# Antisense-Mediated Down-Regulation of Factor V-Short Splicing in a Liver Cell Line Model
## 3 Results
### Characterisation of the F5 Gene in HepG2 Cells  
Since the HepG2 cell line is known to have multiple chromosomal aberrations [33], we performed a preliminary characterisation of the \(F5\) gene in these cells (Figure 2), in order to ensure the suitability of this model for the intended splicing modulation experiments. Figure 1: **Alternative splicing of the \(F5\) gene. ** Schematic representation of the \(F5\) pre-mRNA region spanning exons 12–14. The large exon 13 contains an internal optional intron (light blue), which is usually retained in the mature mRNA (default full-length FV transcript, top), but occasionally spliced out (alternative FV-short transcript, bottom). The consensus scores for the FV-short-specific donor and acceptor splice sites are reported in brackets and the morpholino antisense oligonucleotides (MAOs) designed to target these sites are shown. EX: exon. The number of copies of the \(F5\) gene in HepG2 cells was assessed by MLPA analysis and found to be normal (Figure 2A). Two reference probes yielded ratios of 1.5 and 2 in this analysis, indicating the presence of 3 and 4 copies of the corresponding control loci on chromosomes 6 and 20, respectively (Figure 2A). This is consistent with reported copy number changes in the HepG2 genome [33] and unlikely to interfere with the \(F5\) gene analysis, as the copy numbers of the \(F5\) gene exons are normalised against the average copy number of all 9 reference probes. Sequencing of the whole \(F5\) exon 13 from genomic DNA of HepG2 cells identified several well-known single nucleotide polymorphisms (SNPs) (Figure 2B), but no novel sequence variations. Importantly, none of the identified variants was located at or close to the FV-short-specific donor or acceptor splice sites (Figure 2C). Accordingly, preliminary experiments indicated that HepG2 cells express both full-length FV and FV-short mRNA, with a vast predominance of the full-length transcript, as in normal hepatocytes. Optimisation of Full-Length FV and FV-Short Transcript Amplification and Identification of a New Alternatively Spliced F5 Transcript  
In view of determining the effect of MAO treatment on the alternative splicing of \(F5\), we developed and optimised an RT-(q)PCR protocol for the simultaneous detection and quantification of the full-length FV and FV-short transcripts, taking into account the extreme differences in the size (\(\sim\)2.1 kb) and relative abundance (>100-fold) of the two transcripts. To this end, we used a \(F5\)-specific primer (instead of random hexamers) for the reverse-transcription of total RNA, which greatly improved the detection of the low-abundance FV-short transcript. Moreover, to eliminate the bias associated with the higher amplification efficiency of the much shorter FV-short amplicon, we employed transcript-specific primers generating PCR products of similar lengths. Finally, to prevent competition for the PCR reagents between templates of vastly different abundance, we amplified the full-length FV and FV-short transcripts in separate PCR reactions, which were run in parallel using the same thermal cycles. Our original PCR design comprised a common forward primer in exon 12 and transcript-specific primers in exon 13 (red primers in Figure 3A). This set-up worked well for FV-short, but the primer pair for the full-length FV transcript yielded not only the expected product of 443 bp, but also a lesser lower-molecular weight band, which appeared specific, as it could not be eliminated by manipulating the amplification conditions (Figure 3B). Purification and sequencing of the unexpected PCR product revealed a novel splicing event between the donor splice site of \(F5\) intron 12 and an alternative acceptor splice site located 123 bp into exon 13 (Figure 3B), leading to the in-frame deletion of the first 123 nucleotides of exon 13. Upon translation, this would predict the loss of 41 amino acids (residues 659-699) between the A2 and B domains of the protein.