Download EXPRESSING FOREIGN GENES IN MICE

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Classic Experiment
9.3
EXPRESSING FOREIGN
GENES IN MICE
n the span of three years from 1980 – 1982, the notion of expressing foreign
B
proteins in mice went from an idea to a reality. During this time, several lab-
oratories worked furiously to introduce new genes and express exogenous proteins, first in mouse embryonic stem cells and then in full-grown mice. Ralph
Brinster and Richard Palmiter were among the pioneers in this field when, in
1981, they first demonstrated the robust expression of a viral gene in a transgenic
mouse.
Background
A powerful approach to the study of genes and the proteins they encode is the controlled expression in both cells
and whole organisms. Before the advent of recombinant
DNA techniques, biologists accomplished this by injecting
foreign mRNA into oocytes from frogs and studying the
biological activity of the protein encoded by the foreign
mRNA. In the 1970s and 1980s, the molecular biology
revolution allowed genes to be fused to specific promoters, which would allow them to be expressed in cell line.
Whereas biologists became able to study the gene function in cultured cells, they still wanted to study genes in
a living organism. This requires the expression of a specific foreign gene in embryonic cells, leading to introduction of the foreign gene into the animal’s genome, and examination of its function in the organism.
In the early 1970s, Brinster demonstrated that foreign
genes could be expressed in mice by injecting cancer cells
into an early embryonic form of a developing mouse
known as a blastocyst. This approach, however, made it
difficult to express a specific gene in the desired cell types.
This would require introducing the gene into the mouse
genome. In 1980, biologists demonstrated that this was
possible by injecting a plasmid containing viral DNA into
fertilized mouse oocytes, then detecting the viral sequences
in the newborn mice. This set the stage to determine
whether a functional protein could be expressed from a
foreign gene incorporated into the mouse genome.
The Experiment
Brinster’s challenge was to design the experiment in such
a way that it could be easily and unequivocally demonstrated that the mouse was making the foreign protein. To
accomplish this, Brinster chose to express an easily assayed
enzyme rather than a protein of greater biological interest in his first transgenic mouse. He chose the enzyme
thymidine kinase from the herpes simplex virus (HSV), the
choice of which offered several advantages. First, the gene
came from a human virus; thus its sequence sufficiently
differed from the endogenous mouse gene allowing its integration into the mouse genome to be readily demonstrated. Second, the activity of thymidine kinase can be
easily assayed by following the conversion of radioactively
labeled thymidine to thymidine monophosphate. Finally,
an inhibitor of the HSV thymidine kinase activity that does
not inhibit the endogenous mouse enzyme was available,
allowing the researchers to specifically monitor the activity of the foreign protein.
Genes are expressed from DNA sequences upstream of
the protein-coding region called promoters. Promoters
control where and when a gene is expressed. To express
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a viral gene in a mouse requires that the biologist remove
the gene from the control of the viral promoter and fuse
it to a promoter that is active in mouse cells. Brinster collaborated with Palmiter, who had been studying the promoter of the mouse metallothionein-1 (MT-1) gene.
Palmiter fused the MT-1 promoter to the HSV thymidine
kinase gene. They then could ask whether a viral protein
could be expressed in a mouse.
To generate the transgenic mouse, Brinster and
Palmiter injected the plasmid containing HSV thymidine
kinase fused to the MT-1 promoter into the pro-nuclei of
fertilized mouse eggs, which they then implanted back into
female mice. The scientists mated progeny mice with normal females, and analyzed the resulting progeny for the
presence of the HSV thymidine kinase DNA as well as
thymidine kinase activity.
Using Southern blot analysis, they detected the presence of the MT-1 promoter/thymidine kinase gene fusion,
known as the transgene. They isolated genomic DNA, then
cleaved it with a restriction endonuclease. They proceeded
to separate the DNA by agarose gel electrophoresis—
which separates DNA fragments on the basis of size—
and transferred it to a nitrocellulose membrane. The two
scientists then hybridized a radioactively labeled probe,
specific for the transgene, to the membrane for analysis.
This analysis revealed that the transgene had been successfully integrated into the genomes of four progeny mice.
Next, to determine whether the transgene expressed a
functional protein, Brinster and Palmiter analyzed homogenates from the liver, a tissue where the mouse MT-1
gene is highly expressed, for viral thymidine kinase activity. Liver homogenates from one mouse contained approximately 200 times more thymidine kinase activity
than the liver homogenates of its littermates. This mouse
was one of the four that had the transgene integrated into
its genome. To demonstrate that this increase in activity
was a result of viral thymidine kinase expression they
treated liver homogenates with an inhibitor that specifically blocks the HSV thymidine kinase activity. Thymidine
kinase activity in liver homogenates from the transgenic
mouse was markedly reduced by this inhibitor, whereas
TABLE 9-1
Expression of Viral Thymidine Kinase
in Transgenic Mice
Mouse Transgene DNA
Thymidine Kinase Activity
Inhibitor
23-1
23-2
14500
497,000
Inhibitor
14700
187,000
[Adapted from R. L. Brinster et al., 1981, Cell 27:223–231.]
the activity in homogenates from its non-transgenic littermates was unchanged (Table 8.1). Thus Brinster and
Palmiter confirmed the presence of viral thymidine kinase
activity, and demonstrated that a foreign protein could be
expressed in a mouse.
Discussion
Progress in embryology and molecular biology had left the
field ripe for researchers to experiment with advancing the
expression of foreign proteins in animals. The careful
choice of the easily assayed HSV thymidine kinase gene
put under the control of the metallothionein promoter allowed Brinster and Palmiter to demonstrate the feasibility
of this technique.
The ability to generate transgenic mice has been invaluable to the study of gene function in vivo. Before this
technology was available, researchers had to find naturally occurring mutations in order to analyze gene function in mice. Now, a specific gene can be expressed in
mice. Soon, genes were fused to promoters that allowed
expression in specific tissues. Scientists have generated
transgenic mice to analyze the function of a great number
of genes, allowing them to determine the roles of the genes
in a variety of diseases and biological processes.