By Michael Kriegler (auth.)
A useful handbook of protocols for attaining expression of overseas genes in mammalian cells. It comprises a few very new ideas comparable to PCR-based expression. the writer provides a theoretical creation to the protocols and compares the strengths and weaknesses.
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Extra resources for Gene Transfer and Expression: A Laboratory Manual
Not encode their own SV40 large T antigens but are COS-dependent for DNA replication. As such, the COS expression system warrants description at this point. The COS-type cell is a simian cell that is permissive for SV40 replication and has been genetically engineered to contain all viral and cellular trans-acting factors necessary to support the replication of a recombinant DNA molecule containing an SV40 ori. The development of COS was based on the notion that one could destroy the origin of replication of SV40 without destroying the function of the SV40 early promoter responsible for driving the expression of the viral T antigens.
These sequences have been shown to be responsible for the metabolic instability of these RNAs. However, a similar site is found in the 3' non-coding sequence of J3-globin mRNA, which is highly stable, so there must be some selectivity in AU-rich destabilizing sequences. These sequences have been described in a variety of lymphokine genes, as well as in a number of oncogenes such as c-fos and c-myc. In the case of c-fos, mRNA decay may be due to multiple destabilizing elements. The deletion of the AU-rich region alone is insufficient to maintain mRNA stability: The c-fos coding region itself can incur instability on an otherwise stable molecule.
The vector is composed of three segments. Segment one contains the SV40 origin of replication and enhancer, the adenovirus major late promoter (MLP), a majority of the adenovirus tripartite leader sequences, and an intervening sequence. Segment two contains the dihydrofolate reductase (DHFR) coding region, the SV40 polyadenylation sequence (poly A), and sequences encoding the adenovirus VAl RNA. Segment three contains the bacterial origin of replication and the bacterial f3-lactamase gene conferring ampicillin resistance (Amp') derived from the pUC18 plasmid.
Gene Transfer and Expression: A Laboratory Manual by Michael Kriegler (auth.)
Categories: Methodology Statistics