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8945d_027-028 6/18/03 11:10 AM Page 27 mac85 Mac 85:1st shift: 1268_tm:8945d: 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 8945d_027-028 6/20/03 1:01 PM Page 28 mac117 mac117:1268tm:8945d: 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.