Mammalian Expression Vector for Efficient Cloning of PCR Fragments ( CHO cells were harvested luci...)

Mammalian Expression Vector for Efficient Cloning of PCR Fragments

CHO cells were harvested, luciferase activity was quantitated (figure 2) and the protein of the correct molecular weight was detected by Western blot analysis (data not shown). Luciferase activity and expression from the pCMV-Script vector were equivalent to that from the pcDNA 3.1 vector.

The pCMV-Script vector was stably transfected into CHO cells using Stratagenes Mammalian Transfection Kit. In separate experiments, the pCMV-Script vector and the pCMV-Script vector containing the luciferase gene were transfected into CHO cells, and G418 selection was used to obtain stable cell lines. Luciferase expression from stable cells containing the pCMV-Script vector with the luciferase gene insert was compared to cells containing the pCMV-Script vector with no insert. Detected luciferase activity was 1000-fold over background, measured by a standard luciferase assay (data not shown).

Conclusions

Stratagenes new pCMV-Script cloning kit offers high-efficiency cloning of blunt-ended PCR fragments for expression in mammalian cells. The pCMV-Script vector features (1) the CMV promoter for expression in a variety of cell lines, (2) the kanamycin- and neomycin-resistance gene for selection in either prokaryotic or eukaryotic systems and (3) ligation of insert to the pCMV-Script vector in the presence T4 DNA ligase and Srf I to eliminate background of recircularized vector. The pCMV-Script vector can be used to clone inserts that have been generated by any PCR enzyme. We have tested this vector and demonstrated high-level expression in mammalian cells.

REFERENCES

Bauer, J., et al. (1992) Strategies 5: 62-65.
Sanchez, T., Zheng, C.-F., and Bauer, J. (1996) Strategies 9: 44-46.
Costa, G., and Weiner, M. (1994) Strategies 7: 47-48.

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( CHO cells were harvested luci...)Mammalian Expression Vector for Efficient Cloning of PCR Fragments

Conclusions

REFERENCES