Manuscript (Sam Et Al)

Supplementary data

Construction of the hFIX-expressing plasmids

A 320 bp fragment from the hFIX-cDNA 5'-end was PCR-amplified, using oligonucleotides hKozF9-F (carrying the Kozakelement, GCCACC) and hFIXE4-R as forward and reverse primers, respectively (Table 1). To facilitate thecloning steps, BamHI and DraI restriction sites were introduced at the 5′-ends ofthe forward and reverseprimers, respectively. The PCR was carried out based on the following temperature profile in 30 cycles: 30seconds at 94oC for denaturation, 1 minute at 54oC for annealing and 1 minute at 72oC for extension, followed by a final extension step at 72oC for 5 minutes.

After cloning into a pTZ57R/T vector (Fermentas), the 320 bp fragment from the hFIX-cDNA 5'-end was used to substitute its corresponding fragment in the plasmid pET26-hFIX [11]between the BamHI and DraI sites. Subsequently, the Kozak-containing hFIX-cDNA was subcloned between the BamHI and NotI sites in plasmid pcDNA3.1 (Invitrogen). The resulting plasmid was named pCMV.FIX (Fig. 1). To generate liver-specific hFIX expressing plasmids, the amplified hAAT-derived promoter was sub-cloned between the NruI and HindIII sites in the plasmids pCMV.FIX and pCMV.FIX-II (Haddad-Mashadrizeh et al. 2009)in the places of the CMV enhancer/promoter, resulting in the plasmids pAAT.FIX and pAAT.FIX-II (Fig. 1).

To introduce the liver-specific enhancer (rABE) upstream the hAAT and CMVpromoters, the PCR-amplifiedrABE (mentioned above) was inserted between the BglII and MunI sites in the plasmids, pCMV.FIXand pAAT.FIX, thus leading to the constructs prABE/CMV.FIX and prABE/AAT.FIX, respectively (Fig. 1). In the next step, the rABE/CMV and rABE/AAT fragments from the prABE/CMV.FIX and prABE/AAT.FIX plasmids were isolated and used to substitute the CMV and hAAT promoters between the BglII and HindIII sites in the plasmids pCMV.FIX-II and pAAT.FIX-II, resulting in constructs prABE/CMV.FIX-II and prABE/AAT.FIX-II, respectively (Fig. 1).

Verification of the plasmids constructed during the cloning and subcloning steps were carried out by restriction digestion followed by nucleotide sequence analysis of both strands of the inserted fragments, using the ABI 373A automated sequencer (MWG Biotech). Comparison of the obtained sequences against the GenBankdatabase was performed using the BLAST program.