Ralph Brinster and his associates demonstrated that control genes and DNA sequences could be used from species that were not closely related to express transferred foreign genes.
To manipulate the genome of animals directly has been the dream of biologists and geneticists for more than one hundred years. The ability to control the development and expression of phenotypic characteristics has been a holy grail in the field of genetics. A generation of hard work by hundreds of molecular biologists has brought humankind to the present breathtaking summit in genetics.
Based on techniques for gene splicing worked out in bacteria, and those for maintaining and manipulating embryos, gene transfers have been accomplished in higher animals. Some credit goes to John Gurdon
For twenty-five years, Ralph Brinster at the University of Pennsylvania had been working on manipulating the mouse genome and made significant contributions to the methodology in the field. Seeing the potential of Richard Palmiter’s mouse metallothionein gene, Brinster initiated a collaboration to produce transgenic mice. To produce such mice, three things had to be accomplished: the growing of mammalian embryos in vitro (in glass) up to the blastocyst stage; the uptake of at least one copy of the gene by the embryo; and the insertion of specific regulator DNA flanking sequences whose expression the scientists could control. The first task was accomplished by Brinster in 1963 using mouse embryos. His culture techniques became a standard for mammalian embryologists. His technique, in which the embryo is grown under an oil layer in a medium containing pyruvate, allowed for consistent and reproducible blastocysts that could then be transplanted into pseudopregnant mice. In addition, Brinster found that testicular teratocarcinoma cells could colonize a blastocyst even if they were older, and yet surprisingly, they express characteristics in an appropriate tissue-specific manner. (For example, some carcinoma cells incorporated into the dermis of a white mouse express the fur color of the dark donor mouse.) These same cells would cause lethal tumors if injected into an adult mouse. In addition to the relevance of this experiment for cancer research, it offered the possibility that one might introduce new genes into the germ line.
A similar set of experiments was conducted by Beatrice Mintz
By 1980, most groups were using direct microinjection of DNA and injecting copies of a specific DNA segment with the proper flanking sequences into the nuclei of recently fertilized eggs. Brinster’s group used the latter technique to transfer the human growth hormone gene to mouse embryos. In 1979 and 1980, Brinster and his colleagues, utilizing direct microinjection, tried five different genes: rabbit beta-globin, SV40 virus, HSV thymidine kinase, a sea urchin histone, and Xenopus 5S RNA (ribonucleic acid). Only thymidine kinase showed evidence of incorporation and expression.
Brinster’s group found that having the appropriate RNA polymerase present in addition to other sequences was important. Brinster and Palmiter attached the proper flanking sequences to the mouse metallothionein (MT) gene and then fused a gene for the enzyme thymidine kinase (TK) and its promoter to the MT gene. They then fused this MT-TK gene to the gene for human growth hormone. This triple gene was inducible, assayable, had the proper insertion sequences, and might even be expressed. The mouse metallothionein portion would, they hoped, switch on the thymidine kinase enzyme when the cell was exposed to a heavy metal, such as cadmium. They observed the induced expression of the thymidine kinase in their first group of newborn mice in 1981. This experiment demonstrated that one could use control genes and insertional DNA sequences from species that were not closely related in order to introduce and express transferred foreign genes. Antibody to this new gene inhibited 95 percent of the thymidine kinase, confirming that the incorporated gene was being controlled by MT’s regulator gene.
Between December, 1980, and December, 1981, several groups reported transferring new genes from one animal to another, resulting in the production of transgenic animals. Of these experiments, Brinster and Palmiter’s was the most dramatic: Human growth hormone gene was fused to the MT-TK gene to create a line of giant mice (they grew to twice their normal size). Later, Robert Hammer from Brinster’s laboratory used the same procedure to correct a genetic defect in a line of dwarf mice, thus proving that gene therapy was possible on mouse embryos. Growth hormone acts on most cells (maybe all), and therefore tissue specificity was not an issue in this case, although it might be for other genes. Tissue-specific expression would be desirable for most genes, such as the immune response genes or hemoglobin genes. Brinster’s group is working on the temporal controls for tissue-specific expression of other genes. The ultimate goal is to insert single copies of genes and have them expressed only in the proper tissue at the right time.
The ability to maintain embryos in culture and to alter their genome has provided investigators with a significant opportunity to enhance the knowledge of how living systems develop. The technique of transferring DNA to developing cells will advance basic embryology and open up the possibility of genetic therapy. The techniques described represent the next step after a defective gene has been found. Since this work was accomplished, the genes for several genetic diseases have been localized. Scientists can make the products of some of those genes utilizing biotechnology and, in some cases, can alleviate or ameliorate undesirable phenotypic expressions. Manipulating the phenotypes of plants and animals could improve food output or allow animals to be more resistant to disease. One might even be able to save endangered species.
In the field of medicine, the knowledge of gene regulation and control would aid in the fight against cancer, altered immune conditions, congenital conditions, and possibly some of the consequences of aging. In combination with in vitro fertilization, the possibility of eliminating congenital defects exists. For example, if both parents are carriers of a trait, 75 percent of the offspring will be producing some aberrant form of that protein, and the severity of the disease will be related to the amount of abnormal protein (or lack of) produced. Adding “good” genes might reduce the amount of abnormal peptide incorporated into the desired protein and/or provide more of the good protein.
Experiments indicate that control genes are genetic switches and that many are also generic that is, capable of switching on any gene attached to it. The transfer of these control genes may, in some cases, be all that is necessary to correct some genetic defects or to alter the phenotype. Using control genes sensitive to particular triggers in the environment, scientists could maintain some kind of control over the gene after it has been inserted into the genome, so as to have the gene expressed when appropriate.
The aforementioned accomplishments stimulated a rash of new experiments, as measured by their citation frequency. The successes of these researchers in mammalian gene transfer have stimulated other investigators and have led to an exponential increase in experiments in this field.
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Brinster, Ralph, and Richard Palmiter. “Introduction of Genes into the Germ Line of Animals.” Harvey Lectures 80 (1986): 35. A well-written, concise, and not too technical overview of gene transfer in animals, including landmark experiments, technical difficulties, and prospects for its application in medicine. -
Chiu, Lisa Seachrist. When a Gene Makes You Smell Like a Fish . . . and Other Amazing Tales About the Genes in Your Body. New York: Oxford University Press, 2006. The title of the book and its chapters belie the rigorous work inside. Still, a useful layperson’s guide to genetics. -
Gurdon, John, and Douglas Melton. “Gene Transfer in Amphibian Eggs and Oocytes.” Annual Review of Genetics 15 (1981): 63. Excellent review by a pioneer in genetic material transfer between animals. Well referenced. Contains a thorough discussion of techniques and status of the field at the time. For the general reader. -
Lewin, Benjamin. Genes VIII. Upper Saddle River, N.J.: Prentice Hall, 2004. Detailed upper-level text in molecular genetics, well referenced and illustrated, with a glossary and summaries at the end of each chapter. -
Palmiter, Richard, and Ralph Brinster. “Germline Transformation in Mice.” Annual Review of Genetics 20 (1986): 61. A good historical review of gene transfer in mice covering both the authors’ work as well as that of others. Well referenced and well written. -
Scangos, George, and Charles Bieberich. “Gene Transfer into Mice.” In Advances in Genetics: Molecular Genetics of Development. Vol. 24 of Molecular Genetics of Development, edited by John Scandalios. New York: Academic Press, 1987. A well-referenced review of gene transfer in mice specifically for the serious student.
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