Genetic engineering in animals

Transgenic technologies are now being employed with many animal model systems. We will focus on the three animal models most heavily used for basic genetic research: the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the mouse Mus musculus. Versions of many of the techniques considered so far can also be applied in these animal systems.

TRANSGENESIS IN C. ELEGANS The method used to introduce trangenes into C. elegans is simple: transgenic DNAs are injected directly into the organism, typically as plasmids, cosmids, or other DNAs cloned in bacteria. The injection strategy is determined by the worm's reproduc-

tive biology. The gonads of the worm are syncitial, meaning that there are many nuclei within the same gonadal cell. One syncitial cell is a large proportion of one arm of the gonad, and the other syncitial cell is the bulk of the other arm (Figure 11-32a). These nuclei do not form individual cells until meiosis, when they begin their transformation into individual eggs or sperm. A solution of DNA is injected into the syncitial region of one of the arms, thereby exposing more than 100 nuclei to the transforming DNA. By chance, a few of these nuclei will incorporate the DNA (remember, the nuclear membrane breaks down in the course of division, and so the cytoplasm into which the DNA is injected becomes continuous with the nucleoplasm). Typically, the transgenic DNA forms multicopy extrachromosomal arrays (Figure 11-32b) that exist as independent units outside the chromosomes. More

Syncitial region Gonad

Nuclei

Syncitial region Gonad

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C. elegans

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C. elegans

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One unit of injected recombinant DNA

Extrachromosomal array Integrated array

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Figure 11-32 Creation of C. elegans transgenes. (a) Method of injection. (b) The two main types of transgenic results: extrachromosomal arrays and arrays integrated in ectopic chromosomal locations.

rarely, the transgenes will become integrated into an ectopic position in a chromosome, still as a multicopy array. Unfortunately, sequences may become scrambled within the arrays, complicating the work of the researcher.

TRANSGENESIS IN D. MELANOGASTER Transgenesis in D. melanogaster requires a more complex technique but avoids the difficulties of multicopy arrays. It proceeds by a mechanism that differs from those discussed so far, based on the properties of a transposable element called the P element, which acts as the vector. A transposable element is a DNA segment that is capable of moving from one location in the genome to other locations. We will consider transposable elements and how they move in much more detail in Chapter 13.

For our purposes here, all we need to know is that P elements come in two types (Figure 11-33a):

• One type of element, 2912 bp long, encodes a protein called a transposase that is necessary for P elements to move to new positions in the genome. This type of element is termed "autonomous" because it can be transposed through the action of its own transposase enzyme.

• The transposase has been deleted from the second type of element, called a nonautonomous element. Still, a nonautonomous element can move to a new genomic location if transposase is supplied by an autonomous element. The one requirement is that the nonautonomous element contain the first 200 bp and final 200 bp of the autonomous element, which includes the sequences that the transposase needs to recognize for transposition. Moreover, any DNA inserted in between the ends of a nonautonomous

P element will be transposed as well.

As with C. elegans, the DNA is injected into a synci-tium — in this case, the early Drosophila embryo (Figure 11-33b). More precisely, the DNA is injected at the site of germ-cell formation, at the posterior pole of the embryo. The adults that grow from the injected embryo will typically not express the transgene but will contain some transgenic germ cells, and these cells will be expressed in the offspring.

What type of vector carries the injected DNA? To produce transgenic Drosophila, we must inject two separate bacterial recombinant plasmids. One contains the autonomous P element that supplies the coding sequences for the transposase. This element is the P helper plasmid. The other, the P-element vector, is an engineered nonautonomous element containing the ends of the P element and, inserted between these ends, the piece of cloned DNA that we want to incorporate as a transgene into the fly genome. A DNA solution containing both of these plasmids is injected into the posterior pole of the syncitial embryo. The P transposase

(a) P-elements

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(b) Method of injection

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\ Eventual location of germ Drosophila embryo cells

(c) Integration event

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Figure 11-33 Creation of D. melanogaster transgenes.

(a) The overall structure of autonomous and nonautonomous P transposable elements. (b) Method of injection. (c) The circular P-element vector (right) and a typical integration event at an ectopic chromosomal location (left). Note that the bacterial vector sequences do not become integrated into the genome; rather, in integration, exactly one copy of the DNA segment is contained between the P-element ends.

expressed from the injected P helper plasmid catalyzes the insertion of the P-element vector into the fly genome. The nature of the transposase enzymatic reaction guarantees that only a single copy of the element inserts at a given location (Figure 11-33c).

How can we detect the progeny that develop from gametes that successfully receive the cloned DNA? Typically, they are detected because they express a dominant wild-type transgenic allele of a gene for which the recipient strain carries a recessive mutant allele.

Transposable elements are widely used in transgenesis, in plants as well as insects. Perhaps the best-known plant example is the Activator transposable-element system first described in Zea mays (corn), which has been developed into a transgenic cloning vector for use in many plants.

TRANSGENESIS IN M. MUSCULUS Mice are the most important models for mammalian genetics. Most exciting, much of the technology developed in mice is potentially applicable to humans. There are two strategies for transgenesis in mice, each of which has its advantages and disadvantages:

• Ectopic insertions. Transgenes are inserted randomly in the genome, usually as multicopy arrays.

• Gene targeting. The transgene sequence is inserted into a location occupied by a homologous sequence in the genome. That is, the transgene replaces its normal homologous counterpart.

Ectopic insertions To insert transgenes in random locations, the procedure is simply to inject a solution of bacterially cloned DNA into the nucleus of a fertilized egg (Figure 11-34a). Several injected eggs are inserted into the female oviduct, where some will develop into baby mice. At some later stage, the transgene becomes integrated into the chromosomes of random nuclei. On occasion, the transgenic cells form part of the germ line, and in these cases an injected embryo will develop into a mouse adult whose germ cells contain the transgene inserted at some random position in one of the chromosomes (Figure 11-34b). Some of the progeny of these adults will inherit the transgene in all cells. There will be an array of multiple gene copies at each point of insertion, but the location, size, and structure of the arrays will be different for each integration event. The technique does give rise to some problems: (1) the expression pattern of the randomly inserted genes may be abnormal (called a position effect) be- cause the local chromosome environment lacks the gene's normal regulatory sequences, and (2) DNA rearrangements can occur inside the multicopy arrays (in essence, mutating the sequences). Nonetheless, this technique is much more effi-cent and less laborious than gene targeting.

Gene targeting Gene targeting enables us to eliminate or modify the function encoded by a gene. In one application,

Nucleus

Nucleus

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Single-cell mouse embryo

Micropipette with DNA solution

Single-cell mouse embryo

Chromosome

Integrated array

Figure 11-34 Creation of M. musculus transgenes inserted in ectopic chromosomal locations. (a) Method of injection. (b) A typical ectopic integrant, with multiple copies of the recombinant transgene inserted in an array.

a mutant allele can be repaired through gene replacement in which a wild-type allele substitutes for a mutant one in its normal chromosomal location. Gene replacement avoids both the position effect and DNA rearrangements associated with ectopic insertion, because a single copy of the gene is inserted in its normal chromosomal environment.

Gene targeting in the mouse is carried out in cultured embryonic stem cells (ES cells). In general, stem cells are undifferentiated cells in a given tissue or organ that divide asymmetrically to produce a progeny stem cell and a cell that will differentiate into a terminal cell type. ES cells are special stem cells that can differentiate to form any cell type in the body—including, most importantly, the germ line.

To illustrate the process of gene targeting, we look at how it achieves one of its typical outcomes — namely, the substitution of an inactive gene for the normal gene. Such a targeted inactivation is called a gene knockout. First, a cloned, disrupted gene that is inactive is targeted to replace the functioning gene in a culture of ES cells, producing ES cells containing a gene knockout (Figure 11-35a). DNA constructs containing the defective gene are injected into the nuclei of cultured ES cells. The defective gene inserts far more frequently into nonhomologous (ectopic) sites than into homologous sites (Figure 11-35b), and so

Figure 11-35 Producing cells that contain a mutation in one specific gene, known as a targeted mutation or a gene knockout.

(a) Copies of a cloned gene are altered in vitro to produce the targeting vector. The gene shown here has been inactivated by the insertion of the neomycin-resistance gene (neoR) into a protein-coding region (exon 2) of the gene and had been inserted into a vector. The neoR gene will serve later as a marker to indicate that the vector DNA took up residence in a chromosome. The vector has also been engineered to carry a second marker at one end: the herpes tk gene. These markers are standard, but others could be used instead. When a vector, with its dual markers, is complete, it is introduced into cells isolated from a mouse embryo. (b) When homologous recombination occurs (left), the homologous regions on the vector, together with any DNA in between but excluding the marker at the tip, take the place of the original gene. This event is important because the vector sequences serve as a useful tag for detecting the presence of this mutant gene. In many cells, though, the full vector (complete with the extra marker at the tip) inserts ectopically (middle) or does not become integrated at all (bottom). (c) To isolate cells carrying a targeted mutation, all the cells are put into a medium containing selected drugs — here a neomycin analog (G418) and ganciclovir. G418 is lethal to cells unless they carry a functional neoR gene, and so it eliminates cells in which no integration of vector DNA has taken place (yellow). Meanwhile, ganciclovir kills any cells that harbor the tk gene, thereby eliminating cells bearing a randomly integrated vector (red). Consequently, virtually the only cells that survive and proliferate are those harboring the targeted insertion (green). [After M. R. Capecchi, "Targeted Gene Replacement." Copyright 1994 by Scientific American, Inc. All rights reserved.]

11.5 Genetic engineering

(a) Production of ES cells with a gene knockout

Targeting vector n tk

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Insert neoR into exon 2.

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(b) Targeted insertion of vector DNA by homologous recombination

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Genetic Engineering Humans

Newborn chimeric male (carrying cells from two mouse strains)

Newborn chimeric male (carrying cells from two mouse strains)

Embryo
Transgenic Mice Curly

Figure 11-36 Producing a knockout mouse carrying the targeted mutation. (a) Embryonic stem (ES) cells are isolated from an agouti (brown) mouse strain (A/A) and altered to carry a targeted mutation (m) in one chromosome. The ES cells are then inserted into young embryos, one of which is shown. Coat color of the future newborns is a guide to whether the ES cells have survived in the embryo. Hence, ES cells are typically put into embryos that, in the absence of the ES cells, would acquire a totally black coat. Such embryos are obtained from a black strain that lacks the dominant agouti allele (a/a). The embryos containing the ES cells grow to term in surrogate mothers. Agouti shading intermixed with black indicates those newborns in which the ES cells have survived and proliferated. (Such mice are called chimeras because they contain cells derived from two different strains of mice.) Solid black

Figure 11-36 Producing a knockout mouse carrying the targeted mutation. (a) Embryonic stem (ES) cells are isolated from an agouti (brown) mouse strain (A/A) and altered to carry a targeted mutation (m) in one chromosome. The ES cells are then inserted into young embryos, one of which is shown. Coat color of the future newborns is a guide to whether the ES cells have survived in the embryo. Hence, ES cells are typically put into embryos that, in the absence of the ES cells, would acquire a totally black coat. Such embryos are obtained from a black strain that lacks the dominant agouti allele (a/a). The embryos containing the ES cells grow to term in surrogate mothers. Agouti shading intermixed with black indicates those newborns in which the ES cells have survived and proliferated. (Such mice are called chimeras because they contain cells derived from two different strains of mice.) Solid black

coloring, in contrast, indicates that the ES cells have perished, and these mice are excluded. A represents agouti, a black; m is the targeted mutation, and M is its wild-type allele. (b) Chimeric males are mated with black (nonagouti) females. Progeny are screened for evidence of the targeted mutation (green in inset) in the gene of interest. Direct examination of the genes in the agouti mice reveals which of those animals (boxed) inherited the targeted mutation. Males and females carrying the mutation are mated with one another to produce mice whose cells carry the chosen mutation in both copies of the target gene (inset) and thus lack a functional gene. Such animals (boxed) are identified definitively by direct analyses of their DNA. The knockout in this case results in a curly-tail phenotype. [After M. R. Capecchi, "Targeted Gene Replacement." Copyright 1994 by Scientific American, Inc. All rights reserved.]

the next step is to select the rare cells in which the defective gene has replaced the functioning gene as desired. How is it possible to select ES cells that contain a rare gene replacement? The genetic engineer can include drug-resistant alleles in the DNA construct arranged in such a way that replacements can be distinguished from ectopic insertions. An example is shown in Figure 11-35c.

In the second part of the procedure, the ES cells that contain one copy of the disrupted gene of interest are injected into an early embryo (Figure 11-36a). Adults grown from these embryos are crossed with normal mates. The resulting progeny are chimeric, having some tissue derived from the original lines and some from the transplanted ES lines. Chimeric mice are then mated with their siblings to produce homozygous mice with the knockout in every copy of the gene (Figure 11-36b). Mice containing the targeted transgene in each of their cells are identified by molecular probes for sequences unique to the transgene.

MESSAGE Germ-line transgenic techniques have been developed for all well-studied eukaryotic species. These techniques depend on an understanding of the reproductive biology of the recipient species.

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