Cpd

About 10-30% of offspring will contain foreign DNA in chromosomes of all their tissues and germ line

Breed mice expressing foreign DNA to propagate DNA in germ line

▲ EXPERIMENTAL FIGURE 9-41 Transgenic mice are produced by random integration of a foreign gene into the mouse germ line. Foreign DNA injected into one of the two pronuclei (the male and female haploid nuclei contributed by the parents) has a good chance of being randomly integrated into the chromosomes of the diploid zygote. Because a transgene is integrated into the recipient genome by nonhomologous recombination, it does not disrupt endogenous genes. [See R. L. Brinster et al., 1981, Cell 27:223.]

▲ FIGURE 9-42 Inactivation of the function of a wild-type GTPase by the action of a dominant-negative mutant allele.

(a) Small (monomeric) GTPases (purple) are activated by their interaction with a guanine-nucleotide exchange factor (GEF), which catalyzes the exchange of GDP for GTP (b) Introduction of a dominant-negative allele of a small GTPase gene into cultured cells or transgenic animals leads to expression of a mutant GTPase that binds to and inactivates the GEF As a result, endogenous wild-type copies of the same small GTPase are trapped in the inactive GDP-bound state. A single dominantnegative allele thus causes a loss-of-function phenotype in heterozygotes similar to that seen in homozygotes carrying two recessive loss-of-function alleles.

types of dominant alleles, dominant-negative alleles produce a phenotype equivalent to that of a loss-of-function mutation.

Useful dominant-negative alleles have been identified for a variety of genes and can be introduced into cultured cells by transfection or into the germ line of mice or other organisms. In both cases, the introduced gene is integrated into the genome by nonhomologous recombination. Such randomly inserted genes are called transgenes; the cells or organisms carrying them are referred to as transgenic. Transgenes carrying a dominant-negative allele usually are engineered so that the allele is controlled by a regulated promoter, allowing expression of the mutant protein in different tissues at different times. As noted above, the random integration of exogenous DNA via nonhomologous recombination occurs at a much higher frequency than insertion via homologous recombination. Because of this phenomenon, the production of transgenic mice is an efficient and straightforward process (Figure 9-41).

Among the genes that can be functionally inactivated by introduction of a dominant-negative allele are those encoding small (monomeric) GTP-binding proteins belonging to the GTPase superfamily. As we will examine in several later chapters, these proteins (e.g., Ras, Rac, and Rab) act as intracellular switches. Conversion of the small GTPases from an inactive GDP-bound state to an active GTP-bound state depends on their interacting with a corresponding guanine nucleotide exchange factor (GEF). A mutant small GTPase

(a) In vitro production of double-stranded RNA

Sense transcript

Antisense transcript N

(a) In vitro production of double-stranded RNA

Sense transcript

Antisense transcript N

(b)

Noninjected

Injected

Noninjected

Injected that permanently binds to the GEF protein will block conversion of endogenous wild-type small GTPases to the active GTP-bound state, thereby inhibiting them from performing their switching function (Figure 9-42).

Double-Stranded RNA Molecules Can Interfere with Gene Function by Targeting mRNA for Destruction

Researchers are exploiting a recently discovered phenomenon known as RNA interference (RNAi) to inhibit the function of specific genes. This approach is technically simpler than the methods described above for disrupting genes. First observed in the roundworm C. elegans, RNAi refers to the ability of a double-stranded (ds) RNA to block expression of its corresponding single-stranded mRNA but not that of mRNAs with a different sequence.

To use RNAi for intentional silencing of a gene of interest, investigators first produce dsRNA based on the sequence of the gene to be inactivated (Figure 9-43a). This dsRNA is injected into the gonad of an adult worm, where it has access to the developing embryos. As the embryos develop, the mRNA molecules corresponding to the injected dsRNA are rapidly destroyed. The resulting worms display a phenotype similar to the one that would result from disruption of the corresponding gene itself. In some cases, entry of just a few molecules of a particular dsRNA into a cell is sufficient to inactivate many copies of the corresponding mRNA. Figure 9-43b illustrates the ability of an injected dsRNA to interfere with production of the corresponding endogenous mRNA in C. elegans embryos. In this experiment, the mRNA levels in embryos were determined by incubating the embryos with a flu-orescently labeled probe specific for the mRNA of interest. This technique, in situ hybridization, is useful in assaying expression of a particular mRNA in cells and tissue sections.

Initially, the phenomenon of RNAi was quite mysterious to geneticists. Recent studies have shown that specialized RNA-processing enzymes cleave dsRNA into short segments, which base-pair with endogenous mRNA. The resulting hybrid molecules are recognized and cleaved by specific nucleases at these hybridization sites. This model accounts for the specificity of RNAi, since it depends on base pairing, and for its potency in silencing gene function, since the complementary mRNA is permanently destroyed by nucleolytic degradation. Although the normal cellular function of RNAi is not understood, it may provide a defense against viruses with dsRNA genomes or help regulate certain endogenous genes. (For a more detailed discussion of the mechanism of RNA interference, see Section 12.4.)

Other organisms in which RNAi-mediated gene inacti-vation has been successful include Drosophila, many kinds of plants, zebrafish, spiders, the frog Xenopus, and mice. Although most other organisms do not appear to be as sensitive to the effects of RNAi as C. elegans, the method does have general use when the dsRNA is injected directly into embryonic tissues.

▲ EXPERIMENTAL FIGURE 9-43 RNA interference (RNAi) can functionally inactivate genes in C. elegans and some other organisms. (a) Production of double-stranded RNA (dsRNA) for RNAi of a specific target gene. The coding sequence of the gene, derived from either a cDNA clone or a segment of genomic DNA, is placed in two orientations in a plasmid vector adjacent to a strong promoter. Transcription of both constructs in vitro using RNA polymerase and ribonucleotide triphosphates yields many RNA copies in the sense orientation (identical with the mRNA sequence) or complementary antisense orientation. Under suitable conditions, these complementary RNA molecules will hybridize to form dsRNA. (b) Inhibition of mex3 RNA expression in worm embryos by RNAi (see the text for the mechanism). (Left) Expression of mex3 RNA in embryos was assayed by in situ hybridization with a fluorescently labeled probe (purple) specific for this mRNA. (Right ) The embryo derived from a worm injected with double-stranded mex3 mRNA produces little or no endogenous mex3 mRNA, as indicated by the absence of color. Each four-cell stage embryo is -50 ^m in length. [Part (b) from A. Fire et al., 1998, Nature 391:806.]

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