By Michael Kriegler (auth.)
A sensible handbook of protocols for attaining expression of overseas genes in mammalian cells. It contains a few very new innovations resembling PCR-based expression. the writer supplies a theoretical advent to the protocols and compares the strengths and weaknesses.
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Additional info for Gene Transfer and Expression: A Laboratory Manual
ADENOVIRUS-BASED VECTORS Unfortunately, the development of such cell lines has not led to the development of expression vectors as sophisticated as those described for SV40-based COS transfection. The result of such neglect is that COS-based transfection is the overwhelming favorite for transiently expressing genes at high levels for purposes such as expression cloning. Nevertheless, the MOP and WOP cell lines may be appropriate for your use, particularly if you intend to construct your own polyoma virus expression vector.
To address this problem, a generalized adenovirus vector system was developed. The vector carried a complete structural gene encoding the SV40 T antigen whose expression was driven by its own promoter. The foreign genes of interest, either the polyoma T-antigen genes or the HSVtk gene, were inserted just upstream of the SV40 T-antigen cistron, and just downstream from the adenovirus MLP and tripartite leader. Neither the polyoma T antigens nor the HSVtk gene provides adenovirus helper function.
The gpt-encoding, dominant selectable, vaccinia virus open reading frame vectors described in the text are depicted here. These vectors contain vaccinia virus tk gene sequences flanking the E. 5), and the pUC-derived ampicillin resistance gene and origin of replication. The unique cloning sites into which foreign DNA can be inserted in both plasmids downstream of the initiation codon of the gene encoding the 11K peptide are indicated. Two derivatives of pTKgpt-F1s that shift the reading frame of the inserted sequence one nucleotide (pTKgpt-F2s) and two nucleotides (pTKgpt-F3s) relative to the 11K initiation codon have been generated that permit the insertion of foreign sequences into all three possible reading frames.