Protocol of gene transfer, greatly increasing the
variety of cells that can be used as recipients of foreign genes. One of the
most important developments in recent years in the field of molecular biology
has been the advent of techniques that allow the re-introduction of cloned
genetic sequences into mammalian cells in culture. The pioneering experiments
done by Wigler et al. (1977) used
solely mouse L cells and depended on the expression of an introduced Herpes
simplex virus thymidine kinase
gene. Therefore, recipient cells were required to be mutant cells lacking
endogenous expression of the cellular isozyme for thymidine kinase (Pellicer et
al. 1978).More recently it has
become desirable to use a wide variety of cell types as recipients of the cloned
genes, particularly to study proper regulation of a gene in a homogeneous
background. Because many initial attempts at introducing foreign DNA into cells
other than mouse L cells had been unproductive, the idea arose that only a few
cell types were competent to express cloned DNA. However, it is possible that
these first negative results could have been a result of lack of sensitivity in
the detection of foreign DNA, or that modification in the basic techniques might
be required for the expression of plasmid DNA in various cell types.

 

They set out to devise a sensitive assay system
which could be used to monitor expression of

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foreign DNA shortly after uptake by the cells in
culture. With such a system, expression of DNA can be detected transiently;
that is, while the plasmid DNA is in a non-integrated state. In such
experiments, thousands of DNA templates are being transcribed. This approach
has two distinct advantages over previous experiments, which allowed the detection
of foreign DNA only after it was integrated into the host cell chromatin. First,
transient expression can be monitored within 48 h of DNA uptake, while stable
expression may require 3-4 weeks.
Second, since thousands of templates are transcribed, higher levels of RNA
homologous to the cloned gene are present.

 

The system they discuss is based on the expression
of a bacterial gene, that encoding

chloramphenicol acetyltransferase (CAT), in mammalian
cells. This bacterial enzyme has been well characterized and sensitive assays
exist. By cloning this bacterial gene

under eukaryotic control signals we have constructed
a unique plasmid, pSV2cat, which
allowsthe expression of a bacterial gene in mammalian cells.

 

Briefly discuss
two applications of the transient assay system: the quantitative

analysis
of promoter strength, which has led to the development of expression vectors
that

transform
mammalian cells at very efficient levels, and the use of this system to define
optimum transfection protocols for cells previously thought to be difficult to
transfect, namely undifferentiated teratocarcinoma cells.

 

One of
the first uses of the CAT assay system has been the analysis of putative
promoter

sequences.
By subcloning these control sequences juxtaposed to the CAT gene the relative
levels of transcription from various promoters can be compared by the assay. A
comparison of various promoter activities in different cell types is listed in
table 1. In addition to the above
work,other recent reports have used this assay system to study enhancer
elements.

 

 

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