Generation of control and expanded (CCUG)n repeat expression clones

The (CCTG)_DM2 expansion is part of a complex polymorphic motif (Bachinski et al., 2003, 2009) of the form (TG)_12-26(TCTG)_7-12(CCTG)_3-9(G/TCTG)_0-4(CCTG)_4-15. DM2 expansions can be as large as 40 kb with the CCTG motif uninterrupted (Liquori et al., 2001; Bachinski et al., 2003; Sallinen et al., 2004). Reported normal alleles have repeat tract lengths of up to 26 CCTG motifs with one or more interruptions (Bachinski et al., 2009). The smallest reported DM2 expansions associated with clinically detectable manifestations are between 55 and 100 CCTG repeats (Liquori et al., 2001; Lucchiari et al., 2008; Bachinski et al., 2009). Because this complex polymorphic repeat motif has been shown to have an effect on DNA structure (Edwards et al., 2009), we included the (TG)_n(TCTG)_n tracts in the (CCTG)_DM2 constructs. We took advantage of the repeat-primed PCR (RP-PCR) assay developed in our laboratory (R.K.) for the diagnostic detection of the DM2 expansions (Sallinen et al., 2004; Bachinski et al., 2009). Using this approach, we amplified repeats from a clinically affected, genetically confirmed DM2 patient to produce (TG)_n(TCTG)_n(CCTG)_n repeats with 16 to 189 pure (CCTG)_n motifs. Cloned repeats were verified by sequencing to ensure purity of the expanded (CCTG)_n repeat tract. In order to express the (CCTG)_n repeats in Drosophila, mutant fragments containing 106 repeats with the upstream region were recovered from the TOPO vector and cloned into pUAST (Brand and Perrimon, 1993). The same cloning procedure was used with genomic DNA from a normal individual to generate the control vector containing a normal (CCTG)₁₆ allele. The presence and the length of the (CCTG)_n repeats in the pUAST vector were confirmed by sequencing in both directions: DM2-106, (TG)₂₂(TCTG)₂(CCTG)₁₀₆; and N-16, (TG)₂₀(TCTG)₁₂(CCTG)₁₆.