Cells were washed at the end of each time point and stained for cell surface HLA-A2 expression and then analyzed by flow cytometry. Construction of RNA expression vector and in vitro transcription of mRNA An RNA expression vector was constructed on the backbone of the PGEM 5Z(+) vector (Promega, Southampton, UK). A 76 nucleotide poly-A sequence was cloned into the vector between
the Sph1 and Apa1 sites and a sequence containing a new multiple cloning cassette and the 3′ untranslated region Selleck NVP-HSP990 of the human α-globin gene, to increase the intracellular stability of mRNA transcripts [22], was inserted between the Sac1 and Sph1 sites. Subsequently, the cDNA Selleck AZD9291 sequences encoding the open reading frames of either GPC-3 or enhanced green fluorescent protein (eGFP) were inserted between Nhe1 and Age1 sites in the new cloning cassette, downstream of the SP6 promoter site of PGEM5Z and upstream of the mRNA stabilizing sequences (Figure 1a). The 1.74 kb GPC-3 open reading frame sequence was generated by PCR from reverse transcribed RNA extracted from HepG2 cells using primers 5′-CGAGCTAGCATGGGCCGGGACCGTG and 5′-AGGACCGGTGTGCACCAGGAAGAAGAAGC, which incorporated restriction sites for Nhe1 and Age1, respectively. The vector was sequenced to confirm authenticity. The vector
was linearized using a SnaB1 restriction site, which is immediately downstream of the poly A sequence, and the resulting linear DNA was isolated by gel extraction. Following the manufacturers instructions, the linear DNA (1 μg) served as template in an SP6 mMessage Machine reaction (Ambion, Huntingdon, UK). After 3 hours at 37°C the capped mRNA was extracted and purified using RNAeasy columns (Qiagen, Crawley, UK). Transcripts Ureohydrolase were then analyzed and quantified by denaturing agarose gel electrophoresis before use. Dendritic cell culture and mRNA transfection Fresh heparinised, peripheral blood samples were obtained from HLA-A2 positive, normal subjects, according to a protocol approved by The Kings College Hospital Ethical
Committee (LREC Protocol number 01/248). Informed, written consent was obtained and the study was performed according to the principles of World Medical Association Declaration of Helsinki. DC were derived from PBMC essentially as described by Romani et al [23]. Adherent cells (7 × 106 per well of 6-well plates; Nunc, UK) were cultured at 37°C for 7 days in X-Vivo, 1% autologous plasma, and 800 u/ml GM-CSF plus 500 u/ml interleukin-4 (IL-4) (both from R&D Systems, Abingdon, UK) with cytokine replenishment after 3 days. Immature DC were AR-13324 in vitro transfected with mRNA by electroporation in 400 μL of X-Vivo with no supplements in a 4 mm cuvette using the Easy-ject plus system (Equibio, Ashford, UK) at 300 V and 150 μF and a pulse time of 4 ms.