Textbook Assignment: Chapter 26, Pages 751 - 757, Chapter 27, Pages 774-783.
Major concepts
Introduction: This lecture deals with selected areas of application of recombinant DNA technology in Medicine, Agriculture, and Industry, as described in chapters 26 and 27 of our textbook. We have already dealt with some of the material in these chapters in previous lectures. Because of lack of time in the lecture schedule, these notes deal rather selectively with the remaining topics from these chapters. The material that is presented in this lecture will be included in the final examination.
Biotechnology: Biotechnology is the application of recombinant DNA techniques in commercial enterprises. There are three main areas: 1) Industry; 2) Agriculture; 3) Medicine/Pharmaceuticals.
Industry: The following are examples of the diverse use of biotechnology in industry:
Agriculture: Extensive use of biotechnology has been made to modify both plants and animals used in agriculture. This frequently involves the development of transgenic plants and animals through the insertion of genes from other species into their genomes. Improvements to commercial crops include: disease, drought, insect, and pesticide resistance; improved nutrient content, extended fruit shelf life, and pharmaceutical delivery.
Transgenic plants: Transgenic approaches are beginning to be widely used with plants. Thus, for example, crop plants have been developed that have transgenic resistance to glycophosphate, the active herbicidal ingredient of the weed killer, Roundup. Glycophosphate inhibits an enzyme needed for synthesis of amino acids, without which plants cannot grow. A bacterial gene that confers resistance to glycophosphate has been transfected into crop plants, making them resistant. A Ti plasmid vector (described later in these notes) was used for transfection of cultured tobacco cells, which were then used to regenerate plants. These plants will survive selectively, while weeds will not, in a field sprayed with glycophosphate. Similar procedures have generated "Roundup-ready" soybeans (page 777). Another example is the expression in corn plants of a toxin from Bacillus thuringensis that kills insect pests (page 776). This has become controversial because of fears that Monarch butterflies may be killed by pollen from the corn plants (see internet link in lecture 40).
Commercial production: A variety of transgenic plants are in varying stages of experimentation or commercial production. For example, frost-resistant and salt-tolerant plants that can be grown in otherwise hostile environments are being pursued vigorously. In some cases, unexpected new problems are encountered with genetically engineered crops. One such case is the Flavr Savr tomato, which remains firm for a much longer time after ripening because the activity of the enzyme polygalacturonase has been greatly reduced by expression of an antisense RNA that blocks gene expression by combining with the mRNA. This allows vine ripened tomatoes to remain in good condition for a long enough time to be shipped to stores and displayed without rapidly going bad. However, the methods that were used for packing and shipping tomatoes that were harvested green and ripened artificially later proved to be too rough for ripe tomatoes. This resulted in an unexpected market failure of the first major commercial crop, and a requirement for developing new methods to handle and ship the ripe tomatoes.
Environmental concerns: In many cases, there are also concerns that the transgenes could get into other types of plants. This could, for example generate herbicide resistant weeds. In addition, many of the selective procedures involved in generating recombinant vectors involve the use of antibiotic resistance. There is concern that plasmids carrying antibiotic resistance could be released into the environment causing various types of pathogens to become antibiotic resistant.
Agricultural animals: Biotechnology uses in livestock animals include: the making of transgenic animals for protein production and organ donation, in vitro fertilization and embryo splitting used to propagate animals with highly desirable characteristics, and the use of bovine growth hormone (BGH)(first expressed in E. coli) to increase milk production.
Transgenic animals: Methods have been developed that allow cloned genes to be incorporated into germ line cells of a number of animal species. Most of the pioneering work has been done with mice, but transgenic pigs, sheep, goats and cows have also been developed. The first step is to transfect the gene in question into an embryonal stem cell (ES cell), which is then injected into an early embryo. Embryonal stem cells are cultured cells that have retained totipotent developmental potential and can become any part of the animal, including the germ cells, when injected into a sufficiently early embryo. The resulting chimeric animals are then bred with wild type mice, creating heterozygotes for the transgene. Mating of two of the heterozygotes can then generate 1/4 of the offspring that are homozygous for the transgene. In most cases, this process generates a transgenic animal that expresses the transfected foreign gene without any adverse effects on normally expressed genes from its own genome. Alternatively, it is sometimes possible to obtain random genomoic incorporation of genetic material injected directly into zygotes (page 781).
Growth hormone: In one the pioneering studies on transgenic animals, a mouse with multiple copies of a rat growth hormone gene was found to grow to a much larger size than a normal mouse (see page 781 of our textbook). However, attempts to produce larger pigs with human growth hormone were less successful.
Use of milk gene promoters: One very interesting application of transgenic animals has been to link genes coding for desired proteins to promoters for milk protein genes in transgenic animals. This results in the desired protein being produced in the milk of the animal, sometimes in quite large amounts (page 755). These techniques are beginning to be used, particularly in sheep and goats, to generate rather large amounts of foreign proteins in the milk. One of the advantages is that correct glycosylation of the proteins occurs in these systems. The previous textbook for this course describes the production of alpha-1-antitrypsin in amounts up to 35 grams per liter in milk (pages 479-480, Klug and Cummings, Norlin reserve).
MCDB transgenic mouse facility: A facility for research on transgenic mice has been constructed in the new part of the MCDB building and studies on a variety of transgenic and knockout mice are currently being undertaken by several members of the MCDB faculty.
Medicine/Pharmaceuticals: At present, up to 70% of the commercial biotechnology industry is involved in the production of pharmaceuticals and medically-related products. A few of the many applications are listed below, followed by more detailed descriptions of selected areas:
Treatment of human genetic diseases: Most human genetic diseases are caused by mutations that cause an enzyme or some other type of protein to become non-functional. In theory, production of the missing protein from an expression vector that would function in the patient's cells could cure such diseases. In practice, the procedure is not that simple, largely because most proteins must be expressed selectively in specific tissues and because the amounts that are produced must be carefully controlled. Nevertheless, there have been a few successful treatments. The textbook briefly describes successful treatment of a severe combined immunodeficiency caused by absence of adenosine deaminase activity (pages 756-757). A recent followup report stated that the two original patients in this project had sustained a long-term alleviation of the symptoms resulting from insertion of a functional adenosine deaminase gene into some of their white blood cells and infusion of the cells back into their bodies. Similar possible treatments for a variety of other inherited diseases are being studied extensively.
Vaccines: Another area of major interest is the possible use of proteins generated from cloned genes as antigens for the production of vaccines that carry no risk of accidental transmission of the disease. Our textbook describes a licensed project in which a surface protein from the hepatitis B virus in being produced in yeast cells in commercial quantities (pages 754-755).
Hormones: Examples of pharmaceutical products that are now being produced commercially by recombinant DNA methods include human insulin (pages 752-754) and human growth hormone (boxed example 9.5, pages 275-277, and page 754). The use of expression systems allows synthesis of human forms of these hormones, thus avoiding any possibility of allergic reactions caused by hormones from animal sources, which tend to have slightly different amino acid sequences. Another advantage is avoiding possible transmission of disease from use of human blood products or cadaver material as a source. Prior to the availablity of recombinant DNA products, there were known cases of transmission of AIDS virus in human factor VIII preparations and Creutzfeld-Jacob disease (caused by prions) in human growth hormone preparations. Hormone-like growth factors used for serum-free cell culture are also mostly produced through use of recombinant DNA technology.
Cost effectiveness: One of the limitations of this type of manufacture is that production of proteins through recombinant DNA technology is relatively expensive. The only proteins that are reasonable candidates for commercial production in this manner are those that are needed in relatively small amounts and can be sold at a price high enough to be profitable. Examples would include hormones and other types of regulatory proteins that are used clinically in relatively small amounts, as well as a variety of types of proteins that are needed in small amounts for basic research in molecular biology, such as cellular growth factors.
Post-translational modification: Another limitation is that many proteins must undergo various types of post-translational modification before they are biologically active. This often includes the addition of complex polysaccharides in a process known as glycosylation. Eukaryotic proteins produced in bacterial expression vector systems are not adequately glycosylated, thus limiting the use of prokaryotic systems to those proteins that do not require glycosylation to function. A variety of eukaryotic expression vector systems have been developed to overcome this problem, including use of yeast, cultured insect cells, and cultured mammalian cells to generate the desired products. In addition, transgenic dairy animals are also beginning to be used to produce large amounts of desired proteins in their milk, as described above.
Methods used in DNA technology: Considerations that must be taken into account when introducing new genetic material include the following:
Eukaryotic vectors: Many eukaryotic genes cannot be expressed effectively in prokaryotic cells because of the need for removal of introns or for posttranslational modification of the protein products. Vectors that are designed for use in eukaryotic cells do not differ greatly in principle from those used in prokaryotic cells. They must be capable of entering their host cells and replicating autonomously in those cells. They must also have a site into which a foreign coding sequence can be introduced without seriously disrupting their ability to function. In many cases, the vectors are based on viruses or plasmids that occur naturally in the host cells. Frequently a bacterial origin of replication (and a means for circularization if needed) are added so that the vectors can be propagated as plasmids in bacterial cells prior to having foreign DNA cloned into them. Some so-called shuttle vectors have been designed specifically to be capable of replication both in prokaryotic and eukaryotic hosts, so that cloned DNA sequences can be transferred readily back and forth between the two.
Yeast artificial chromosomes: Yeast artificial chromosomes (lectures 13 and 18; textbook pages 310-312) allow large DNA sequences to be cloned in yeast cells. Because the yeast cella are eukaryotic, they are able to accomplish many of the post-translational modifications of proteins that bacteria are incapable of. Thus, yeast expression vectors are often used for commercial production of proteins that need such modificaitons (textbook pages 754-755).
Animal virus vectors: Modified animal viruses can also be used to introduce cloned DNA sequences into animal cells. These include retroviruses that have single-stranded RNA viral genomes, but undergo reverse transcription to form double stranded DNA, which becomes incorporated into the host genome after infection (see page 756). A variety of other types of viruses can also be used. One example is a modified form of SV40, which is a double-stranded DNA virus that in its unmodified form can cause malignant transformation of some types of cultured cells. Highly engineered vectors that incorporate elements from more than one type of virus have also been developed.
Plant vectors: The Ti (tumor-inducing) plasmid from the soil bacterium Agrobacterium tumefaciens is widely used as a plant vector. In nature, this plasmid integrates a portion of its DNA into the plant cell genome and causes a tumorous growth on the plant. Attenuated plasmids that are no longer capable of tumor formation can be used to introduce foreign genes into single plant cells, which can then be grown into complete plants by appropriate hormonal manipulation. Our textbook describes this procedure on pages 774-777 and in figures 27.7 through 27.11.
Methods of transfection: Many different methods have been developed to "force" the uptake of transfecting vectors. In some cases, direct uptake of foreign DNA can be encouraged by treatment of the cells with calcium phosphate. Another technique that sometimes works is direct microinjection of DNA into the nuclei of individual cells. Foreign DNA that has been encapsulated into an artificial lipid membrane called a liposome is often incorporated quite effectively by fusion of the liposome with the cellular plasma membrane. The biolistic (biological balistic) process coats microprojectiles with DNA and literally shoots them into cells or organelles. The process of electroporation uses brief exposure to high voltage electricity to punch small holes in the plasma membrane that heal after foreign DNA has entered. All of these various procedures are rather widely used, and new ones are being developed regularly.
Transfection vs. transformation: In eukaryotic cells, the word transformation is normally used only to describe a process that causes the cell to become malignant (cancerous). To avoid confusion, the introduction of a foreign gene that does not cause malignancy is referred to as transfection (page 756). In prokaryotic cells, the term transformation is commonly used to describe a genetic change caused by uptake of foreign DNA, as described in lecture 8 (pages 188-190 of textbook).
Reporter genes: After a gene has been cloned, it is often desirable to determine both how its expression is regulated and where it is expressed in a multicellular organism. This is frequently done by attaching the promoter for the gene in question to a so-called reporter gene. To study regulation of expression, the reporter construct is then transfected into a cell type that normally expresses the gene in question. By manipulating the promoter sequence, and also by manipulating that transcription factors are expressed in the host cell, investigators can then use the reporter gene to determine how their manipulations have altered expression. Common reporters include an easily assayed enzyme called chloramphenicol acetyltransferase (CAT), beta-galactosidase (lac Z), and green fluorescent protein. To study patterns of expression, the reporter construct is transfected into an intact organism (usually by formation of a transgenic animal or plant, as described above). Whenever and wherever the gene of interest is expressed, the reporter gene will also be expressed since it is under the control of a similar promoter. When beta-galactosidase is used as the reporter, blue staining with X-gal can be used to identify sites of expression. Similarly, fluorescence can be used to determine sites of expression of green fluorescent protein, which is rapidly becoming a highly popular reporter gene. Blue/white selection of colonies of bacteria carrying recombinant vectors is another example of the use of a reporter gene (Lecture 14 and pages 263-265) Our textbook discusses the use of reporter genes for studies on developmental genetics on page 687.
Removing or inactivating genetic material: As described in the following paragraphs, two major techniques are used to prevent the expression of specific genes. The first is gene knockout, which results in permanent inactivation of a specific gene. The second is the use of antisense technology, which leads to DNA:RNA or RNA:RNA hybridization of the mRNA for a specific gene, followed by degradation of the double-stranded form.
Gene knockouts: Gene knockout experiments in mice and other species are currently providing major insight into the functions of many different genes. Techniques have been developed for permanent inactivation of a specific gene in cultured embryonal stem cells (ES cells) of mice. When injected into early embryos, some of these cells become germ cells, such that some of the gametes that are procuded when the animals mature contain only the inactivated gene. These gametes produce heterozygous progeny in crosses with wild-type mice. Crosses of two of the heterozygous progeny can produce homozygous embryos that do not have any functional copies of the knockout gene.
Developmental roles of specific genes: Studies on knockout mice are providing valuable information about the functions of specific genes, including their roles during early embryonic development. They are also revealing a surprising degree of redundancy in some control systems. For example, the c-src gene can become a cancer-causing oncogene when it is excessively activated by loss of a regulatory function, and it also plays an important role in normal cellular multiplication when properly regulated. However, total inactivation of this gene in knockout mice had only minor effects on development to term and live birth of relatively normal animals. Ultimately it was shown that two other genes with similar functions could adequately replace nearly all of the functions of the c-src gene. When all three of these genes were knocked out, cellular multiplication could not occur normally, causing early embryonic lethality. Many similar cases are known with other gene families.
Antisense technology: As an alternative to a gene knockour, single stranded DNA or RNAS oligonucleotides can be used to bind a specific mRNA, creating a double stranded DNA/RNA or RNA/RNA hybrid, which is then degraded by the cell. Oligonucleotides are made specifically to be complementary to the mRNA of interest, such that only that mRNA is bound and all other cellular functions remain normal. Once the mRNA is degraded, translation of the protein product cannot take place. Alternatively, it is possible to introduce into the cellular genome a DNA seuence with an appropriate promoter that will transcribe an antisense RNA in a particular tissue. This is the technique that has been used in the FlavrSavr tomato to block expression of polygalacturonase, as described earlier in these notes.