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APPENDIX Gene Transfer Methods Applicable to Agricultural Organisms PhyIlis B. Moses INTRODUCTION The transfer of genes from one organism to another is a nat- ural process that creates variation in biological traits. This fact underlies all attempts to improve agriculturally important species, whether through traditional agricultural breeding or through the techniques of molecular biology. In both cases, human beings manipulate a naturally occurring process to produce varieties of organisms that display desired traits, for example, food animals with a higher proportion of muscle to fat, or disease-resistant corn. The major differences between traditional agricultural breed- ing and molecular biological methods of gene transfer lie neither in aims nor in processes, but rather in speed, precision, reliabil- ity, and scope. When traditional, or classical, breeders cross two sexually reproducing plants or animals, they mix tens of thou- sands of genes in the hope of obtaining progeny with the desired trait or traits. Through the fusion of sperm en c] egg, each parent contributes half of its genome (an organism's entire repertoire of genes) to its offspring, but the composition of that half varies in each parental sex cell and hence in each cross. In addition, because the traits desired usually come from only one parent and may be controlled by one or a few genes, many crosses are necessary before the "right" chance recombination of genes results in expression of the trait in the offspring. Even then, the progeny usually have to be crossed back to the parental variety to ensure stable adoption of 149
150 AGRICULTURAL BIOTECHNOLOGY the new trait. Sometimes undesired traits derived from one parent of a new, improved variety persist whereas the desired traits are lost. Such are the difficulties and limitations of classical breeding. Molecular biological methods of gene transfer alleviate some of these problems by allowing the process to be manipulated at a more fundamental level. Instead of gambling on recombination of large numbers of genes, scientists can insert individual genes for specific traits directly into an established genome. They can also control the way in which these genes express themselves in the new variety of plant or animal. In short, by homing in on desired traits, molecular gene transfer can shorten the breeding time for new varieties and, in addition, lead to improvements not possible by traditional breeding. Laboratory methods to move individual genes between organ- isms capitalize on naturally occurring mechanisms of gene transfer other than sexual reproduction. These include uptake of DNA by cells and cell-to-cell transfer of packaged genetic material such as viruses. Scientists began by studying these mechanisms in sim- ple systems- bacteria and the viruses that infect them. Research has progressed at a remarkable rate. Now scientists can trans- fer genes into organisms as diverse as soybeans and sheep. Much work remains, however, to perfect gene transfer and its attendant technologies of embryo culture and plant regeneration. Scientists have relied heavily on favorite mode! organisms such as the bacterium Echerischia cold and the fruit fly Drosophila melanogaster, because of their ease of manipulation and the large body of scientific knowledge accumulated about them. Mode! systems are critical to the progress of research. Nevertheless, molecular biologists must extend their techniques to commercially important agricultural organisms. Movement in this direction will not replace all traditional agricultural breeding with molecular gene transfer. It will, however, expand the array of methods available to improve agriculturally important species. General Considerations Gene transfer occurs naturally among bacteria by a variety of mechanisms. Scientists learned in the 1950s and 1960s to exploit these mechanisms to study gene regulation in bacteria and in
APPENDIX: GENE TRANSFER METHODS 151 the 1970s developed additional artificial gene transfer methods for bacteria. It turned out to be a relatively simple matter to get some types of bacteria to take up pieces of DNA from their surrounding medium. Genes contained on the new pieces of DNA could be stably inherited and expressed to give new characteristics to the host bacteria. Scientists then devised special conditions that improved DNA uptake, maintenance, and gene expression in the new hosts. Gene transfer is now a routine laboratory procedure for bacterial strains such as E. coli. The goals of gene transfer experiments with other organisms are the same as those of earlier work with bacteria to study gene regulation and to obtain stable inheritance and expression of new characteristics. The difference is that these other organisms are more complicated biological entities than are bacteria. Hence the experimental problems and procedures are more complicated. It has proved necessary to devise some very special conditions and tools to move DNA into the cells of other organisms. The explosion of knowledge in molecular biology is the direct result of certain basic biological discoveries that permit scientists to handle genes as macromolecules. Researchers can identify, iso- late, cut, and splice genes and transfer them from one species to another. Enzymes, obtained mainly from bacteria, enable scien- tists to perform the first four steps on genes from any organism, by procedures that are now standard in molecular biology. The fifth step, gene transfer, must be worked out individually for different organisms. Different species of animals, plants, and microbes vary widely in the ease with which gene transfer can currently be carried out. Plants have in general been more difficult to deal with than animals or microbes. The technology is improving rapidly, however, and it is likely that most organisms will in time be tractable targets for gene transfer. This report surveys the scientific status and short-term prog- nosis for gene transfer systems applicable to animals, plants, and microbes of agricultural importance. The development of these methods has hinged on scientists' understanding of underlying molecular mechanisms in organisms. These mechanisms are then exploited, as was originally done for bacteria, to develop methods
152 AGRICULTURAL BIOTECHNOLOGY for moving genes between organisms. This permits both funda- mental studies of gene function and the endowment of an organism with new, desired characteristics. Many factors must be considered in the design of gene transfer systems. The first requirement is an easily detected "tag" for the gene so that its progress into a new host can be traced. Sometimes the uniqueness of the foreign gene is sufficient: The gene can be identified by the new characteristic it confers or its physical pres- ence can be detected by a probe for its particular DNA sequence. Unfortunately, such direct identification methods are sometimes either impracticable or inconvenient and time consuming. In these cases the foreign gene can be tagged by attaching it to another gene whose presence in the host is easily and rapidly detectable. Genes for drug resistance are often used as tags. Host cells that incorporate these genes-along with the foreign gene can survive a drug treatment, whereas cells that have not taken up the genes will die. Another important consideration is the efficiency of gene . transfer. The probability of success must be high enough for transfer of the gene to be detected with a reasonable frequency. If drug resistance or other selection schemes are used, a lower fre- quency may be acceptable. In such cases many cells can be treated for gene transfer, but only those few that actually incorporate the foreign genes survive the selective treatment and are recovered. Special vectors can improve the efficiency of gene transfer. Foreign genes attached to the vector will be carried by it into the host cell. Vectors are often derived from circular DNA molecules called plasmids, or from viruses. Different transfer systems have particular features that can limit the size of foreign DNA segments that they are capable of transferring. Segment sizes are measured in base pairs, the funda- mental chemical units of DNA. A typical gene may be composed of anywhere from 1,000 to 50,000 base pairs. A few techniques for gene transfer can handle segments of DNA at the upper end of this range, but most current methods are limited to segments at the lower end. Because it may be desirable to transfer more than one gene at a time, scientists are working to develop more and better vectors that can handle multiple genes. When a DNA molecule is used as a vector for foreign genes, its own size sometimes limits how much extra DNA it can carry. Small
APPENDIX: GENE TRANSFER METHODS 153 vectors, themselves less than 10,000 base pairs, are more often limited than are large vectors, which may be well over 100,000 base pairs. However, larger vectors require extra manipulations to equip them with foreign DNA. The final state of foreign genes inside the host cell is also im- portant. Genes can be maintained on vectors that are independent, self-replicating "minichromosomes," or they can be integrated into the larger chromosomes of the host cell. Depending on the exper- iment's purpose, independent maintenance or integration of new genes may be preferable. However, to ensure stable inheritance of transferred genes in intact animals or plants, the genes must usually be integrated. Related to the state of genes is the question of gene copy number, that is, how many identical copies of a foreign gene end up in the cell. Again, depending on the experiment's purpose, many copies or only one copy may be desired. For example, if gene transfer is used to engineer a cell line to manufacture large amounts of a commercially valuable protein, a high copy number, self-replicating minichromosome would be used. On the other hand, if the purpose is to equip an animal or plant with a new gene for disease resistance, only one or two copies of the gene might be needed in the organism's own chromosomes, where it would be properly expressed and inherited by succeeding generations. Transferred genes must be regulated so that their protein products are made In appropriate amounts at the correct time and in the right place. Genes are normally controlled by certain sequences in the surrounding DNA. These sequences in turn are affected by various factors within cells, for example, hormones. Transferred genes can be regulated by their normal control se- quences. Alternatively, scientists can equip them with new control sequences to either mimic the natural situation or achieve new effects. Before permanent genetic modification of an organism is at- tempted, it is important to study the gene of interest under various conditions to understand its normal function and regulation and to engineer any beneficial changes. These studies are conveniently done using a "transient expression" system, by which the activity of transferred genes can be rapidly measured inside cells, without waiting for stable, long-term genetic modification of the cells.
154 A GRICULTURAL BIO TECHNOLOGY The flexibility and rapidity of initial studies are also enhanced by "shuttle vectors," which are designed to replicate in both ani- mals or plants and in bacteria. By using shuttle vectors, scientists can easily grow and isolate genes in quantity from bacteria, mod- ify them in vitro, and then quickly transfer them into animal or plant cells to test their function. The transferred genes can also be reisolated from the anneal or plant cells, put back into bacteria, and grown in quantity again for further use. DIRECT DNA UPTAKE The earliest and still most widely used method for introducing DNA into animal cells grown in culture in the laboratory is direct uptake- of DNA from the surrounding culture medium. The con- ditions are in principle the same as those used for bacterial cells: DNA must enter the cell and become stably maintained and inher- ited in the cell line in such a way that its new genetic information is expressed to confer a new trait on the cell. The mechanics differ because animal cells diner structurally from bacterial cells. On the one hand, animal ceils have only a membrane surrounding their contents, whereas bacterial cells (and plant, fungal, and yeast celIs) have both a membrane and a wall. The rigid cell wall of the latter organisms often must be removed to allow DNA to enter the cell. On the other hand, most of the genetic information ~ animal, plant, fungal, and yeast cells is sequestered in the nucleus, an organelle surrounded by its own membrane. (Organisms that have cell nuclei are known as eucaryotes.) New genetic material usually must pass through this second membrane ~ order to be permanently added to a eucaryotic cell. Bacteria (known as procaryotes) lack an organized nucleus and usually accept new DNA more easily. The major advantages of direct DNA uptake (facilitated by chern~cal or electrical treatments, as will be described) are its simplicity and applicability to many organisms and cell types. Hundreds of thousands of cells may be simultaneously treated, in contrast to m~croinjection of DNA into individual ceils (described later), which ~ laborious and time consuming. Because it is so simple and rapid, direct uptake is extremely useful for basic studies of gene expression in cell culture. These studies are important for characterizing a genes function, before researchers attempt
APPENDIX: GENE TRANSFER METHODS 155 elaborate and time-consuming gene transfer experiments in whole animals or plants. Foreign genes introduced by direct uptake are expressed in their new host cells after a short period, usually 1 or 2 days. Direct DNA uptake thus quickly reveals the function of newly isolated or engineered genes during this period of "transient expression." For long-term studies the genes must integrate into the cellos own chromosomes, or be carried ~ by the uptake of new chromosomes, to ensure that they are stably inherited. Integration occurs at a high frequency after direct DNA uptake into animal cells because so many copies of the foreign genes have been introduced. (Main- tenance on new chromosomes is discussed in the sections on Cell Fusion and Vector-Mediated Gene Transfer.) In addition, gene transfer into cultured cells by direct DNA uptake is user! for the commercial production of genetically en- gineered proteins. Drugs, hormones, food additives, and other valuable substances can be manufactured by cells into which the appropriate genes have been transferred. Human insulm for treat- ment of diabetics is now manufactured in bacteria in this way. The limitations of direct uptake, particularly for animals, cen- ter on the fact that intact organisms usually are not suitable re- cipients. Thus, gene transfer into an an~rnal embryo usually must be accomplished by other means. For plants this is not a strict limitation, as many species can be regenerated Into whole plants from a single cultured cell. Chemical Treatment Chemical treatments can induce animal cells to take up DNA from their medium; most frequently these ceBs are in culture rather than in living animal. In the supplest and most popular method, cells are mixed with DNA that has been precipitated with calcium phosphate (Graham and van der Eb, 1973~. This treatment compacts the DNA, so ceils take up many copies of the foreign genes. Alternatively, the chemical DEA~dextran may be used to facilitate DNA uptake (McCutchan and Pagano, 1968~. Cells in culture are relatively unspecialized and often do not correctly regulate genes as would the specialized organs of an in- tact animal. Researchers have therefore developed a technique to introduce DNA directly into intact organs, such as the liver or
156 AGRICULTURAL BIOTECHNOLOGY spleen, of living animals. Calcium phosphate-precipitated DNA is injected directly into the organs, in combination with Tow concen- trations of enzymes that allow the DNA to enter (Dubensky et al., 1984~. This technique enables researchers to quickly study an isolated gene's function in the differentiated, specialized cells of an intact organ, which more accurately reflect the gene's proper function in an animal. A variation of the organ transformation technique involves injecting the calcium phosphate-precipitated DNA intraperitoneally, where it is taken up and expressed by ani- mal tissues, such as those of the liver and spleen (Benvenisty and Reshef, 1986~. Plant cells have been difficult to transform by chemical meth- ods, but recently breakthroughs have been made. Polyethylene glyco} has been used to obtain direct uptake and stable mainte- nance of DNA by protoplasts from a species of wheat, Triticum monococcum (Lorz et al., 1985), another monocot grass, Lolium muitifoTum (Potrykus et al., 1985a), and the dicots oilseed rape, tobacco, and petunias (Potrykus et al., 1985b). The frequency of integration of DNA after direct uptake is sometimes lower than for vector-mediated gene transfer into plants (discussed later), but there are no species restrictions on the type of host cell. However, protoplasts are used as recipients, so they must be capable of re- generating into plants for direct uptake to yield genetically altered species for agriculture. Insect, fungal, yeast, and bacterial cells are all amenable to variations of calcium phosphate or other chemical treatments for direct DNA uptake. Often, direct uptake is used to introduce vector DNA molecules containing engineered genes. Direct uptake procedures simply place foreign genes inside the cell; vectors can help to integrate the genes into the cell's chromosomes or stably maintain the genes within the cell on the vector's minichromosome (see the section on Vector-Mediated Gene Transfer). Electroporation A newer method that is being widely adopted is electropora- tion (Neumann et al., 1982; Potter et al., 1984~. Cells are mixed with DNA in solution and subjected to a brief pulse of electri- cal current. It is thought that the current pulse creates transient pores in the cell's membrane that allow DNA to enter efficiently.
APPENDIX: GENE TRANSFER METHODS 157 Electroporation may work for any type of cell, even those that have resisted DNA uptake by chemical treatments, for example, cells of the immune system. Electroporation can introduce DNA into protoplasts of both major categories of plants dicots (e.g., carrots and tobacco) and rrlonocots (e.g., corn; Fromm et al., 1985~. Electroporation pro- vides a transient gene expression system for plants. As discussed previously, transient expression systems are very useful for pre- liminary characterization of new genes. The lack of such a system for plants had previously held up progress in characterizing plant genes. Electroporation also permits stable integration of genes into plant chromosomes. It has been used successfully to stably transform corn and tobacco cells (Fromm et al., 1986; Schocher et al., 1986; Shillito et al., 1985~. DNA MICROINJECTION DNA can be injected directly into single living cells using very fine glass pipettes (hollow needles). Experimenters use an elabo- rate apparatus consisting of a microscope and delicate microma- nipulators to view the cell, hold it steady, and inject a solution containing DNA. As with chemical or electrical uptake methods, foreign genes can be in the form of isolated molecules or attached to vectors. A disadvantage compared to direct uptake is that rela- tively few individual cells can be injected; however, the frequency of successful incorporation of DNA per injected cell is higher. Animals Microinjection has been very successful for delivering foreign genes into mouse embryos at an early stage of development. Usu- ally DNA is injected directly into a particular structure, the male pronucleus, of a fertilized mouse egg. This is the most receptive structure to the incorporation of foreign DNA. The embryos are subsequently reimplanted into foster mothers for development to term. Foreign genes are incorporated into the developing cells' chromosomes and are often present in every cell of the mature animal. Animals given new genes by this procedure are called "transgenic." Their new genes are usually passed on normally to their progeny. These foreign genes can be expressed, that is, make their protein products, which can confer new characteristics on
158 A GRICULTURAL BIO TECHNOLOGY the animal. The now classic example is transgenic mice contain- ing foreign genes for growth hormone. Expression of these genes caused the mice to grow to up to twice their normal size (Hammer et al., 1984; PaIm~ter et al., 1983~. Many other animal genes have now been transferred into fer- tilized mouse eggs by m~cro~njection and correctly expressed in the resulting mature mice. These include the chicken transferrin gene expressed in the liver (McKnight et al., 1983~; a mouse im- munogIobuTin gene expressed in the spleen (Brinster et al., 1983~; the rat elastase gene expressed in the pancreas (Swift et al., 1984~; the rat skeletal muscle myosin gene expressed in skeletal muscle (Shari, 1985~; a chimaeric mouse/human ,B-gIobin gene in blood, bone marrow, and spleen (Chada et al., 1985~; and a swine histo- compatibility gene (Frels et al., 1985~. Traits of potential economic value to the farmer that might be transferred by m~croinjection Include increased levels of certain circulating hormones, antibiotic resistance, and ~mmunogIobulins (antiboclies) for "genetic vaccination" against pathogens. As noted previously, the introduction and expression of such genes has been successful in mice. A necessary supporting technology for in vitro microinjection of mammalian embryos is embryo transfer into surrogate mother animals, for in viva development of the embryos to term. Embryos of each livestock species must be handled in a slightly different manner, which must be experimentally determined. Hammer and his collogues (1985) reported the successful pro- duction of transgenic farm animals (rabbits, sheep, and pigs) by microinjection. The same foreign gene for growth hormone used to produce transgenic mice was used for these other species. New techniques were needed to visualize pronuclei for microinjection, because of differences in the fertilized eggs of each species. The m~- croinjected gene was integrated into the chromosomes of all three species, and was expressed in some of the transgenic rabbits and pigs. Scientists have been very successful in m~croinjecting genes into embryos of the laboratory fruit fly Drosophila melanogaster for studies on the molecular biology of this insect. Rubin and Spradling (1982) pioneered this approach with their transposable P-element vector (discussed in the section on Vector-Mediated Gene Transfer). This vector or others similar to it might be
APPENDIX: GENE TRANSFER METHODS 159 adapted for both beneficial and harmful insects of agronomic im- portance. Researchers routinely m~croinject genes into frog eggs, which are very large and metabolically active cells, for basic studies on gene expression in animals. More recently, m~croinjection was used to transfer DNA into the chromosomes of developing fish eggs (Chourrout et al., 1986; Zhu et al., 1985~. Projects are aimed at basic studies of fish molecular biology and questions of how fish respond to their environment at the molecular level, as well as at aquacultural applications. Both bovine and fish growth hormone genes have been in- jected into fish eggs. It has already been shown that injection of the purified protein hormones augments fish growth (Gill et al., 1985; Sekine et al., 1985~. Transferred genes should be even more effective than purified hormones in promoting fish growth. Re- searchers have injected metaDothione~n genes from both mammals and fish into fish eggs, with the goal of engineering fish resistant to toxic metals. They have Injected "antifreeze" genes obtained from winter flounder (also found in all antarctic fishes) to increase the cold tolerance of commercially valuable fish. Plants Microinjection can be used to deliver genetic material into plant cells. Segments of DNA, whole chromosomes, and even cellular organelles such as chioroplasts, which contain their own DNA molecules, can be micro~jected by methods used for animal cells, although certain physical properties of plant cells complicate the technique. Key elements for protoplast m~croinjection include m~cro- scopic resolution of the cell nucleus, which ~ enhanced by staining with dyes; immobilization of the cell by a holding pipette, embed- ding within agarose, or adhesion to glass surfaces; and efficient cell culture techniques. Researchers can successfully transform up to 14 percent of the cells they m~croinject with DNA (Crossway et al., 1986~. This high frequency might be increased further by using rn~cro~njection ~ conjunction with specially developed vec- tors, derived from the Ti plasmid or plant transposable elements (see sections on these vectors). Because of the high transformation frequency possible with microinjection, a direct selection scheme
160 AGRICULTURAL BIOTECHNOLOGY (e.g., drug resistance) is unnecessary. Furthermore, specific host- range requirements associated with the Ti plasmid or viral vectors are obviated. Although at present the recipient plant species must be amen- able to cell culture and regeneration from protoplasts, suspension cultures or pollen grains may be used in the future, which would bypass the problem of regeneration. Alternatively, DNA may be injected into the developing floral side-shoots of plants, where it can pass into germ cells. Researchers have reported that the cereal rye (a monocot) can be transformed in this way (de la Pena et al. 1987~. Microinjection of individual chromosomes or cellular organel- les (e.g., chIoroplasts, mitochondria, and nuclei) could potentially produce improved cultivars with new traits such as herbicide resis- tance or cytoplasm~c mate sterility. Transfer of traits by microin- jection would be more direct, precise, and faster than by breeding or cell fusion (described in the next section), because microinjec- tion transfers a specific, limited amount of genetic information. There would be less need for selection or backcrossing, which are often time-consuming, difficult processes. Most agronomic traits are polygenic, that is, they are caused by the interplay of several different genes in the plant. Genetic studies often reveal that these genes are linked in blocks on specific segments of chromosomes. Classical plant breeding can sometimes transfer such traits between species via interspecific crosses, but these crosses are not always successful. Transfer of individual chro- mosomes would permit researchers to introduce traits that result from the interaction of several genes linked on that chromosome. Chromosome microinjection would also enable the transfer of traits that are encoded by single genes that have not yet been identified and isolated. Much of the sophisticated biochemistry and genetics of single-gene traits known for animals and used to isolate important genes is lacking for plants. Consequently, few plant genes of agronomic importance have been isolated. Whole- chromosome transfer may allow scientists to genetically engineer plants that would not be tractable at this time by more sophisti- cated gene-splicing (recombinant DNA) techniques. Attempts are being made to transform plant cells by microinjection of isolated chromosomes (Greisbach, 1983, 1987~.
APPENDIX: GENE TRANSFER METHODS CELL FUSION 161 Cell fusion combines the entire genetic contents of two cells, producing hybrid cells that often express certain traits from both parents. The parent cells can be from different species or from different types of the same species. E`usion is usually mediated by chemicals such as polyethylene glyco} or dimethylsulfoxide, although newer techniques use electrofusion. Animal Cells Cell fusion is the basis for the manufacture of monoclonal an- tibodies. Monoclonal antibody-producing cell lines (hybridomas) are created by fusing antibody-producing B-celIs from animals with myeloma cells, which grow indefinitely in culture. The pure, highly specific antibodies thus obtained are important reagents for research, medicine, and agriculture. Diagnostic kits and vaccines for animal health based on monoclonal antibodies are already on the market (Gamble, 1986~. Diagnosis of plant pathogens such as viruses, bacteria, fungi, and nematodes can also be facilitated by tests based on monoclonal antibodies; commercial products should be available in the near future (Gamble, 1986~. Certain agricultural applications have been held back by lack of suitable myeloma lines for fusion with B-celis from farm an- imals, as opposed to standard laboratory animals such as the mouse. However, this problem can be surmounted by creating hy- bridomas by direct DNA uptake. DNA from B-celIs and myeloma cells is simultaneously introduced into recipient cells by calcium phosphate coprecipitation or by electroporation (Gamble, 1986~. This approach obviates the need to fuse interspecific cell lines, and thus solves the problem of finding suitable myeloma lines for different livestock species. Fusion of animal cell lines in culture is also exploited to map genes to specific chromosomes, an important step in locating genes to use in transfer experiments ant] in breeding strategies. Gene maps for mice and men are quite advanced. Those for livestock lag behind, but efforts are starting, notably for swine (Fries and RuddIe, 1986~. To map these genes, swine cells are fused to mouse cells in culture. The interspecies cell hybrids reject most of the swine chromosomes. Ideally, a set of cell lines, each harboring
162 AGRICULTURAL BIOTECHNOLOGY a single different swine chromosome, is made. Known DNA se- quences are used as probes for particular genes with those se- quences. These probes bind to defined lengths of DNA from the fused cells. Because swine and mouse chromosomes can be distin- guished by small differences in DNA sequences (known as restric- tion fragment length polymorphisms), differences in the lengths of DNA containing the gene detected by the probe indicate whether that gene is on a swine or a mouse chromosome of the hybrid cell. Location on a swine chromosome pinpoints the gene to that single particular swine chromosome, which is the only swine chromosome in the hybrid cell. Gene mapping is expected to play an important role in finding genes for transfer of complex traits in livestock, such as lactation, fertility, growth, and disease resistance. Plant Cells In eucaryotic cells the cytoplasm that part of the cell sur- rounding the nucleus-contains organelles that have their own separate DNA. In plants, protoplast fusion is used to transfer genes from both the nucleus and the cytoplasm. Fusion combines the genomes of two parents, as in traditional breeding, but results can sometimes be obtainer! faster, even though the fusion product must be backcrossed to the recipient line for several generations to create a new, stable line possessing the one trait desired from the donor. Protoplast fusion can be used for transferring genes that are hard to identify, isolate, and clone or for polygenic traits. Furthermore, protoplast fusion can be used for plants that cannot be crossed sexually (although plants regenerated from such fused hybrids may sometimes be sterile). Most commonly, cells from closely related plants are fused in order to transfer one particular trait from the donor plant into the recipient. For example, a single dominant nuclear gene for resistance to tobacco mosaic virus (Evans et al., 1981) and a polygenic trait for hornworm resistance (Bravo and Evans, 1985) were transferred into tobacco lines by this method. Traits from a wild species can be introduced into a related cultivated species. Cells of wild and cultivated potato plants were fused to transfer the wild species' resistance to potato leaf roll virus (Austin et al., 1985~. The hybrids were fertile, bore tubers like those of the cultivated species, and were resistant to the virus.
APPENDIX: GENE TRANSFER METHODS 163 Cytoplasmic (mitochondrial and chIoroplast) traits can be transferred by fusing a donor cell whose nucleus has been inacti- vated, usually by irradiation, with an intact recipient cell to form a "cybrid." Initially, the cybrid contains the active nucleus of the recipient cell along with mitochondria and chIoroplasts from both the donor and recipient cells. However, progeny cells that contain mitochondrial or chloroplast genotypes from one parent only quickly segregate. Plants are then regenerated from cells that harbor the desired donor cytoplasm~c genotypes. Both cytoplas- mic male sterility (m~tochondria) and resistance to the triazine class of herbicides (chIoroplast) have been transferred into a single Brassica line via cybrid formation (Pelletier et al., 1983~. VECTOR-MEDIATED GENE TRANSFER A vector is a molecule of DNA that is attached to a foreign gene to facilitate its transfer, maintenance, and expression within the target cell. Vectors offer many advantages: high frequency of gene transfer, transfer into specific cell types, more control over the final copy number of a transferred gene, and certain properties that make them easy to track, permit them to be stably maintained in the target cell, and enable them to express foreign genes. Vectors can, therefore, greatly improve gene transfer. However, different species and cell types may require different types of vectors, and often much work must go into creating an appropriate vector system before genes can be transferred into a specific organism. Animal Viruses SV40 AND ADENOVIRUS The first vectors developed for animal cells were derived from simple DNA viruses, which were relatively easy to manipulate by recombinant DNA techniques. Extra DNA, coding for foreign genes and for special markers (~tags~) to track their progress, are inserted into the virus's chromosome. These passenger genes can be expressed via their own regulatory sequences or, sometimes more efficiently, via those of the virus. The first animal virus used was SV40 (sirn~an virus 40; Hamer et al., 1979; Mulligan et al., 1979~. Fundamental studies on SV40 by Paul Berg and his coworkers laid the groundwork for their and
164 AGRICULTURAL BIOTECHNOLOGY other groups' subsequent development of it and other viruses as vectors for gene transfer, and earned Berg a Nob e! Prize in 1980. SV40 can exist within the host cell both as an independent circular molecule or as a segment integrated in the host's DNA. This versatility, along with its well-characterized life cycle and gene regulation, have given researchers great flexibility in designing vector systems based on SV40. SV40's drawbacks are that it normally infects only cells of certain species (notably primates) and is severely limited in the amount of DNA it can carry. Only about 2,500 base pairs (the size of one small animal gene) can be added to this virus, and even this addition must be compensated for by deleting some of its own DNA. Adenoviruses infect a wider variety of mammalian species than does SV40. Their DNA is a very long, linear molecule, which like SV40 can either replicate to give a high copy number of independent molecules or insert itself into the host's DNA in a low copy number. The molecular biology of adenoviruses has been well studied and like that of SV40, has provided fundamental insights into eucaryotic gene regulation. Adenovirus vectors have several advantages over SV40 and retroviruses (which are discussed later). Adenovirus can acco- modate large, complete passenger genes with their own control sequences. Furthermore, two different genes at widely separated locations can be accomodated on the same vector molecule, per- mitting separate and distinct control of the two passenger genes within one cell. In addition, hybrid viruses composed of both ade- novirus and SV40 can give even greater flexibility in control of gene expression and extend the host range for gene transfer (van Doren and Gluzman, 1984~. Several developments with SV40 and adenoviruses are of par- ticular interest. These viruses have been used to transfer genes into cells of diverse origin, notably mouse and human bone marrow cells (KarIsson et al., 1985~. Transient expression" with a recom- binant SV40 vector was obtained at much higher frequency than with the calcium phosphate procedure. However, the recombinant SV40 vector did not integrate into the cells' chromosomes. With adenovirus-mediated transfer, one to three copies of foreign genes were transferred intact at very high frequency and maintained sta- bly in the host cells' chromosomes. This low-copy number, stable
APPENDIX: GENE TRANSFER METHODS 165 integration is desirable for certain studies of gene regulation and for permanent genetic modification of animals. Viral vectors can also be used for large-scare production of specific proteins in cultured animal cells. Although proteins can sometimes be efficiently manufactured in bacterial or yeast cells, many animal proteins are not correctly processed and assembled by ceils of simpler organisms. In these cases it may be more efficient to manufacture proteins in cultured animal cells. To be economically feasible, protein manufacture by recom- binant DNA technology must yield large amounts of the desired product. Researchers have developed SV40 and adenovirus vec- tors that meet this requirement by expressing any inserted gene at a high level (Ready et al., 1985; Yamada et al., 1985~. The researchers made these "expression vectors" by connecting viral regulatory sequences that normally cause high-level production of proteins needed in huge quantities by the virus (e.g., coat proteins, which encase the thousands of viruses produced during infection of a cell) to genes for commercially desired proteins such as the hormone human choriogonadotropin, which is important in main- taining pregnancy. The expression vectors exploit the facts that many copies of viral DNA accumulate inside the cell and that each of these copies produces great quantities of the desired protein. BOVINE PAPILLOMA VIRUS Bovine papilloma virus (BPV) is another DNA virus under study and development as a vector for transferring mammalian genes (Server et al., 1981, 1982~. This virus does not integrate its DNA into the host cell's chromosome. Instead, the vector with its passenger DNA is maintained as an extrachromosomal DNA molecule, which usually replicates to give about 100 copies of the transferred gene in every cell. The extrachromosoma] maintenance and high copy number are advantageous for "transient expression" assays, detailed studies on gene expression, and production of proteins in quantity. An additional attribute is that BPV can carry large amounts of DNA-up to 20,000 base pairs. The circular shape of BPV's DNA and its ability to maintain itself as an independent chromosome have enabled scientists to further engineer BPV (as well as SV40) vectors to replicate in both marnrnalian and bacterial cells. Researchers use these Shuttle
166 AGRICULTURAL BIOTECHNOLOGY vectors" to move cloned genes back and forth between mammalian and bacterial cells for ease of study and manipulation. Drawbacks to BPV vectors are that the engineered DNA molecules are sometimes unstable, only a few types of cells (usually epithelial) can serve as hosts, and applications may be limited to cultured cells. Furthermore, in contrast to SV40, the basic biology of BPV is only now being characterized. Thus, researchers need to pursue fundamental studies on BPV's life cycle and regulatory mechanisms before optimal BPV vectors can be designed. VACCINIA VIRUS Vaccinia is a very large and complicated DNA virus. It is famous for its role as the vaccine used to eradicate the deadly human disease smallpox in this century. Although vaccinia is similar enough to the smallpox (variola) virus to immunize ~.F~.in~. it, vaccinia itself does not cause disease. Poxviruses are unique in that they set up shop in the cell's cytoplasm, unlike other viruses, which head for the cell's nucleus. Vaccinia expresses its genes in the cytoplasm using its own en- zymes, which respond to vaccinia's regulatory sequences but can- not recognize those of the host cell. Therefore, when Vaccinia is used as a vector for foreign genes, these genes are expressed only if they are hooked up to vaccinia's own regulatory sequences. Among its advantages is vaccinia's ability to grow easily in cell culture. By inoculation into the skin, it can also infect a wide range of animal hosts, making it a versatile vector. Moreover, sirn~lar poxviruses could be used as vectors for additional species. Because Vaccinia is so large, it can. accomodate more inserted DNA than any other virus amounts greater than 25,000 base pairs are stable (Smith and Moss, 1983~. This is more than 10 times the carrying capacity of SV40, and covers the size of several genes. Vaccinia has two natural safety features: it does not integrate into its host's DNA. O and it cannot become latent (i.e., persist in a dormant state for a Tong period). In addition, the virus can be attenuated further by genetic engineering. Scientists can insert passenger genes into the virus's gene for the enzyme thymidine kinase, thereby inactivating it. Because this enzyme is needed for optimal growth of the virus, Vaccinia recombinants cannot spread as easily as the normal virus (Buller et al., 1985). In
APPENDIX: GENE TRANSFER METHODS 167 addition, viruses without thymidine kinase can survive treatment with a drug that kills the normal virus, enabling rapid laboratory detection of the desired recombinants. Because the large vaccinia DNA molecule is too cumbersome to handle in vitro, foreign genes must be transferred onto the vac- cinia vector by a two-step process. First a small circular "insertion vector" is built in vitro. This vector contains the foreign gene, sur- rounded by cloned DNA from vaccinia's thyrn~dine kinase gene. Second, animal cells are infected with normal vaccinia virus, and then insertion vector DNA is added to the infected cells by direct DNA uptake. Inside the cells an exchange occurs between the thymidine kinase sequences on the insertion vector and the identi- cal (homologous) sequences on the viral DNA, placing the foreign gene into the viral DNA. The foreign gene interrupts the thymi- dine kinase gene, inactivating it as described in the preceeding paragraph (Mackett et al., 1982~. The most important use of the vaccinia vector will be for the production of vaccines against viruses and parasites that have re- sisted conventional vaccines. Furthermore, a single recombinant vaccinia virus can carry antigenic genes from several disease agents or several strains of a virus like influenza. Thus vaccinia can im- munize against several diseases in one shot (Perkus et al., 1985~. Importantly, vaccinia vaccines not only stimulate antibody pro- tection but also confer long-lasting cellular immunity (Benn~nk et al., 1984~. Recombinant vaccinia vaccines for major diseases of livestock (e.g., vesicular stomatitis virus, swine gastroenteritis) and for ra- bies, influenza, herpes simplex, hepatitis B. and some elements of malaria have already been successful In animal tests (Cremer et al., 1985; Mackett et al., 1985; Moss et al., 1984; Paoletti et al., 1984; Wiktor et al., 1984~. Because of its wide host range, vaccinia can immunize a large variety of animal species. Like the original smallpox vaccine, the vaccines would be cheap, easy to manufac- ture, dispense, and administer, and stable without refrigeration as freeze-dried preparations ideal for field use. RET ROVIRU S ES Retroviruses are a fondly of viruses that contain RNA as their primary genetic material. On infection of a host cell, the RNA ~
168 A GRI C UL TUNA L BI O. TECHN'OL O G Y copied into DNA, which then inserts itself into the host cell's chro- mosome, becoming a stable part of the host's genetic information. Retroviruses have been found in association with many animals, including humans, and probably exist for all agriculturally impor- tant animal species. There are several particular advantages to retroviral vectors (Anderson, 1984~. They can infect a high percentage of the target cells, integrate in one copy at a single site in the cell's genome, and reliably express the foreign gene. Other methods often lead to the transfer of multiple copies of the gene, which may interfere with its correct expression. Retroviruses are currently the focus of intense research on both their basic biology and their use as vectors. For example, engineered retroviruses can infect bone marrow cells in culture. These transformed cells can then be transplanted back into the animal. A gene introduced in this way may be able to correct a genetic defect in an animal or human, although it would not be inherited by the individual's progeny. However, infection of germ line cells of early embryos of animals should allow heritable traits to be transferred for breeding purposes in agriculture. The first key experiments in the use of retroviral vectors con- centrated on the transfer of genes for drug resistance into blood- producing cells of the mouse (Joyner et al., 1983; Williams et al., 1984) and of genes for the enzyme human hypoxanthine phospho- ribosy~transferase (HPRT), whose absence causes Lesch-Nyhan syndrome, into mouse or human ceils (Miller et al., 1983, 1984a; Willis et al., 1984~. The HPRT gene functioned in both mouse and human cells in culture, as well as in live mice. Further experiments demonstrated efficient transfer of a rat growth hormone gene into mouse cells by retroviruses and correct expression of the gene by its own regulatory sequences (Miller et al., 1984b). More recently, ,6-gIobin genes were transferred into and correctly expressed in lines of transgenic mice (Soriano et al., 1986~. This demonstrates that retroviruses can deliver genes into the germ cells of early embryos so that the genes are inherited normally and function in intact animals. The engineering of safe retroviral vectors involves some genetic tricks to ensure that the virus will not be able to reinfect other cells or spread to other organisms after the desired transfer of genes. In constructing the vector some of the retrovirus's own
APPENDIX: GENE TRANSFER METHODS 169 genes are replaced with foreign passenger genes, depriving the virus of the ability to replicate itself. To overcome this handicap, a so-called "helper virus" is used, which provides gene products that the engineered retrovirus can no longer make. These essential products are the enzyme for replication and the proteins for the virus coat. For the purpose of safety and efficiency the helper virus is debilitated by the removal of a small portion of the genetic material necessary to its reproduction. The helper is maintained only as an integrated "provirus" in a cell line; it is a permanent part of the cell's DNA and cannot become infectious. The handicapped vector retroviruses that carry foreign genes are propagated in this cell line, aided by the replication and coat proteins manufactured by the helper provirus. Vector viruses are then purified away from the cells containing the helper provirus. These purified vectors now can enter other target cells and integrate the foreign gene into the target cells' genome, but that is all they can do-without the helper provirus they cannot replicate in the target cells to produce more infectious viruses. Thus the retroviral vector is a gene delivery system, not an infectious agent. The vector can be further disabled by engineering a defective regulatory sequence at one end of its genome. Such vectors inte- grate into the host's chromosome, and then become stuck. Even in the presence of the helper virus, they cannot express their viral genes, replicate further, or move out of the cell's chromosome. Foreign genes transferred in by these vectors are expressed from their own regulatory sequences. Retroviral gene transfer vectors applicable to agricultural an- imals have been developed. One system based on a turkey retro- virus efficiently delivers genes into avian and some mammalian cells (Watanabe and Temin, 1983~. Another retrovirus system can introduce genes into a broad range of mammalian species, including farm animals (Cone and Mulligan, 1984~. Thus, just a few retroviral vectors may serve for genetic engineering of many livestock species.
170 AGRICULTURAL BIOTECHNOLOGY BACULOVIRUSES Baculoviruses, which infect lepidopteran insects, should have uses in agriculture for manipulation of both beneficial and harm- ful species. They have already been used to express human ,B-interferon (Smith et al., 1983), c-myc protein (Miyamoto et al., 1985), interIeukin 2 (Smith et al., 1985), and bacterial ,3- galactosidase (Pennock et al., 1984) in cultured insect cells, and human a-interferon in silkworm larvae (Maeda et al., 1985~. Baculoviruses have some similarities to vaccinia virus in the way they are engineered for gene transfer (Miller et al., 1986~. Their large, double-stranded DNA genome may accomodate up to 100,000 extra base pairs of DNA, due to the virus's extendable rod-shaped structure. Insertion of genes into such a large DNA molecule is accomplished via small insertion vectors, as described previously for vaccinia. Viral and insertion vector DNA are simul- taneously introduced into insect cells by direct uptake using cal- cium phosphate. Homologous recombination in viva then places the foreign genes from the insertion vector into the baculovirus genome. Foreign genes are most conveniently inserted into the virus's gene for polyhedrin. This strategy has several benefits. First, insertional inactivation of the polyhedrin gene gives an easily de- tected recombinant virus phenotype, because these viruses form areas of infected cells that look different from those made by the normal virus. Second, viruses with a defective polyhedrin gene cannot be transmitted between host insects; they can move only from cell to cell within a single insect or cell culture. Thus the re- combinant baculoviruses have a built-in safety feature. Third, the regulatory sequence (promoter) of the polyhedrin gene can express foreign proteins at high levels, as over 20 percent of the infected cell's messenger RNA and protein are normally made from this gene. Foreign genes cloned in baculoviruses can also be expressed from their own promoters. A bacuTovirus, high-level expression system could be used to manufacture commercially useful proteins, as bacuToviruses can be mass-produced in insect cell cultures. Baculoviruses might be particularly advantageous for the manufacture of insect-derived substances such as pheromones, which can be used for biological control of insect pests.
APPENDIX: GENE TRANSFER METHODS 171 Baculoviruses infect many lepidopteran insect species and can themselves be used as insecticides. Their effectiveness as biological insecticides may be augmented by genetic engineering, for exam- ple, by introduction of insect-specific toxin genes. Because bac- uloviruses infect only invertebrates, with different baculoviruses being relatively specific for certain lepidopteran insect hosts only, they should not spread indiscriminantly to other insects, animals, or plants. Plant Viruses CAULIFLOWER MOSAIC VIRUS Only small steps have been taken with viral vectors for plants, in contrast to the great strides in virally mediated gene transfer into animals. There are no known plant retroviruses and only a few, small DNA viruses. The best-studied virus is cauliflower mosaic virus (CaMV), a small double-stranded DNA virus that infects cruciferous plants, such as cabbage and mustard. CaMV is transrn~tted in nature by aphids, but its DNA can infect plants if simply rubbed onto their leaves. CaMV causes systemic infection and replicates abundantly throughout the plant. It thus should transfer many copies of a gene per cell into all tissues of a mature plant. furthermore, powerful CaMV gene regulation sequences can promote high-level expression of foreign genes. In fact, CaMV promoters are being used to augment the expression of plant genes transferred via other systems, as most plants recognize these pro- moters even when they are cletached from the rest of CaMV. The biggest obstacles to the development of a CaMV vector have been the severe limitation on the virus's size and thus on the quantity of DNA that can be inserted, and the instability of the genetically engineered virus. This instability may be caused both by the packaging limitation on extra DNA and by the way the virus replicates. Furthermore, CaMV does not integrate into plant genomes under normal conditions of infection. Some success in introducing foreign genes into plants using CaMV has been reported, however. Bacterial drug resistance genes were expressed and stably propagated in Ca~V-infected turnip plants (Brisson et al., 1984~.
172 AGRICULTURAL BIOTECHNOLOGY G EMINIVIRUSES Geminiviruses are single-stranded DNA viruses of plants that are transmitted by insects, such as leafhoppers. Viruses in this group infect many crops, including the monocots wheat and corn and the dicots beans, tobacco, and tomatoes. Work on developing a vector system based on these viruses is in progress (Krid] and Goodman, 1985; Lazarowitz, 1987~. Recently published experiments indicate that geminiviruses can be combined with the Agrobacterium Ti plasmid delivery sys- tem (described in a subsequent section) to obtain "agroinfection" of corn plants with the geminivirus maize streak virus (Grirnsley et al., 1987~. This dual system may prove useful in introducing engineered geminivirus vectors into plants, because often their DNA is not infectious unless transmitted as as intact virus by the natural insect mechanism. These experiments also demonstrated that AgTobacterium can transfer DNA to corn, a monocot, which was thought not to be amenable to the Ti plasmid gene transfer system. RNA VIRUSES Although there are many known plant RNA viruses, progress has been limited by the fact that manipulations developed to recombine DNA cannot be done on RNA directly. However, sci- entists can construct complementary DNA copies of RNA virus genomes. These copies can be used to construct a vector that will carry a foreign gene. The DNA can then be transcribed back into RNA, enabling the engineered virus to infect cells. Brome mosaic virus (BMV), which infects monocots includ- ing the important cereal crops has been developed as a vector in this way by Ah~quist and coworkers (Ah~quist et al., 1984; Ah~quist and Janda, 1984; French et al., 1986~. These researchers achieved transfer and expression of a bacterial drug resistance gene in barley protoplasts. The vector replicated rapidly within the cells, and the foreign gene, under the control of the p owe rfu] BMV promoter, was expressed at high levels within 20 hours of infection. Although the plant remains infected with the virus, symptoms of infection vary greatly for different virus/host combinations. Sometimes symptoms are very mild: Wheat in some parts of the world is always infected with BMV to little effect, whereas
APPENDIX: GENE TRANSFER METHODS 173 infected barley suffers stunted growth. Desirable vectors would produce mild or no symptoms in the host plant and would not affect the plant's productivity in the field. These characteristics might be further improved by genetic engineering of the vector virus. Control of viral disease, rather than introduction of a new trait, may be possible through exploitation of a natural phe- nomenon involving RNA viruses and their associated viral satel- lites. These are small nucleic acids that require the helper func- tions of a bona fide virus to replicate. They often attenuate the disease symptoms caused by that virus. Because satellites replicate rapidly at the expense of their helper, this molecular parasitism may provide a basis for viral disease control. Chinese scientists have placed RNA satellites in pepper plants in the field, but with- out gene transfer into the chromosome. The plants resisted viral infection (Tien and Chang, 1983~. Incorporation of satellite genes into the plant's chromosome could build in protection against disease symptoms caused by the helper virus (Kaper and Tousignant, 1984~. To this end, British scientists have tranferred DNA copies of a viral satellite into the genome of tobacco plants, using the Ti plasmid system for gene transfer. The DNA copies functioned to produce satellite RNA (BauIcombe et al., 1986~. Further testing is needed to determine whether the plants are resistant to viral disease. In a related development, American scientists engineered a single gene of the RNA virus tobacco mosaic virus (TMV) into tobacco and tomato plants via the Ti plasmid vector. Expression of this gene by the host plants made them resistant to infection by TMV (Abe! et al., 1986~. TMV causes large Tosses worldwide on cash crops such as tobacco, tomatoes, and bell peppers. Viral satellites might also serve to transfer foreign genes into plants directly, in the manner described for BMV. There are several potential advantages to RNA virus vector systems for plants. First, upon infection with cloned DNA or in vitro RNA transcripts, the plant should express the new trait im- mediately, in contrast to the Ti plasmid system (discussed in a subsequent section), in which a long regeneration process is usu- ally necessary to obtain a transformed plant. Second, expression of the virus as an extrachromosomal, self-replicating RNA molecule means that gene expression wfl! not be influenced by "position
174 AGRICULTURAL BIOTECHNOLOGY effects" due to insertion in undesirable places in the plant's chro- mosomes. Third, gene expression via the strong viral promoter, coupled with template replication to give many gene copies, would allow the production of large amounts of specific gene products within plant cells. Fourth, RNA viruses suitable to this strategy probably can be found for any host plant. One possible problem with the use of RNA vectors, or any vector that replicates through an RNA intermediate (e.g., CaMV, retroviruses, and some transposons isee the next section]), is the high error rate associated with RNA replication. This might cause mutations detrimental to the foreign genes or to the vector itself during its replication cycle (van VIoten-Doting et al., 1985~. Transposable Elements Transposable elements (also called "transposons") can move from place to place within an organism's genome and take extra pieces of DNA along for the ride. These elements have some phys- ical and functional properties in common with retroviruses, but they do not spread from cell to cell by infection and therefore are not considered to be viruses. Barbara McClintock first recognized transposable elements in corn 40 years ago, for which she won a Nobel prize in 1983. Transposable elements have since been found to be widespread in nature: examples have been described in bacteria, yeast, ne- matodes, fruit flies, mice, corn, soybeans, and snapdragons. It is likely that they will be found to exist for all species. Their ap- parent ubiquity in nature may make transposons especially useful for genetic modification of agronom~caIly important insects and plants. Already, transposons have been used to modify Pseu- domonas fluorescent bacteria that live on corn roots by insertion of an insecticidal gene from Bacillus thuringiensis (Obukowicz et al., 1986~. In addition, studies of gene function aided by the use of transposable elements are very important for understanding basic aspects of gene expression in insects and plants. This knowledge is essential to the application of genetic engineering. The jumping abilities of transposable elements have been used to isolate important genes from corn (Fedoroff et al., 1984~. This is done by inducing the transposable element to jump into the corn gene of interest, thereby inactivating the gene and producing
APPENDIX: GENE TRANSFER METHODS 175 a mutant plant. When DNA from the mutant plant is compared to DNA from a normal plant, the characteristic sequence of the transposable element identifies its location, thus acting as a tag for the mutant gene. The DNA surrounding the transposable element is then cloned, yielding copies of the gene of interest. Although these copies are inactive because of the insertion of the transposable element, their sequences can be used as probes to find the active gene copy from a normal plant. This gene isolation strategy contrasts with gene transfer via transposable elements in that for isolation the transposable element is inserted into the gene, whereas for transfer the gene is inserted into the transposable element. The major elements described in plants are Ac, Mu, and Spm in corn, Tgml in soybeans, and Taml in snapdragons. Researchers are trying to adapt these elements as vectors, particularly because they are so prevalent in the monocot crop corn, which has resisted most efforts to transfer genes via the most highly developed plant vector, the Ti plasmid. Engineered as vectors, transposable ele- ments might be microinjected into corn embryos to transfer genes into the germ line, bypassing problems encountered with the in- troduction of DNA into cultured corn cells and the subsequent regeneration of plants. The transposable P-element of the fruit fly Drosophila melano- gaster has proved a very powerful too! for gene transfer in this or- ganism (Rubin and Spradling, 1982; Spradling and Rubin, 1982~. The principle of gene transfer is much the same as that described previously for retroviruses. The gene of interest is inserted into the P-element vector. This disrupts some functions of the P-element required for transposition, but these functions can be provided by a second, helper copy of the P-element. The helper has been engineered so that it cannot transpose itself but can still produce enzymes that cause transposition of the vector. Because transpos- able elements are not infectious in the way viruses are, both vector and helper P-elements must be microinjected into Drosophila em- bryos. The transposase enzyme of the helper acts upon DNA sequences located at the ends of the vector element, causing the vector to insert itself into the host's chromosomes. Large segments of DNA can be transferred in this manner. Transfer is efficient and stable and can be accomplished in the germ cells of the embryo,
176 AGRICULTURAL BIOTECHNOLOGY allowing the new trait to be inherited by future generations. Suc- cessfu] transfer, inheritance, and expression have been achieved with a wide variety of Drosophila genes. It may be possible to adapt the P-element or a similar system to other insects of agro- nomic importance. The Ti Plasluid The most successful gene transfer vector developed thus far for plant cells is the Ti plasmic! found in the soil bacterium Agrobac- terium tumefaciens. Plasmids are circular DNA molecules that ex- ist independently of the cell's main chromosomes; the Ti plasmid is a naturally occurring variety that is quite large. Agrobacterium infects most species of dicots and causes a tumorous disease called crown gall. The disease is instigated by natural gene transfer of part of the bacterium's Ti plasmid, called T-DNA, into the plant's chromosomes. Plant cells acquire new properties as a consequence of the transferred genes. Besides metabolic changes that incite their uncontrolled growth into a tumor, the cells are programmed to manufacture certain chemical compounds called opines, which are used by the parasitic AgrobacteTium as food. Thus Agrobac- [erium [umefaciens is a natural genetic engineer that forces a plant to do its bidding! It inserts its bacterial genes to create tumors composed of altered plant cells that provide it with specialized food. Researchers have adapted the Ti plasmid to transfer foreign genes into plants and to obtain stable and heritable expression of the genes in normal, nontumorous plants. In order to be able to regenerate plants from cells transformed with T-DNA in culture, they modified the Ti plasmid to eliminate its tumor-promo/in properties. Transferred genes can be expressed under the control of their own normal regulatory signals, or T-DNA signals can be used to turn on the foreign genes. A strategy similar to that used for vaccinia is used to insert foreign genes within the T-DNA of the large Ti plasmid: transfer of engineered genes from a small plasmid insertion vector to the Ti plasmid by in viva homologous DNA recombination within Agrobacterium cells. The T-DNA containing the foreign genes is then transferred from the Ti plasrnid within Agrobacterium into the chromosomes of plant cells by its natural process (de Block et
APPENDIX: GENE TRANSFER METHODS 177 al., 1984; Fraley et al., 1983; Herrera-Estrella et al., 1983; Horsch et al., 1984, 1985~. Alternatively, a strategy like that of helper retroviruses or P-elements is used. In this case two separate plasmids are placed within Agrobacterium, one containing foreign genes cloned within the T-DNA's border sequences that enable the DNA segment to move, the other providing the helper functions that catalyze movement. Again, foreign genes contained between T-DNA border sequences are transferred into the plant cell (An et al., 1985; Bevan, 1984; Hoekema et al., 1983~. Plants currently amenable to Ti plasrn~d vectors include petu- nias, tobacco, soybeans, carrots, tomatoes, alfalfa, and oilseed rape. Genes transferred include the small subunit of the plant pho- tosynthetic enzyme ribulose 1,5-bisphosphate carboxylase (Broglie et al., 1984; Herrera-EstrelIa et al., 1984), the bean storage protein phaseolin (Mural et al., 1983; Sengupta-Gopalan et al., 1985), the corn storage protein zein (Matzke et al., 1984), the wheat photo- synthetic chlorophyll a/b binding protein (Lamppa et al., 1985), and a bacterial enzyme for resistance to the herbicide glyphosate (Coma) et al., 19853. Although many experiments focus on a basic understanding of plant gene regulatory mechanisms, experiments with herbicide and pest resistance genes are already introducing agronomic mod- ifications into dicotyTedonous crops such as soybeans, tomatoes, turnips, tobacco, and oitseed rape. Likewise, the nutritional im- provement of seed crops is an important goal. One commercial firm is attempting to transfer the gene for a methionine- and cysteine-rich protein found in Brazil nuts to soybeans to improve their nutritional balance (Altenbach et al., in press). Until recently it was thought that the Ti plasmid could not be used to transfer genes into monocots. This class of plants is not naturally infected by Agrobacterium. However, it now appears that at least some monocots can be transformed by DNA trans- ferred from the Ti plasrn~. T-DNA transfer and expression was demonstrated for asparagus and lilies (Hernalsteens et al., 1984; Hooykaas-van SIogteren et al., 1984), and more recently for corn (Graves and Goldman, 1986~. In addition, the Ti vector can de- liver DNA of a plant virus into corn plants (Grimsley et al., 1987~. It will be an important breakthrough if the powerful Ti system
178 AGRICULTURAL BIOTECHNOLOGY can be usefully applied to the major monocot cereal crops corn, wheat, and rice. An important factor in the use of the Ti plasmid system, as well as in direct DNA uptake and cell fusion methods, is the ability to regenerate whole plants from transformed cells. This still has not been accomplished for several major crops, but recent progress with rice is very encouraging (AbdulIah et al., 1986; Fujimara et al., 1985; Yamada et al., 1986~. Current efforts are also directed toward methods to introduce T-DNA into pollen grains, seeds, and seedlings, routes that bypass the steps of protoplast culture and regeneration. Similar to the Ti plasmid, the Ri plasmid from Agrobacterium rhizogenes can be used to transfer genes into plants (David et al., 1984; Tepfer, 1984~. This plasmid induces root proliferation in affected tissue. The roots are organized plant tissue, in contrast to Ti-induced tumors, which are masses of undifferentiated cells. The fast-growing root cultures are themselves useful for tests of new herbicides and pesticides developed to control pathogens that attack roots (Mugnier et al., 1986~. Furthermore, the Ri plasmid vector can transfer new genes that confer resistance on the plant to herbicides, pesticides, or to the pathogens themselves. Fungal and Bacterial Plasmids Plasmids occur naturally In yeast, fungi, and bacteria. Sci- entists have used plasmid vectors extensively for basic research on the molecular biology of strains of these organisms commonly studied in the laboratory. With recombinant DNA techniques, re- searchers can cut and splice genes into small plasmids quite easily. Likewise, they can combine useful parts from different plasmids to create new plasmas vectors better suited to a particular gene transfer operation. Small plasmids can be introduced into cells by direct DNA uptake. Once inside the cell they replicate and stably maintain themselves and can express foreign genes that have been engineered into them. Furthermore, under certain conditions plas- mids can transfer the foreign genes they carry into the host cells' chromosomes, where the genes can also be maintained and ex- pressed. Thus plasmids are versatile vectors for gene transfer into procaryotes (bacteria) and simple eucaryotes (yeast and fungi).
APPENDIX: GENE TRANSFER METHODS 179 Until recently, transformation systems were lacking for fungi of agricultural and industrial importance. For instance, the fungal corn pathogen Cochtiabolus heterostrophus contains a toxin gene that might be manipulated to create a weed control agent or to develop resistant strains of corn. Progress was stymied, however, until the development of a plasmid-based gene transfer system for C. helerostrophus (Turgeon et al., 1985~. Work on other pathogenic fungi is also progressing. The systems for pathogenic fungi rely on elements of a plasmid vector developed for the laboratory model fungus Aspergitius nidulans (Yelton et al., 1984~. Pathogenic and beneficial fungi and bacteria are important candidates for agronomically valuable gene transfer strategies. Pathogenic fungi and bacteria can be used as biological con- tro! agents for pest insects or weeds. Isolation and transfer of pathogenicity genes has a twofold purpose: construction of im- proved agents for biological control, and discovery of resistance genes in the plant or insect that counteract the pathogenicity. The use of transposons and plasmids to isolate and study pathogenic genes from fungi and bacteria that attack crop plants will lead to an understanding of the molecular bases of many agronom~cally critical diseases and suggest ways to combat them. Beneficial fungi and bacteria may be improved and their host range extended to help other plants and animals. In addition, transformation of beneficial fungi and bacteria should prove ad- vantageous for introducing improved traits for commercial pro- duction of special metabolites such as antibiotics and pigments and for food processing and waste disposal. For example, studies on bacteria with a natural capacity to degrade toxic herbicide and pesticide residues should yield improved strains that may prove useful in detoxifying the environment (Ghosal et al., 1985; Serdar and Gibson, 1985~. Another important aspect of bacterial gene transfer is basic and applied research on strains of Rhizobium that fix nitrogen for legumes. These studies have the following goals: improved strains of Rhizobium, engineered strains of other bacteria that can fix nitrogen for other crops such as cereals, and perhaps even crops that can fix nitrogen themselves. Rhizobium might also be used for the commercial production of ammonia. Bacteria, as described at the outset, are generally easy tar- gets for gene transfer. However, details must often be worked
180 AGRICULTURAL BIOTECHNOLOGY out for species that differ significantly from the laboratory model Escherichia cold An example of bacterial gene transfer for agricultural pur- poses is the transfer of an insecticidal toxin gene from Bacillus thuringiensis to a Pseudomonas puorescens strain that colonizes corn roots, to extend the number of plant hosts that can be pro- tected against pest insects by the bacterial toxin. B. thuringiensis itself has been marketed as an insecticide for many years. Af- ter ingestion, its toxin ~ activated in the insect's gut. There are different strains of B. thuringiensis that make toxins capable of killing over 100 different lepidopteran and dipteran pests. These toxins are harmless except to targeted insects, and delivery via bacteria with a specific range of plant hosts ensures a high level of specificity for the pesticide. Scientists at Monsanto Company have transferred the B. thuringiensis toxin gene into P. pnorescens via a plasmid and also into the P. puorescens chromosome via a transposon (Obukowicz et al., 1986; Watrud et al., 1985~. The new biological insecticide is intended to protect corn against the black cutworm. P. puorescens does not persist in the field, so the genetically engineered bacteria should kill off insects after application early in the growing season and then die. A second strategy is to transfer the toxin gene into crops, to make them self-protecting. Scientists at the Belgian company Plant Genetic Systems engineered the B. thuringiensis toxin gene into plants, which then expressed the toxin and resisted insect predators (Vaeck et al. 1987~. A novel vector for introduction of genes into plants may re- sult from studies on corynebacteria. These microbes colonize grasses, including wheat, corn, and sorghum. Some species are pathogenic to plants, others are harmless. Corynebacteria have their own plasmids, into which foreign genes could be inserted, and their proteins produced by the corynebacteria could easily be secreted through bacterial cell walls. Thus, foreign proteins expressed within corynebacteria might be made reaclily available to the plant host. Candidate products include insecticides, her- bicides, antibiotics, and growth regulators. Japanese researchers have transformed certain food strains of corynebacteria and intend to use genetic engineering to improve their commercial production of amino acids. Transformation of field strains has proved more
APPENDIX: GENE TRANSFER METHODS 181 difficult but should soon be feasible (A. Vidaver, personal commu- nication). PROSPECTS Molecular biologists have made tremendous strides since the early 1970s in experimental gene transfer and expression. Tech- nology and the knowledge on which it is based continue to advance rapidly. It is truly remarkable that within 15 years, gene transfer has evolved from an esoteric technique practiced by a few bacte- rial and viral geneticists to a popular procedure that researchers in disparate biological fields use for wide-ranging studies. This review has described major gene transfer methods with immediate potential for agricultural research. Diverse techniques are available: direct uptake of DNA, microinjection of DNA, cell fusion, and gene delivery by an array of vectors. Although details differ among animals, plants, and bacteria, underlying principles do not. Thus progress with one organism may have application to other systems by analogy. A recent example is electroporation, di- rect DNA transfer in a highly charged electric field. First achieved in 1982 with cultured mammalian cells, it has been widely adopted and further adapted for plant cells. Likewise, general strategies for structuring and manipulating vectors may be applied to many or- ganisms. For instance, helper viruses that contribute essential life- support functions for defective viruses are a paradigm now adapted for gene transfer vectors derived from viruses, transposons, and plasmids. There are still important problems that must be solved, how- ever, in order to design optimal gene transfer systems. Molecular biologists still lack knowledge about many detailed mechanisms governing DNA uptake, integration into chromosomes, and gene regulation. Current approaches for DNA transfer therefore rely largely on experience and observation. They might be vastly improved by a more thorough understanding of the underlying molecular mechanisms. The most serious problem for modification of animals and plants is that of obtaining correctly regulated gene expression in the appropriate tissues of the target organism. Regulation and stability of introduced genes is unfortunately still variable, although much progress is being made. For example, papers have
182 A GRI C UL TURA L BI O TECHNOL O G Y reported correct tissue-specific and developmental regulation of a gIobin gene (for the blood protein hemoglobins in transgenic mice (Chada et al., 1985; Magram et al., 1985~. The transferred gIobin gene was turned on for the first time in fetal blood-forming cells, and was expressed in adult mice only in the blood-forming tissues bone marrow and spleen. This pattern mimics normal regulation of the mouse gIobin gene. However, scientists have not yet devised a way to get genes to integrate at a specific site in animal or higher plant cell chro- mosomes, which would help in obtaining correct regulation and stability of introduced genes. Furthermore, with some current techniques genes are frequently inserted into chromosomes as mul- tiple copies, confounding these problems. DNA rearrangements, lethal insertions, male-sterile mutations, and mosaic organisms in which not all cells contain the new gene sometimes result. Moreover, changes In a gene's expression sometimes occur after transmission of the new gene to progeny. Researchers are working toward the ideal of targeted inser- tion of one stable gene copy that will be sexually transmitted and correctly expressed in all progeny. Fundamental studies of DNA recombination in mammalian ceils that may lead to targeted in- tegration are being carried out (Shau! et al., 1985; Thomas and Capecchi, 1986~. Another strategy to obtain correct gene regula- tion, for instance of gIob~n, is to insert a very large chromosomal segment that contains the gene surrounded by its usual neighbor- ing genes. Genes within such a cluster may be correctly regulated by complex sequences in the surrounding DNA. Currently, very large segments of DNA are difficult to handle and require special vectors to accomodate them. In most cases, researchers wish to keep inserted genes silent during early embryogenesis, and then activate them at the ap- propriate time in the organism's development. However, inserted genes are not always controlled correctly, even when their own regulatory sequences are still attached to them. For example, an inserted growth hormone gene controlled by its own promoter was not regulated correctly in transgenic mice, causing the female mice to be sterile. In contrast, when the same growth hormone gene was attached to the promoter from another gene, metallothionein, it could be turned on or off by raising or Towering the amount of trace metals in the transgenic animals' diets. Researchers could
APPENDIX: GENE TRANSFER METHODS 183 thus control both the amount of growth hormone made by trans- genic animals and the time at which it was made. Transfer of genes into cultured plant cells, from which trans- genic plants will be regenerated, presents an additional problem- the traits targeted for the mature plants may not be expressed in cultured cells and conversely, successful expression in cultured cells may not carry over to plants regenerated from these cells. For ex- ample, a salt-tolerant cell in culture may not yield a salt-tolerant plant when regenerated. The problem of selecting traits in cell culture extends to all plant gene transfer techniques performed on cultured cells, including direct DNA uptake, DNA microinjection, cell fusion, and vector-mediated methods. Scientists are therefore working on adapting existing systems to deliver genes into pollen grains, seeds, and seedlings, which can develop normally into ma- ture plants. This strategy has the additional acivant age of obvi- ating the need for plant cell culture and regeneration techniques for each individual species, which have been stumbling blocks for gene transfer into some agronomically important plants, notably the monocots corn and wheat, with recent progress being made for rice. Development of vectors for these species has also lagged behind, although adaptation of the Ti plasrn~d used for dicots, or transposons from monocots, may prove feasible. Alternatively, direct DNA uptake or rn~croinjection of pollen or embryos might be used. The problems of uptake and subsequent localization of DNA still impede research with some organisms, although these prob- lems are being overcome, for example, in the pathogenic fungi. These problems extend also to compartments of eucaryotic cells other than the nucleus that contain their own DNA mitochondria (the cell's energy powerhouses) and chIoroplasts of green plants (which harness the energy of sunlight through the process of pho- tosynthesis). For instance, the chIoroplast's DNA encodes proteins essential to photosynthesis, and often related to these, proteins involved In herbicide resistance. An important goal yet to be achieved is the directed transport of new DNA into the plant chIoroplast, although there is some experimental evidence to sug- gest that the Ti plasmas might be used (de Block et ale, 1985~. Other possible methods include microinjection of DNA directly into chIoroplasts and introduction of new genes on plasmas that would be stably maintained in chIoroplasts.
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