on Mmu16, explaining the absence of Chmp2b -/-;TgAPP J20 progeny from the stage 2 cross.
TLA also allowed us to assess transgene sequence integrity. We found three SNPs (hAPP transgene sequence: 624 G > A, 979 G > A, 10649 G > A) and four indels (TG: 185 G > +1T, TG: 1168 G > +8GGCGGGAC, TG: 1423 C > +1G, TG: 5932 C > -1T) within the integrated transgene construct; however, all were silent mutations or found within intronic sequence. Furthermore, TLA analysis showed at least one transgene copy is truncated at the 3' end (TG: 12088), ablating the ampicillin cassette, and is fused to the 5' of another transgene copy to form a concatemer (TG:12088 fused to TG:3).

Assessment of ZBTB20 expression
The integration site co-ordinates localize the J20 transgene insertion and deletion entirely within intron 1 of the gene Zinc-finger and BTB domain containing 20 (Zbtb20, transcript NM_001285805.1) on Mmu16. Zbtb20 is a member of the BTB/POZ family of transcriptional repressors and functions primarily as a transcriptional repressor (Xie et al., 2008); it is important for hippocampal development and function, the site of greatest Aβ deposition in aging J20 animals (Mucke et al., 2000). Moreover, missense mutations in this gene are associated with Primrose syndrome, a cause of intellectual disability with autism (Cordeddu et al., 2014;Mattioli et al., 2016). Additionally, haploinsufficiency of the gene has been suggested as an important factor in del3q13.31 syndrome, a cause of developmental delay and intellectual disability (Rasmussen et al., 2014). To determine whether transgene insertion has affected Zbtb20 transcription in J20 animals in hippocampal tissue, we investigated mRNA and protein levels of ZBTB20 in wildtype and J20 animals.
Firstly, a predesigned RT-qPCR assay was chosen from IDT to overlap the exon 8-9 boundary within the protein coding region of the Zbtb20 transcript, downstream of the transgene insertion site. Importantly this junction is present in all predicted RefSeq protein coding transcript isoforms. We detected significantly less transcript in J20 animals compared to wildtype using this assay (Figure 3a). To determine whether reduction in Zbtb20 transcript results in a reduction of ZBTB20 protein in J20 animals, we assayed total hippocampal protein by western blot for ZBTB20 with an affinity purified polyclonal antibody, and observed that no change in ZBTB20 protein level in adult J20 hippocampal tissue could be determined by this method. We aimed to validate this result with another antibody (ab48889, Abcam) however, we were unable to determine a specific band at the correct molecular weight (data not shown). Because of the postnatal lethality demonstrated in Zbtb20 -/mice (Sutherland et al., 2009) and the complexity in producing control adult Zbtb20 -/hippocampal tissue, we were not able to validate this result using an appropriate negative control.

Discussion
Using TLA we have located the insertion site of the J20 APP transgene on Mmu16 within intron 1 of the Zbtb20 gene. We have also found a 41kb deletion of intronic sequence flanking the insertion site. Due to the key role of Zbtb20 in hippocampal cell differentiation and function, we determined its expression in hippocampal tissue, and found that while Zbtb20 transcript expression is reduced in J20 hippocampus, we could find no evidence that protein expression is altered, although we note the current technical limitations of our study because of the lack of an appropriate negative control. In a similar study investigating the transgene insertion site of the R6/2 Huntingdon's disease model mouse, Jacobsen et al. discovered that the HTT exon 1 transgene inserted within intron 7 of the Gm12695 gene, causing an almost 30 fold increase of expression of this gene compared to wildtype in cortical tissue irrespective of CAG-repeat length polymorphisms, showing that the genomic aberration caused by foreign DNA insertion within intronic sequence can alter gene regulation (Jacobsen et al., 2017). Notably, disruption of the expression of nearby genes may also occur in gene-targeted systems. For example, two of the four lines of mice that are null for the Prion protein gene Prnp, exhibited late onset ataxia and neurodegeneration --this was subsequently found to be the result of aberrant upregulation of a gene (Prnd) downstream of Prnp, which occurred as a result of induced exon skipping (Moore et al., 1999). In the J20 model, hippocampal Zbtb20 transcription is perturbed without concomitant protein reduction. This is perhaps not as surprising as it seems; recent data indicate mRNA levels explain only around 40% of variability in protein levels (Wilhelm et al., 2014), with protein abundance being primarily dependent on translational control (Schwanhäusser et al., 2011;Schwanhäusser et al., 2013).
It may be important to assess expression in multiple neuronal cell types and other tissues throughout development in the J20 model, considering that ectopic expression of Zbtb20 in the subiculum and post-subiculum results in aberrant CA1 type development in those regions with associated CA1-specific markers (Nielsen et al., 2010) and the gene has been shown to have a role in liver function (Sutherland et al., 2009). The transgene sequence itself is intact in the J20, excluding several single nucleotide mutations; however, these are either silent or found within the transgene construct's three intronic regions and are unlikely to affect the J20 transgene. TLA analysis and in-house copy number qPCR (data not shown) indicate that this transgene has integrated multiple times in an array on Mmu16.
Multiple transgene insertion may be liable to recombination events, causing loss of some copies of the transgene, resulting in delayed onset of phenotype in affected animals. This potential confound can be allayed by undertaking a genomic copy-number qPCR to confirm copy-number consistency between individuals. The Jackson Laboratory have published their assay for general use.
Genetic engineering can result in unintended disruption of the genome, which can confound interpretation of phenotype if the genomic alterations cause changes in protein expression. Transgenic models have been and continue to be powerful tools for biomedical research, but knowledge of the insertion site and the local effects on gene expression will inform phenotyping studies.

Grant information
The western blot results provided in the paper do not prove that the "ZBTB20 protein is unaltered in adult J20 hippocampal tissue". Knock-out controls and molecular size markers are missing to prove that the showed band is not an unspecific signal. Moreover, the lack of appropriate controls (i.e. different quantity of protein extract) do not allow to validate the use of Figure 2B as a quantitative Western Blot. See why in the publications 1 to 3 Finally, as recommended in PLOS ONE for example ( ), the author should provide: http://journals.plos.org/plosone/s/submission-guidelines Original uncropped and unadjusted blots and gels, including molecular size markers, should be provided in either the figures or the supplementary files.
The image should include all relevant controls, and controls should be run on the same blot or gel as the samples.
All blots and gels that support results reported in the manuscript should be provided. Please provide ZBTB20 additional antibody results (which are data not shown in the paper) as additional files. Add reference of this additional antibody in material and methods.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly No competing interests were disclosed. Competing Interests: Referee Expertise: Neuroscience I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.