br Molecular Therapy br applies
applies to other solid tumors, which would make them very attractive anticancer agents.
MATERIALS AND METHODS
PANC-1, MIA PaCa-2, Capan-2, HPAF-II, Hs766T, A549, HEK293, and HEK293T cell lines were obtained from the Amer-ican Type Culture Collection (ATCC). Luciferase-expressing cells (CP15-Luc) were established by transducing the parental cell line CP15 (derived from CP15 tumors) with a recombinant retrovirus pLHCluc.45 NP-18 cells were established and provided by Dr. Capellà.46 Cells were cultured in DMEM (Gibco-BRL) supple-mented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 mg/mL), and streptomycin (100 mg/mL) (Gibco-BRL), and they were maintained in a humidified Necrosulfonamide of 5% CO2 at 37 C. Interspecies contamination and cell morphology were evalu-ated by microscopic observation. Cell lines obtained from the ATCC were immediately expanded and frozen. Every 2 months cells were plated again from the original batch. Cells were not authenticated by the authors. Interspecies contamination was tested by PCR routinely.
Expression and Reporter Plasmids
miRVec plasmids were obtained from NKI human miRNA library (miRLib) bacteria cells grown in Luria-Bertani broth (LB) containing ampicillin (100 mg/mL). The miRLib library was generated by Voorhoeve et al.47 and commercialized by Source BioScience LifeSciences. psiCHECK-2_ELF4_wt, psiCHECK-2_ELF4_mut1, psiCHECK-2_KLF8_wt, psiCHECK-2_KLF8_mut1, and psiCHECK-2_KLF8_mut2 were generated by cloning the corresponding 30 UTR fragments (genomic DNA: ELF4, 45,233–45654 bp; KLF8, 53,454– 54,679 bp) into psiCHECK-2 vector. The 30 UTR fragments contained XhoI and NcoI restriction sites at their 50 and 30 ends, respectively, and they were purchased as G-Blocks (Integrated DNA Technologies). G-blocks were digested with XhoI and NcoI restriction enzymes (Roche) and introduced into the psiCHECK-2 vector, digested with the same restriction enzymes. Plasmid constructions were tested by colony PCR and validated by Sanger sequencing using the primer set 1 (Table S3).
Adenovirus Generation and Titration
pAdwtE hTR, pAdwtE miR-759, pICOVIR15 miR-99b, and pICOVIR15 miR-485 were generated by an adapted recombineering protocol, based on homologous recombination in bacteria using a positive-negative selection screen with the RpsLNeo cassette.48,49 Primer sets used for the recombination step contain long tails homol-ogous to the insertion point at the adenoviral genome (Table S3). The RpsLNeo cassette was amplified by PCR from the pJetRpsL plasmid (with primer set 2), and it was introduced by homologous recombina-tion downstream of the adenoviral fiber gene at the adenoviral genome (pAdwt). The EGFP gene was then amplified (with primer set 3). The RpsLNeo cassette was replaced with the EGFP amplicon by homologous recombination, generating the adenoviral genome pAdwtE. The RpsLNeo cassette was then amplified by PCR from
the pJetRpsL plasmid (with primer set 4), and it was introduced by homologous recombination 3 bp upstream of the R-ITR in the reverse strand of the corresponding adenoviral genome (pAdwtE or pICOVIR15).
Sequences encoding for hTR, miR-759, miR-99b, or miR-485, as well as the CMV promoter regulating their expression, were ampli-fied from the corresponding miRVec plasmid by PCR (with primer set 5). The RpsLNeo cassette was replaced by each one of the amplicons by homologous recombination. pAdwtE miRLib was generated by replacing the RpsLNeo cassette with a mixture of
243 amplicons amplified from miRVec plasmids by PCR (with primer set 5). Plasmid constructions were verified by PCR (with primer sets 6 and 7), EcoRI enzymatic digestion, and Sanger sequencing. The pAdwtE, pAdwtE hTR, pAdwtE miR-759, pICOVIR15, pICOVIR15 miR-99b, and pICOVIR15 miR-485 plas-mids were transfected into HEK293 cells to obtain a first round of viral particles. Viruses were propagated in A549 cells and purified
by cesium chloride density gradient centrifugation according to standard techniques.50 Adenoviral concentrations were calculated based on optical density (vp/mL) or on viral infectious units (IFU/mL), as previously described.51
AdwtE miRLib Bioselection and Identification of Isolated Clones
pAdwtE miRLib plasmids were transfected in HEK293 cells to obtain a first viral homogenate and titrated based on viral IFU, as previously described.51 PANC-1 and MIA PaCa-2 cells were in-fected with 1 IFU/cell and 5 IFU/cell of the viral homogenate, respectively. When cytopathic effect (CPE) was observed, 10% of the supernatant from PANC-1 and 30%–70% from MIA PaCa-2 were used to infect new cells; 20 rounds of infection were carried out. Individual adenoviral clones were isolated by plaque assay in PANC-1 cells (66 clones) or in A549 cells (45 clones) (for MIA PaCa-2 bioselected viruses).52 Plaque assays from MIA PaCa-2 bio-selected clones was carried out in A549 cells since MIA PaCa-2 cells do not grow well at high confluency, a characteristic that is recom-mended for plaque assay. The largest isolated clones were selected and tested for miRNA identification. Encoded miRNAs were iden-tified by Sanger sequencing using the primer set 7 (Table S3) in the purified viral genomes.