The U87 and GBM8401 cells were transiently transfected with the PON2 cDNA construct in 6-cm cell culture dishes using serum free Opti-MEMi medium and Lipofectamine 2000 reagent following manufacturer's instructions (Invitrogen). After 24 h, the medium of the transfected cells was replaced with regular medium. Subsequently, the expression of PON2 protein in U87 and GBM8401 cells was examined using Western blot analysis. PON2 cDNA was constructed into pcDNA 3.0 expression vector (Promega), hence, pcDNA 3.0 (Promega) was used as vector control.
Pcdna 3.0 expression vector
Manufactured by Promega
The pcDNA 3.0 expression vector is a plasmid used for the expression of recombinant proteins in mammalian cells. It contains a cytomegalovirus (CMV) promoter for high-level expression, a multiple cloning site for insertion of the gene of interest, and a neomycin resistance gene for selection of stably transfected cells.
Automatically generated - may contain errors
2 protocols using pcdna 3.0 expression vector
1
PON2 Overexpression in U87 and GBM8401 Cells
2
PON2 Gene Cloning and Expression
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Total RNA (1 μg) was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) and oligodeoxythymidine to synthesize template cDNA. The PON2 cDNA was amplified via a polymerase chain reaction for 30 cycles at 95°C for 1 min, 58°C for 1 min, and 72°C for 1 min. The PON2 open reading frame was ligated into a pcDNA 3.0 expression vector (Promega) and the resulting construct was sequenced to confirm the presence of the gene. The PON2 open reading frame primers were displayed in Table 1 .
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