Gonitel et al. 10.1073/pnas.0800048105. |
Fig. 6. Progression of regional CAG repeat instability is R6/2 and HdhQ150 mouse models. In the R6/1 and R6/2 transgenic mouse models the same human genomic fragment containing exon 1 of the HD gene has integrated into different locations in the mouse genome. The HdhQ150 knockin model was generated by inserting a repeat of 150 CAGs into the endogenous mouse Hdh gene. Therefore, by comparing these three models, the behavior of the CAG repeat has been examined at three different genomic locations. (A) Each row is a compilation of traces representing tissues taken from the same R6/2 mouse at 14 weeks of age (end-stage disease). The three regions exhibit differing levels of instability: the emergence of the radical component of CAG repeat distribution can be seen in the striatum and is consistent among the three mice. In contrast, the brainstem and the cortex show little if any instability. (B) Again, each row is a compilation of traces representing tissues taken from the same HdhQ150 knockin mouse at 3, 6, and 9 months of age. Similar to the R6/1 model, mosaicism can already be observed by 3 months and progressively develops toward expansion with age. The differing levels of instability exhibited by the three regions are comparable to those that develop in R6/1. Generally, the overall shape of the distribution of the repeats is consistent between mice for the same brain region with the variance of the radical component increasing with age.
Fig. 7. Reproducibility of CAG repeat profiles generated by independent PCRs of the same neuronal DNA sample. The traces in each column visualize products obtained by performing multiple PCRs on DNA pools of striatal neuronal cells dissected from R6/1 mice. The profiles of the CAG distribution have little variation among the reactions. This indicates that in each case the amount of DNA that goes into each reaction is sufficient to be representative of the total neuronal population of the CAG alleles in the striatum.
Fig. 8. MSH3 is present in neuronal cells in the human and mouse striatum. Sections of R6/1 and WT mice aged 9 months and from HD and control human striatum were immunoprobed with antibodies to NeuN to identify neurons and to MSH3. All nuclei were visualized by using TO-PRO-3. MSH3 is predominantly found in neurons in both the mouse and human striata. Arrows, neuronal cells; arrowheads, nonneuronal cells.
Fig. 9. Sensitivity of the CAG repeat size detection. (A) Detection of the SP-PCR products by Southern blot. DNA from the striatum of an R6/1 mouse aged 9 months was used at two concentrations: 20 pg per PCR in lanes 1-16 and 40 pg in lanes 17-26. Most bands represent products from single molecules. (B) The same PCR products as in lanes 1-16 were run on the ABI377. The peaks detected by GeneScan correspond perfectly to the bands on the Southern blot (see SI Methods).
Fig. 10. CAG repeat profiles generated by bulk PCR and SP-PCR are comparable. Bulk PCR (100 ng of DNA) and SP-PCR (6 pg of DNA per reaction; number of products = 108) performed on striatal DNA from an R6/1 mouse aged 9 months produce similar CAG repeat size distributions.
SI Methods
DNA Extraction, RNA Extraction, and CAG PCR Amplification.
Amplification of the CAG repeat from R6/1 and R6/2 mouse DNA and human DNA was performed with a FAM-labeled forward primer (GAGTCCCTCAAGTCCTTCCAGCA) and reverse primer (GCCCAAACTCACGGTCGGT) in 20-ml reactions containing 0.2 mM dNTPs, 10% DMSO, AM buffer (67 mM Tris·HCl, pH 8.8/16.6 mM (NH4)SO4/2 mM MgCl2/0.17 mg/ml BSA) and 1 unit of AmpliTaq DNA polymerase (Applied Biosystems). Cycling conditions were as follows: 4 min at 94°C 13 times, 30 s at 94°C; 30 s at 71°C to 65°C in increments of -0.5°C per cycle; 30 s at 65°C 22 times, and 30 s at 94°C; 30 s at 65°C; 30 s at 65°C for 10 min at 65°C. HdhQ150 DNA was amplified in 20 ml containing 0.1 mM dNTPs, 2 M Betaine (Sigma), Detloff buffer [15 mM Tris·HCl, pH 8.8/15 mM Tris·HCl, pH 9.0/16 mM (NH4)2SO4/2.5 mM MgCl2/0.15 mg/ml BSA/0.007% 2-mercaptoethanol], 10 ng/ml forward primer 40224FD (CCCATTCATTGCCTTGCTG), 10 ng/ml reverse primer 40225 (GCGGCTGAGGGGGTTGA), and 0.5 units of AmpliTaq DNA polymerase (Applied Biosystems). Cycling conditions were 5 min at 94°C, 29 times (30 s at 94°C, 30 s at 53°C, 3 min at 72°C), 5 min at 72°C.Repeat Sizing by ABI 377.
PCR amplification conditions were optimized to detect both small-pool and conventional PCR using the ABI377 sequencer (SI Fig. 9). A total of 2 ml of each FAM-tagged PCR product together with GeneScan2500 ROX internal size standard were denatured at 94°C for 5 min with 2 ml of HiDi formamide (Applied Biosystems). Denatured products were loaded onto a 4.25% acrylamide gel (AccuGel; National Diagnostics; 7 M urea; 12 cm) and run on ABI Prism 377 in TBE buffer for 1.5 h. The ABI Prism 377XL collection software operated the run module "GS run 12A-1200" with the following parameters: 3,000 V, 50 mA, 150 W, oven temperature 51°C. Data from the ABI377 were analyzed by using GeneScan 3.1.2 (Applied Biosystems) software.Southern Blotting.
SP-PCR products were run on 27-cm 1.5% MetaPhor agarose (FMC Bioproducts) gels overnight. Southern blot hybridization was according to the method of Church and Gilbert (1). The probe was an XcmI-BstEII fragment containing 18 CAG repeats derived from a human promoter/ exon 1 HD construct (GenBank accession no. L34020) (SI Fig. 9).1. Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991-1995.