The uninterrupted repeat tract was generated by rolling circle amplification and cell-free cloning as previously described.23 HT1080 cells were cotransfected with LC15-F-CTG800 and plasmid PhiC31o, expressing the phiC31 integrase, in order to obtain single-copy genomic integrations of the expanded repeat.13,24 selleck catalog Following puromycin selection, we obtained more than 15 stably transfected clones and then selected a clone with robust transgene expression, as determined by formation of nuclear foci of CUGexp RNA. Figure 1 Antisense oligonucleotides (ASOs) reduce RNA foci in HT1080 cells. (a) Diagram of pLC15-F construct for expression of expanded-CUG repeats in the DMPK 3�� UTR. The attB site supports genomic integration by PhiC31 integrase. CMV/CBA, CMV enhancer/chicken …

Design of ASOs To examine antisense effects on instability, we used oligonucleotides having a phosphorothioate backbone and locked nucleic acid (LNA) modification, a chemistry that increases hybridization affinity and nuclease resistance.25 LNA ASOs were previously shown to exhibit activity in HT1080 cells when added to the culture media without transfection reagents.22 We used two 18-mer ASOs having the identical (CAG)6 sequence but differing in the distribution of LNA-modified nucleotides. One design was a 4-10-4 gapmer in which a central stretch of 10 DNA monomers is flanked by four LNA nucleotides on either end (Figure 1b). The other design was a mixmer in which eight LNA nucleotides were interspersed throughout the oligonucleotide (Figure 1b).

Both ASOs were designed for strong hybridization to CUGexp RNA (or CTGexp DNA) but only the gapmer is competent to activate RNase H.25 RNase H-active and inactive ASOs have similar capacity to reduce CUGexp transcripts Next, we examined the effects of CAG-repeat ASOs on CUGexp transcripts in HT1080 cells. Previous studies demonstrated that transcripts with expanded-CUG repeats are retained in nuclear foci.26,27 Consistent with these observations, 49% of the stably transfected HT1080 cells showed nuclear foci of CUGexp RNA, a feature never observed in nontransfected cells (Figure 1c,d). One week after addition of ASOs to the culture media (final concentration 1 ��mol/l) the frequency of cells showing nuclear foci was reduced to 6.0% or 6.5% by gapmer or mixmer ASOs, respectively (Figure 1c,d). We performed quantitative real-time reverse transcriptase-PCR Drug_discovery (qRT-PCR) to determine the extent of target knockdown, using an assay that detects the combined output from transgene and endogenous DMPK alleles. The expression of DMPK 3�� UTR in stably transfected cells was 20-fold higher than in nontransfected cells (Figure 2a).