The P8 gene codon that was optimized for expression in mammalian cells was synthesized by Cosmogenetech, Inc. The p8 DNA fragment (236 bp) was digested with EcoRI/NotI and cloned into the pCI-neo vector via the EcoRI/NotI site (Promega, Madison, WI) (Table 1). The construct was then transformed into E. coli DH5α for amplification. All restriction enzymes were purchased from New England BioLabs (Ipswich, MA).
Colorectal cancer (DLD-1) cells were transfected with plasmid DNA (pCI-neo and pCI-neo-p8). The day before transfection, DLD-1 cells were plated in 6-well plates at a density of 7 × 105 cells per well. After incubating overnight, cells were transfected using Lipofectamine 3000 (Invitrogen) in accordance with the manufacturer’s instructions [31 (link)]. The transfected cells were selected in RPMI 1640 containing antibiotics. (G-418) (Sigma, St. Louis, MO, USA).
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