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A New Lambda Vector for Mammalian Expression

st plaque size. We constructed a cDNA library in the EcoR I and Xho I sites of the Lambda ZAP-CMV vector using poly(A)+ RNA from human HeLa cells. This library contained approximately 3 x 106 primary plaques. The phage titer of the amplified library was 2 x 1010 plaque-forming units (pfu)/ml. To assess the library quality (background level and insert size), we mass excised and selected phagemid colonies by antibiotic selection on kanamycin plates. Plasmid DNA was prepared from 12 randomly picked colonies, and the DNA was digested with either EcoR I and Xho I or Not I and Kpn I restriction enzymes. All 12 clones contained a cDNA insert, ranging in size from 0.5 kb to 2.5 kb, with an average size of 1.4 kb (Figure 3). The titer of the amplified library in the Lambda ZAP-CMV vector remained constant upon storage at 4C, -80C, and in the presence of 7% DMSO.

Luciferase Expression in the pCMV-Script -EX Vector

Figure 4

To demonstrate expression levels in the pCMV-Script-EX vector, we inserted the firefly luciferase gene into the BamH I site of the MCS. This same luciferase gene was inserted into the parental pCMV-Script vector. These two constructs were used for parallel transfections of Chinese hamster ovary (CHO) cells. Cell lysates were prepared and assayed for luciferase activity. In Figure 4, cells transfected with each construct show comparable luciferase activity. We also detected luciferase protein in both samples by Western blot analysis (data not shown). To further demonstrate the functionality of the pCMV-Script-EX vector, we transfected the pCMV-Script-EX vector with luciferase gene construct in
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