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Modern Genetic Analysis Chapter 11. Applications of Recombinant DNA Technology
Recombinant DNA Technology in Eukaryotes

The techniques for gene manipulation and cloning were first developed in bacteria but are now applied routinely in a variety of model eukaryotes. As stated in Chapter 2, the genomes of eukaryotes are larger and more complex than those of bacteria, so modifications of the techniques are needed to handle the larger amounts of DNA and the array of different cells and life cycles of eukaryotes. Although eukaryotic genes are cloned and sequenced in bacterial hosts, it is often desirable to introduce such genes back into the original eukaryotic host or into another eukaryotein other words, to make a transgenic eukaryote.

Transgenic Eukaryotes

DNA is introduced into a eukaryotic cell by a variety of techniques, including transformation, injection, viral infection, or bombardment with DNA-coated tungsten particles (Figure 11-9 on the following page). As we learned in Chapter 10, when exogenously added DNA that is originally from that organism inserts into the genome, it can either replace the resident gene or insert ectopically. If the DNA is a transgene from another species, it inserts ectopically. (Vectors that replicate autonomously in eukaryotic cells are rare, so in most cases chromosomal integration is the route followed.)

The possibility of transgenic modification of eukaryotes such as plants and animals (including humans) opens up many new approaches to research because genotypes can be genetically engineered to make them suitable for some specific experiment. (An example in basic research is in the use of reporter genes. Sometimes it is difficult to detect the activity of a particular gene in the tissue where it normally functions. This problem can be circumvented by splicing the promoter of the gene in question to the coding region of a gene, known as a reporter gene, whose product is easily detectable. Wherever and whenever the gene in question is active, the reporter gene will announce that activity in the appropriate tissue. Examples will be given later in the chapter.)

Furthermore, because plants, animals, and fungi form the basis for a large part of the economy, transgenic "designer" genotypes are finding extensive use in applied research. A particularly exciting application of transgenesis is in human gene therapy, the introduction of a normally functional transgene that can replace or compensate for a resident malfunctioning allele.

MESSAGE
Transgenesis, the design of a specific genotype by the addition of exogenous DNA to the genome, has increased the scope of breeding in basic genetic research and in commercial applications.

We now turn to some of the specialized recombinant DNA techniques used in the eukaryotes bakers' yeast, plants, and animals and in human gene therapy.

Genetic Engineering in Baker's Yeast

The yeast Saccharomyces cerevisiaehas become the most sophisticated eukaryotic model for recombinant DNA technology. One of the main reasons is that the transmission genetics of yeast is extremely well understood, and the stockpile of thousands of mutants affecting hundreds of different phenotypes is a valuable resource when using yeast as a molecular system. In yeast, another important advantage is the availability of a circular 6.3-kb natural yeast plasmid. This plasmid, which has a circumference of 2 m m, has become known as the "2-micron" plasmid. It forms the basis for several sophisticated cloning vectors. This plasmid is transmitted to the cellular products of meiosis and mitosis.

The simplest yeast vectors are derivatives of bacterial plasmids into which the yeast locus of interest has been inserted (Figure 11-10a). When transformed into yeast cells, these plasmids insert into yeast chromosomes generally by homologous recombination with the resident gene and by either a single or a double crossover (Figure 11-11). As a result, either the entire plasmid is inserted or the targeted allele is replaced by the allele on the plasmid. Such integrations can be detected by plating cells on a medium that selects for the allele on the plasmid. Because bacterial plasmids do not replicate in yeast, integration is the only way to generate a stable modified genotype with the use of these vectors.

If the 2- m m plasmid is used as the basic vector and other bacterial and yeast segments are spliced into it (Figure 11-10b), then a construct having several useful properties is obtained. First, the 2- m m segment confers the ability to replicate autonomously in the yeast cell, and insertion is not necessary for a stable transformation. Second, genes can be introduced into yeast, and their effects can be studied in that organism; then the plasmid can be recovered and put back into E. coli, provided that a bacterial replication origin and a selectable bacterial marker are on the plasmid. Such shuttle vectors are very useful in the routine cloning and manipulation of yeast genes.

With any autonomously replicating plasmid, there is the possibility that a daughter cell will not inherit a copy, because the partitioning of plasmid copies to daughter cells is essentially a random process depending on where the plasmids are in the cell when the new cell wall is formed. However, if the section of yeast DNA containing a centromere is added to the plasmid (Figure 11-10c), then the nuclear spindle that ensures the proper segregation of chromosomes will treat the plasmid in somewhat the same way and partition it into daughter cells at cell division. The addition of a centromere is one step toward the creation of an artificial chromosome. A further step has been made by linearizing a plasmid containing a centromere and adding the DNA from yeast telomeres to the ends (Figure 11-10d). If this construct contains yeast replication origins (autonomous replication sequences, ARS), then it constitutes a yeast artificial chromosome (YAC), which behaves in many ways like a small yeast chromosome at mitosis and meiosis. For example, when two haploid cellsone bearing a ura+ YAC and another bearing a ura YACare brought together to form a diploid, many tetrads will show the clean 2:2 segregations expected if these two elements are behaving as regular chromosomes.

Centromeric plasmids can be used to study the regulatory elements upstream of a gene (Figure 11-12). The relevant coding region and its upstream region can be spliced into a plasmid, which can be selected by a separate yeast marker such as ura3+. The upstream region can be manipulated by inducing a series of deletions, which are achieved by cutting the DNA, using a special exonuclease to chew away the DNA in one direction to different extents, and then rejoining it. The experimental objective is then to determine which of these deletions still permits normal functioning of the gene. Proper function is assayed by transforming the plasmid into a recipient in which the chromosome locus carries a defective mutant allele and then monitoring for the return of gene function in the recipient. The results generally define a specific region that is necessary for normal function and regulation of the gene.

In such regulatory studies, it is often more convenient to use a reporter gene instead of the gene of interest. Therefore, if the regulation of gene X is of interest, the promoter region of gene X is spliced to the reporter gene. A gene that has been extensively used as a reporter in yeast is the bacterial lacZ gene, which codes for the enzyme b -galactosidase. This enzyme normally breaks down lactose, but it can also break down an analog of lactose, X-Gal (5-bromo-4-chloro-indolyl- b , D-galactoside) to yield 5-bromo-4-chloroindigo, which is bright blue. The blue color is expressed as a blue yeast colony whenever it is active. (In Chapter 10, we learned that this same enzyme is used to select for integrants into the pUC18 plasmid.) Generally, the fusions are constructed in such a way that the promoter of gene X plus a few codons from the structural gene X are fused in the correct reading frame to the region coding for the enzymatically active part of b -galactosidase. These constructs can be transformed on a nonintegrative vector.

Yeast artificial chromosomes have been extensively used as cloning vectors for large sections of eukaryotic (especially human) DNA. Consider, for example, that the size of the region encoding blood-clotting factor VIII in humans is known to span about 190 kb and that the gene for Duchenne muscular dystrophy spans more than 1000 kb. Furthermore, the large size of mammalian genomes in general means that libraries made in bacterial vectors would be huge. Yeast artificial chromosomes carry much longer inserts, as many as 1000 kb, and the library size is correspondingly smaller. We return to this topic in Chapter 12.

MESSAGE
Yeast vectors can be integrative, can autonomously replicate, or can resemble artificial chromosomes, allowing genes to be isolated, manipulated, and reinserted in molecular genetic analysis.g

Genetic Engineering in Plants

Because of their economic significance, plants have long been the subject of genetic analysis aimed at developing improved varieties. Recombinant DNA technology has introduced a new dimension to this effort because the genome modifications made possible by this technology are almost limitless. No longer is breeding confined to selecting variants within a given species. DNA can now be introduced from other species of plants, animals, or even bacteria.

The Ti plasmid.

The only vectors routinely used to produce transgenic plants are derived from a soil bacterium called Agrobacterium tumefaciens. This bacterium causes what is known as crown gall disease, in which the infected plant produces uncontrolled growths (tumors, or galls), normally at the base (crown) of the plant. The key to tumor production is a large (200-kb) circular DNA plasmidthe Ti (tumor-inducing) plasmid. When the bacterium infects a plant cell, a part of the Ti plasmida region called T-DNAis transferred and inserted, apparently more or less at random, into the genome of the host plant (Figure 11-13). The functions required for this transfer are outside the T-DNA on the Ti plasmid. The T-DNA itself carries several interesting functions, including the production of the tumor and the synthesis of compounds called opines. Opines are actually synthesized by the host plant under the direction of the T-DNA. The bacterium then uses the opines for its own purposes, calling on opine-utilizing genes on the Ti plasmid. Two important opines are nopaline and octopine; two separate Ti plasmids produce them. The structure of Ti is shown in Figure 11-14.

The natural behavior of the Ti plasmid makes it well-suited for the role of a plant vector. If the DNA of interest could be spliced into the T-DNA, then the whole package would be inserted in a stable state into a plant chromosome. This system has indeed been made to work essentially in this way, but with some necessary modifications. Let's examine one protocol.

Ti plasmids are too large to be easily manipulated and cannot be readily made smaller, because they contain few unique restriction sites. Consequently, a smaller, intermediate vector initially receives the insert of interest and the various other genes and segments necessary for recombination, replication, and antibiotic resistance. When engineered with the desired gene elements, this intermediate vector can then be inserted into the Ti plasmid, forming a cointegrate plasmid that can be introduced into a plant cell by transformation. Figure 11-15a shows one method of creating the cointegrate. The Ti plasmid that will receive the intermediate vector is first attenuated; that is, it has the entire right-hand region of its T-DNA, including tumor genes and nopaline-synthesis genes, deleted, rendering it incapable of tumor formationa "nuisance" aspect of the T-DNA function. It retains the left-hand border (L) of its T-DNA, which will be used as the crossover site for incorporation of the intermediate vector. The intermediate vector has had a convenient cloning segment spliced in, containing a variety of unique restriction sites. The gene of interest has been inserted at this site in Figure 11-15. Also spliced into the vector are a selectable bacterial gene (spcR) for spectinomycin resistance; a bacterial kanamycin-resistance gene (kanR), engineered for expression in plants; and two segments of T-DNA. One segment carries the nopaline-synthesis gene (nos) plus the right-hand T-DNA border sequence (R). The second T-DNA segment comes from near the left-hand border and provides a section for recombination with a homologous part of region L, which was retained in the disarmed Ti plasmid. After the intermediate vectors have been introduced into Agrobacterium cells containing the disarmed Ti plasmids (by conjugation with E. coli), plasmid recombinants (cointegrates) can be selected by plating on spectinomycin. The selected bacterial colonies will contain only the Ti plasmid, because the intermediate vector is incapable of replication in Agrobacterium.

As Figure 11-15b shows, after spectinomycin selection for the cointegrates, bacteria containing the recombinant double, or cointegrant, plasmid are then used to infect cut segments of plant tissue, such as punched-out leaf disks. If bacterial infection of plant cells takes place, any genetic material between the left and right T-DNA border sequences can be inserted into the plant chromosomes. If the leaf disks are placed on a medium containing kanamycin, the only plant cells that will undergo cell division are those that have acquired the kanR gene from T-DNA transfer. The growth of such cells results in a clump, or callus, which is an indication that transformation has taken place. These calli can be induced to form shoots and roots, at which time they are transferred to soil where they develop into transgenic plants (Figure 11-15b). Often only one T-DNA insert is detectable in such plants, where it segregates at meiosis like a regular Mendelian allele (Figure 11-16). The insert can be detected by a T-DNA probe in a Southern hybridization or can be verified by the detection of the chemical nopaline in the transgenic tissue.

Expression of cloned DNA.

The DNA cloned into the T-DNA can be any DNA that the investigator wants to insert into the subject plant. A particularly striking foreign DNA that has been inserted with the use of T-DNA is the gene for the enzyme luciferase, which is isolated from fireflies. The enzyme catalyzes the reaction of a chemical called luciferin with ATP; in this process, light is emitted, which explains why fireflies glow in the dark. A transgenic tobacco plant expressing the luciferase gene also will glow in the dark when watered with a solution of luciferin (see photograph at the beginning of this chapter). Such manipulation might seem like an attempt to develop technology for making Christmas trees that do not need lights, but in fact the luciferase gene is useful as a reporter to monitor the function of any gene during development. In other words, the upstream promoter sequences of any gene of interest can be fused to the luciferase gene and put into a plant by T-DNA. Then the luciferase gene will follow the same developmental pattern as the normally regulated gene does, but the luciferase gene will announce its activity prominently by glowing at various times or in various tissues, depending on the regulatory sequence.

Other genes used as reporters in plants are the bacterial GUS ( b -glucuronidase) gene, which turns the compound X-Gluc blue, and the bacterial lac ( b -galactosidase) gene, which turns X-Gal blue. Cells in which these reporters are expressed turn blue, and this blueness can be easily seen either by the naked eye or under the microscope.

Transgenic plants carrying any one of a variety of foreign genes are in current use, and many more are in development. Genetics in Process 11-1 presents examples. Not only are the qualities of plants themselves being manipulated, but, like microorganisms, plants are also being used as convenient "factories" to produce proteins encoded by foreign genes.

Genetic Engineering in Animals

Some of the animals most extensively used as model systems for DNA manipulation are Caenorhabditis elegans (a nematode), Drosophila, and mice. Versions of many of the techniques considered so far can also be applied in these animal systems.

Transgenic animals.

There are several ways of producing transgenic animals. An example is the production of transgenic Drosophila by the injection of plasmid vectors containing P elements into the fly egg (described in detail in Chapter 13). Transgenic Drosophila provide us with another illustration of the use of the bacterial lacZ gene as a reporter in the study of gene regulation during development. The lacZ gene is fused to the promoter region of a Drosophila heat shock gene, which is normally activated by high temperatures. This construct is then used to generate transgenic flies. Subsequent to heat shock, the flies are killed and bathed in X-Gal. The resulting pattern of blue tissues provides information on the major sites of action of the heat shock gene (Figure 11-17). Another approach to the production of transgenic mammals is by injecting special plasmid vectors into a fertilized egg (discussed later in this chapter).

In both these cases, because it is the egg that is initially made transgenic, the extra DNA can find its way into germ-line cells, is then passed on to the progeny derived from these cells, and behaves from then on rather like a regular nuclear gene. Like plants, animals are being manipulated not only to improve the qualities of the animal itself, but also to act as convenient producers of foreign proteins. For example, mammalian milk is easily obtained, so it is a convenient medium for the collection of proteins that are otherwise more difficult to obtain without sacrificing the animal (Figure 11-18 on the following page).

Gene disruptions and gene replacement in mice.

Mice are the most important models for mammals generally. Furthermore, much of the general technology developed in mice can be applied to humans. Two key techniques are the abilities to disrupt a gene (perhaps for a reverse genetics study) and to replace one allele with another. We shall consider examples of these techniques.

Gene disruptions are sometimes called "knockouts." An organism carrying the gene knockout can then be examined for altered phenotypes. Knockout mice are invaluable models for the study of mutants similar to those found in humans. For instance, knockout mice that lack vital DNA repair enzymes (see Chapter 7) have been created to study whether these enzymes control cancer rates.

Figures 11-19 and 11-20 illustrate, step-by-step, the generation of a knockout mouse. First, a cloned, disrupted gene is used to produce embryonic stem (ES) cells containing a gene knockout (Figure 11-19a). Although recombination of the defective part of the gene into nonhomologous (ectopic) sites is much more frequent than its recombination into homozygous sites (Figure 11-19c), selections for site-specific recombinants and against ectopic recombinants can be used, as shown in Figure 11-19d. Second, the ES cells that contain one copy of the disrupted gene of interest are injected into an early embryo (Figure 11-20 on page 360). The resulting progeny are chimeric, having tissue derived from either recipient or transplanted ES lines. Chimeric mice are then mated to produce homozy-gous mice with the knockout in each copy of the gene (Figure 11-20).

MESSAGE
Fungal, plant, and animal genes can be cloned and manipulated in bacteria and reintroduced into the eukaryote cell, where they generally integrate into chromosomal DNA.

The technology for mammalian gene knockouts is similar to that for gene replacement. In gene therapy, a mutant allele is replaced by a wild type, the gene replacement providing a cure for the mutant condition.

© 1999 by W. H. Freeman and Company. All rights reserved.