"Genetic Materials" may be broadly defined as those substances which program for and guide the manufacture of cellular (and viral) constituents and the responses of cells and viral particles to environmental changes. The genetic material of all living cells and viruses (except the so-called "RNA viruses") comprises a long chain, polymeric substance known as deoxyribonucleic acid ("DNA"). The repeating units of the DNA polymer are known as nucleotides. Each nucleotide consists of one of four nucleic acids (adenine, guanine, cytosine and thymine) bound to a sugar (deoxyribose) which has a phosphate group attached. Ribonucleic acid ("RNA") is a polymeric nucleotide comprising the nucleic acids, adenine, guanine, cytosine and uricil, bound to a ribose molecule having an attached phosphate group.
Most simply put, the programming function of genetic materials is generally effected through a process whereby DNA nucleotide sequences (genes) are "transcribed" into messenger RNA ("mRNA") polymers which, in turn, serve as templates for formation of structural, regulatory and catalytic proteins from amino acids. Protein synthesis is thus the ultimate form of "expression" of the programmed genetic message provided by the DNA sequence of a gene.
Certain DNA sequences which usually "precede" a gene in a DNA polymer provide a site for initiation of the transcription into mRNA. These are referred to as "promoter" sequences. Other DNA sequences, also usually "upstream" of a gene in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcription initiation. These other sequences are referred to as "regulator" sequences. Thus, sequences which precede a selected gene (or series of genes) in a functional DNA polymer and which operate to determine whether the transcription (and eventual expression) of a gene will take place are collectively referred to as "promoter/regulator" DNA sequences.
The promoter and regulator sequences of genes are clearly susceptible to enormous structural and functional variation and, in fact, only a few such sequences in rather simple genetic systems have been thoroughly structurally and operationally characterized. Promoter/regulator sequences, in general, serve to regulate gene transcription in response to chemical (and sometimes, physical) environmental conditions in and around the cell. Many generalized "models" for the action of promoter/regulator operation in gene transcription and eventual expression in simple, prokaryotic systems have been proposed. One such model posits a "repressor" gene and a regulator sequence or "operator" sequence near the promoter of another gene. According to this model, transcription of the repressor sequence results in expression of a repressor protein which selectively binds to the operator sequence to effectively preclude gene transcription of the selected gene. An environmental "signal" (e.g., increased concentration of a chemical acted upon by the protein product of the gene in question) may operatively inactivate the repressor protein, blocking its ability to bind to the operator sequence in a way which would interrupt transcription of the gene. Increased concentrations of a substrate could be seen as operating to "induce" synthesis of the protein which catalyzes its breakdown.
Another generalized model of operation of promoter/regulator sequences in the regulation of gene transcription posits formation of an initially inactive form of repressor protein by the repressor DNA sequence. Such an inactive form could not bind to an operator DNA sequence (and disrupt selected gene transcription) until it is combined with some other substance present in the cell. The other substance could be, for example, a compound which is the product of a reaction catalyzed by the protein coded for by the selected gene. Increased concentrations of such a reaction product in the cell would thus operate to repress the potential overproduction of proteins responsible for the product's synthesis. In these examples, the regulator protein functions to inhibit transcription. Other regulatory proteins have been described which potentiate or activate transcription of specific DNA sequences. Thus, there are examples of both negative and positive control proteins and corresponding regulatory DNA sequences.
Similar "models" for the operation of promoter/regulator DNA sequences in eukaryotic cells have been proposed. See, e.g., Brown, "Gene Expression in Eukaryotes", Science, 211, pp. 667-674 (1981).
Among the basic problems of genetic engineering is the isolation and preparation of multiple copies of selected gene sequences of interest, together with the promoter or promoter/regulator DNA sequences which normally affect their transcription in the cells from which they are isolated. Another basic problem of genetic engineering is the insertion and stable incorporation of DNA sequences into cells in a manner which will permit external regulation of the transcription of the gene sequences and their expression.
Significant advances in the isolation and copying of selected DNA sequences have been made possible by the use of restriction endonuclease enzymes (which are capable of effecting site-specific cuts in DNA polymers) and ligating enzymes (which serve to fuse DNA sequences together). DNA sequences of interest are usually incorporated into "vectors" of plasmid or viral origin that allow selective replication in a suitable host cell (for example, bacteria, yeast, or mammalian cells). When these vectors with DNA sequences of interest are introduced into cells of higher animals or plants, they may either be maintained as extrachromosomal elements or incorporated into the chromosomes.
Most genetic engineering activity to date has been directed toward the stable incorporation of exogenous DNA in prokaryotic cells such as bacteria and in the simpler eukaryotes such as yeasts, molds and algae. The hoped-for result of these experiments has been to provide not only a source of multiple copies of selected genes, but the large scale transcription and expression of commercially significant gene in the form of proteinaceous products. See, e.g., Cohen, et al., U.S. Pat. No. 4,237,224; Manis, U.S. Pat. No. 4,273,875; and Cohen, U.S. Pat. No. 4,293,652. Work involving eukaryotic cells of higher organisms, such as plants and animals, has generally involved cells which are capable of continuous growth in culture.
It is recognized that animal cells are in several respects better hosts for recombinant animal genetic material than unicellular plants, and the more similar the animal species from which the gene is excised and the host cell is derived, the greater the likelihood that a functional product will be expressed and correctly processed. Different animal species frequently produce analogous proteins, and often considerable genetic homology is carried over from one species to another. Accordingly, an animal cell is much more likely than a bacteria or yeast cell to be able to perform the post-translational processing steps necessary for the gene-coded protein to be biologically active and much more likely to correctly translate a foreign gene having interrupted coding sequences. Techniques have been developed for introducing foreign genetic material into the genomes of animal tissue cells. These include attaching the foreign genetic material to a cloning vector, such as a modified virus or cosmid, and transfecting the cloning vector into the cell. The genetic material introduced into the cell by means of the vector will frequently incorporate into the genome.
An obstacle to using animal tissue cell cultures, particularly those obtained from higher animals, for the expression of recombinant gene product is the general inadaptability of tissue cell culture to large scale "farming". Unlike bacteria or yeast cells, which rapidly proliferate in a favorable environment, animal tissue cells are much less adaptable to mass production techniques. The cells which comprise the tissues of higher animals reproduce slowly and in many cases, having reached a mature stage, do not reproduce at all. The division of animal tissue cells is often very much influenced by the environs of other cells. Even if a tissue culture is initially provided with a generally ideal environment, the proliferation of cells changes that environment, frequently unfavorably to further proliferation. Tissue cultures are also subject to infection, and a major infection could wipe out a considerable investment of time and effort.
Because normal animal tissue cells generally have proliferation deficiencies, it is preferred to use animal cells that have been altered naturally or artificially to more freely proliferate. A number of tumor cells lines, either naturally or artificially produced, have been characterized and are available for genetic alteration. However, even with these cells, proliferation does not approach the rate achievable with bacteria or yeast cells, and their continued rapid proliferation requires that their environment be continuously monitored and adjusted.
The possibility of obtaining correctly processed animal gene products in substantially greater quantity than can be obtained in minute quantities by direct isolation from animal tissue has very important applications, particularly with respect to therapeutic applications but also with respect to agricultural needs. For example, growth hormone, a substance which is produced in the anterior lobe of the mammalian pituitary gland and regulates the growth of mammals, if produced in substantial quantities could be used therapeutically to treat genetic disorders, such as dwarfism. If produced in even greater quantities and relatively inexpensively, growth hormone could be used to increase the muscle mass or reduce the growing period of domestic animals. Other mammalian proteins whose large scale production would be of immediate benefit include human calcitonin, human growth hormone releasing factor and human blood clotting factors. Other mammalian proteins could be expected to have similar usefulness.
Of significant interest to the background of the invention are numerous publications of prior investigations relating to: (1) regulation of mammalian gene expression; and (2) introduction of purified genes into eukaryotic cells.
Specifically incorporated by reference herein for purposes of indicating the background of the invention and illustrating the state of the prior art are the following publications of co-inventor Palmiter and his co-workers: Durnam, et al., "Isolation and Characterization of the Mouse Metallothionein-I Gene", P.N.A.S., 77, pp. 6511-6515 (1980); Durnam, et al., "Transcriptional Regulation of the Mouse Metallothionein-I Gene by Heavy Metals", J. Biol. Chem., 256, pp. 5712-5716 (1981); Mayo, et al., "Gluocorticoid Regulation of Metallothionein-I mRNA Synthesis in Cultured Mouse Cells", J. Biol. Chem., 256 2621-2624 (1981); Hager, et al., "Transcriptional Regulation of Mouse Liver Metallothionein-I Gene by Glucocorticoids", Nature, 291, pp. 30-342 (1981); Glanville, et al., "Structure of Mouse Metallothionein-I Gene and its mRNA", Nature, 292, pp. 267-269 (1981); and Beach, et al., "Amplification of the Metallothionein-I Gene in Cadmium Resistant Mouse Cells", P.N.A.S., 78, pp. 2210-2214 (1981). The foregoing all deal with the DNA sequence specifying production of low molecular weight, metal-binding protein found in one or more forms in most vertebrae tissues. More particularly, the publications treat mouse metallothionein genes as well as their promoter/regulator DNA sequences and the responsiveness of the promoter/regulator sequences to metals and steroid hormones.
Additional publications of Palmiter and his co-worker which are incorporated by reference herein are: McKnight, et al., "Transferring Gene Expression, Regulation of mRNA Transcription in Chick Liver by Steroid Hormones and Iron Deficiency", J. Biol. Chem., 255, pp. 148-153 (1980); and Palmiter, et al., "Steriod Hormone Regulation of Ovalbumin and Conalbumin Gene Transcription, A Model Based Upon Multiple Regulatory Sites and Intermediary Proteins", J. Biol. Chem., 256, pp. 7910-7916 (1981).
Also incorporated by reference herein is a publication of Brinster and his co-workers dealing with microinjection of plasmids into germinal vesicles of mouse oocytes or pronuclei of fertilizes mouse ova, Brinster, et al., "Mouse Oocytes Transcribe Injected Xenopus 5S RNA Gene", Science, 211, pp. 396-398 (1981).
Also incorporated by reference herein are publications of Evans and co-workers dealing with hormone releasing factor mRNA sequences and also dealing with the cloning of rat growth hormone genes and their introduction into and expression in mammalian cells: Harpold, M. M., P. R. Dobner, R. M. Evans and F. C. Bancroft. Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence. Nucleic Acids Research 5, 2039-2053 (1978); Harpold, M. M., P. R. Dobner, R. M. Evans, F. C. Bancroft and J. E. Darnell, Jr. The synthesis and processing of a nuclear RNA precursor to a rat pregrowth hormone messenger RNA. Nucleic Acids Research 6, 3133-3144 (1979); Soreq, H., M. Harpold, R. M. Evans, J. E. Darnell, Jr. and F. C. Bancroft. Rat growth hormone gene: Intervening sequences separate the mRNA regions. Nucleic Acids Research 6, 2471-2482 (1979); Doehmer, J., Barinaga, M., Vale, W., Rosenfeld, M. G., Verma, I. M. and Evans, R. M. Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector: Expression and regulation. Proc. Natl. Acad. Sci. U.S.A. 79, 2268-2272 (1982); Evans, R. M., Birnberg, N. C. and Rosenfeld, M. G. Glucocorticoid and thyroid hormone transcriptionally regulate growth hormone gene expression. Proc. Natl. Acad. Sci. USA, 79, 7659-7663 (1982); Mayo, K. E., Vale, W., Rivier, J., Rosenfeld, M. G. and Evans, R. M. Expression cloning and sequence of a cDNA encoding human growth hormone releasing factor. Nature, In Press (1983); Barinaga, M., Yamomoto, G., Rivier, C., Vale, W., and Evans, R. M. Growth hormone releasing factor transcriptionally regulates growth hormone expression. Nature, In press (1983); Verma, I., Doehmer, J., Barinaga, M., Vale, W., Rosenfeld, M. and Evans, R. M. In: Eukaryotic Viral Vectors, Expression and Regulation of Rat Growth Hormone Gene in Mouse Fibroblasts (1982); and Palmiter, R. D., Brinster, R. L., Hammer, R., Trumbauer, M., Rosenfeld, M. G., Birnberg, N. C. and Evans, R. M. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature, 300, 611-615 (1982).
Also pertinent to the background of the present invention and incorporated by reference herein, are the publications of Illmensee, et al., Cell, 23, pp. 9-18 (1981) and Gordon, et al., P.N.A.S., 77, pp. 7380-7384 (1981) which respectively treat injection of nuclei into enucleated mouse eggs and introduction of plasmids containing the herpes thymidine kinase gene and SV40 (Simian viruses) in mice. Finally, the recent publication of Wagner, et al. appearing in P.N.A.S., 78, pp. 5016-5020 (1981) and treating incorporation of the human B-globin gene and a functional viral thymidine kinase gene into developing mice, is pertinent to the background of the present invention.