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Mammalian Expression Vector for Efficient Cloning of PCR Fragments

t PCR cloning kit was verified using a test gene. To reduce the number of potential mutations that result during PCR amplification and to generate blunt-ended DNA fragments, we chose Pfu DNA polymerase for PCR amplification of the ampicillin-resistance gene. The pCMV-Script vector was digested with Srf I restriction endonuclease and ligated to the PCR-amplified ampicillin-resistance gene in the presence of Srf I to eliminate the background of recircularized vector. This mixture was transformed into XL10-Gold cells, and 50 l of the 500 l transformation mix was plated on LB agar plates containing kanamycin. In order to determine the percentage of colonies that contained the insert, 100 of the resulting 250 colonies were streaked onto an LB agar plate with ampicillin. Of the recovered transformants, 73% contained the ampicillin-resistance-gene insert.

Testing Mammalian Expression

The pCMV-Script vector was tested in both transient and stable transfection assays. The green fluorescent protein gene (GFP) and the firefly luciferase gene were used for transient transfection assays. The GFP gene was cloned into the pCMV-Script vector, and the resulting construct was transfected using standard lipid transfection methods into Chinese hamster ovary (CHO) cells. The expression of the GFP gene product was detected by Western blot analysis (data not shown).

Figure 2

Similarly, the luciferase gene was cloned into the pCMV-Script vector and used to transfect CHO cells. In order to compare expression levels of genes inserted into the pCMV-Script vector with expression levels in other available vector products, the luciferase gene was also inserted into the pcDNA 3.1 vector. Each construct and appropriate controls were used to transfect CHO cells. Forty-eight hours following transfection,
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