The common house mouse, Mus musculus, has played a prominent role in the study of genetics ever since Carl Correns, Hugo De Vries, and Erich Von Tschermak independently rediscovered Mendels laws at the beginning of the twentieth century. Because these three scientists, as well as Mendel himself, performed their research entirely on plants, many in the scientific community questioned whether Mendels laws could explain the basis for inheritance in animals, especially humans. The reason for this skepticism is easy to see. People, for example, differ in the expression of many commonly inherited traits such as skin color, eye color, curliness of hair, and heightthat show no evidence of transmission according to Mendels laws. We now know that these traits result from the interaction of many genes with multiple alleles that each segregate according to Mendels rst law even though the traits themselves do not. At the beginning of the twentieth century, however, a demonstration of the applicability of Mendels laws to animal inheritance required the analysis of simple traits controlled by single genes. M. musculus has many features that enhance its value as a model organism for genetic analysis, and foremost among these is the availability of hundreds of singlegene mutations. These mutations arose during the mouses long history of domestication as a pet. Over the centuries, dealers in what became known as the fancy mouse trade selected and bred mice with numerous coat colors and other visible mutations, rst in China and Japan, later in Europe (Fig. E.1a). In contrast to the variation that occurs naturally in wild populations, new traits that appear suddenly in captive-bred mice are almost always the result of single-gene mutations. Early animal geneticists made note of this fact and used fancy mice to demonstrate that Mendels laws apply to mammals and, by extrapolation, to humans. In addition to providing a ready source of single-gene mutations, the house mouse has several other features that make it the mammal of choice for genetic analysis. Mice have a very short generation time of just eight to nine weeks. They are small enough so that thousands can live in relatively small rooms. They have large litters of eight or more pups. They breed readily in captivity. Fathers do not harm their young. And after centuries of articial selection, domesticated mice are docile and easy to handle (Fig. E.1b). But why study a mammal at all when animals like fruit ies and nematodes are even smaller and more amenable to genetic analysis? The answer is that a major goal of current biological research is the understanding of human beings. And although many features of human biology, especially at the cellular and molecular levels, are common to a broad spectrum of life-forms, the most advanced organismlevel human characteristics appear in a limited subset of animals. In fact, many aspects of human development and disease are common only to placenta-bearing mammals such as the mouse. Thus, the mouse provides a powerful model system for investigating the genetic basis of simple and complex human traits, especially those related to development and disease (Fig. E.2). Two general themes emerge from our presentation of M. musculus. First, because of the many similarities between mouse and human genomes, researchers can use
Reference
A member of the 129 strain of inbred mice commonly used in targeted mutagenesis studies.
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(a)
(a)
(b)
(b)
Figure E.1
The mouse is a model system for human biology. (a) Examples of visible phenotypes caused by single-gene
mutations. (b) Mother mouse with her pups.
Figure E.2
Hirschsprung disease: A human developmental disease that causes deformities of the colon (a) and (b) a mouse
homology analysis to identify and locate the same genes in both species. In this context, homologs are genes or regulatory DNA sequences that are similar in different species because of descent from a common ancestral sequence. Second, genetic analysis in mice exemplies the combined use of molecular (that is, recombinant DNA) and classical breeding techniques to identify and understand the function of complex genetic systems. Our genetic portrait of the house mouse describes: An overview of M. musculus in the laboratory, including a look at the mouse genome, the mouse life cycle, and two powerful transgenic protocols: the addition of specic genes to the mouse genome by nuclear injection and the removal of specic genes from the mouse genome by targeted mutagenesis. The uses of transgenic technology in determining the function of gene products, characterizing regulatory regions, establishing links between mutant phenotypes and particular transcription units, and creating a mouse model for a human disease. The Hox genes: a comprehensive example.
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TABLE E.1
Trait
Average weight Average length Genome size
Humans
77,000 g (170 lb) 175 cm ~3,000,000,000 bp ~25,000 22 autosomes X and Y Average, 38 weeks (8.9 months) Average, 624728 weeks (1214 years) Average, 28 days Average, 78 years
Haploid gene number Number of chromosomes Gestation period Age at puberty Estrus cycle Life span
E.1
during the 75 million years that mice and humans have been evolving apart from a common ancestor, their genomes have broken apart and rearranged some 170 times (17.6 Mb 170 about 3000 Mb the size of the mammalian genome). Conversely, if the proper genome-scale scissors and glue were available, one could break the mouse genome into about 170 pieces and reassemble those pieceslike a puzzlein the form of the human genome. In addition to its powerful evolutionary implications, conserved synteny is a useful tool for practicing geneticists. Once a researcher has mapped a locus in one species, he or she can look at a homology map and immediately identify its likely map position in the other species. Of course, for genes that have already been cloned, it is possible to use DNA-DNA hybridization, or computer analysis of a wholegenome sequence, to pick out gene homologs from the other species. But for loci characterized only by their phenotypic expression, conserved synteny enables geneticists to move back and forth between the analysis of a trait in humans and the analysis of a model for that trait in mice. The discovery of a locus that predisposes female mice to excessive consumption of 10% ethanol (the concentration of alcohol found in many wines) provides an example of the use of conserved synteny for locating human homologs of mouse genes. Mouse geneticists used DNA markers (as described in Chapter 11 of the main textbook) to map the Alcohol-preference-2 (Alcp2) locus to the middle of mouse chromosome 11. Now that the whole mouse genome has been cloned and sequenced, the genes as transcription units in Alcpz region have been identied, but which one is actually Alcpz is not yet known. However, even though researchers have not yet identied the specic gene, scrutiny of a conserved synteny homology map shows that the most likely location for the human homolog of Alcp2 is on the short arm of human chromosome 17, close to the centromere (Fig. E.3). With this information about the likely location of an alcohol-preference locus,
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Female Oogonia
Spermatogonia
Fetus Implantation Blastocyst Zygote Fertilization Sperm Egg Mature secondary oocyte Diploid phase Haploid phase
Ovulation Spermatozoa
Figure E.4
Alcp2
The mammalian life cycle. The life cycles of males and females are shown separately in the traditional format. Notice that primary germ cells are formed in the female before birth but in the male after birth. Notice also that male germ cells are self-renewing, while in females, no new germ cells are formed after birth.
Mouse chromosome
Mouse loci
Figure E.3
Conserved synteny: An example with mouse chromosome 11. The mouse chromosome, on the left, shows
The mammalian life cycle, like that of all sexually reproducing species, can be visualized as continuous circles (Fig. E.4), with any point along the circumferences marking the start. For ease of presentation, we begin with the haploid phase in both males and females.
positions of loci with mapped homologs in the human genome. The map locations of human homologs are on the right.
human geneticists can now look for linkage between DNA markers on human chromosome 17 and a phenotypic expression of a predisposition to alcohol abuse.
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From this time on, the female progresses through an estrus cycle that lasts about 4 days in mice and about 28 days in humans. During each cycle, primary oocytes (810 in mice, usually only 1 in women) are stimulated to complete the rst meiotic division and extrude the rst polar body at the end of this division; the resulting secondary oocyte begins the second meiotic division but stops at metaphase and is released from the ovary in a process known as ovulation. Following ovulation, the secondary oocyte passes into an oviduct (called a fallopian tube in humans), where for a brief time, known as estrus, it remains alive and receptive to fertilization. In nature, most mammals die while they still have the ability to reproduce. Many human females, however, live long enough to pass through menopause, during which they stop cycling through estrus, no longer ovulate, and thus lose the ability to reproduce.
Fertilization
Just before and during the estrus phase of the estrus cycle, female mammals of nonhuman species release speciesspecic chemical signals, or pheromones. In a behavioral response to these pheromones, a male will copulate with a female and ejaculate semen containing millions of sperm into her reproductive tract. The sperm swim from the vagina into the uterus and thence up the oviducts. Only 100 or fewer sperm survive this journey to the waiting eggs. Fertilization is a multistep process illustrated in Fig. E.5. First, surviving sperm bind to the zona pellucidathe thick solid shell composed of glycoproteins that surrounds the egg proper. The act of binding induces each sperm to release special proteases that enable it to burn its way through the zona
pellucida into the space that surrounds the egg membrane. Although multiple sperm can make it into this space, usually only one fuses with the egg. This fusion causes rapid electrochemical changes in the egg membrane that prevent the entry of additional sperm and activate the newly fertilized egg to enter the pathway of animal development. After fusion, the fertilized egg, or zygote, contains two haploid pronuclei. The two pronuclei never merge; instead, replication occurs within both of the pronuclei. The onecell embryo carries two replicated pronuclei right up to the moment of the rst mitosis, at which time the membranes of the two pronuclei break down, and the two sets of chromosomes, one from the paternal pronucleus, the other from the maternal pronucleus, align along the midplane of the fertilized egg and thence segregate chromatids into the two daughter cells. For the purposes of analysis, scientists divide mouse development into two distinct stages of unequal length, separated by the process of embryonic implantation into the uterus: a preimplantation stage that lasts 45 days in mice, and a postimplantation stage that lasts about 16.5 days in mice. During the preimplantation phase, the embryo is a free-oating object within the females body. It is easy to remove this naturally free-oating preimplantation embryo from the animal, culture it in a petri plate, and expose it to genetic manipulation before placing it back in the reproductive tract of an adult female for development to a newborn animal. After implantation, however, such manipulation is no longer possible because the embryo, if removed from the adults body, cannot be returned. The accessibility of the preimplantation embryo provides the basis for many of the genetic manipulations researchers use to study mammalian development.
Fertilized egg = zygote = 1-cell embryo Fertilization Zona pellucida Maternal pronucleus sperm head Ovulation Sperm One sperm penetrates 8-cell embryo Paternal pronucleus expands First cleavage
2-cell embryo
4-cell embryo
Second cleavage
16-cell embryo:
Third cleavage
Fourth cleavage
Figure E.5
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For Most of the Preimplantation Stage, the Embryonic Cells Remain Undifferentiated
The preimplantation stage starts with the zygote (Fig. E.5). Development proceeds slowly in the beginning, with the rst 22 hours devoted to the expansion of the highly compacted sperm head into a paternal pronucleus that matches the size of the eggs maternal pronucleus. After the paternal pronucleus has completed its expansion and replicated its chromosomes and the maternal pronucleus has replicated its chromosomes, the embryo undergoes the rst of four equal divisions, or cleavages, that increase the number of cells from 1 to 16 over 60 hours. The period of these four equal divisions is called the cleavage stage. During this stage, all the cells in the developing embryo are equivalent and totipotent, that is, they have not yet differentiated and each one retains the ability, or potency, to produce every type of cell found in the developing embryo and adult animal. This is very different from the developmental patterns found in most nonmammalian animal species, including Caenorhabditis elegans, where totipotency disappears as early as the two-cell stage. Because of the mouse cells totipotency, cleavage-stage embryos can be divided into smaller groups of cells that each have the potential to develop into a normal individual. Identical human twins, or more rarely, identical triplets or quadruplets, are examples of the outcome of this process. (Twinning is impossible in C. elegans or Drosophila melanogaster.) In the laboratory, scientists have obtained completely normal mice from individual cells that they dissected out of the fourcell-stage mouse embryo and placed back into the female reproductive tract (Fig. E.6a). This experimental feat demonstrates the theoretical possibility of obtaining four identical clones from a single embryo of any mammalian species. Another more bizarre consequence of the equivalency of cleavage-stage cells is the formation of chimeras, which are the opposite of clones (Fig. E.6b). The term chimera comes from the Greek word for a mythological beast that is part lion, part goat, and part serpent. Geneticists use the term to designate an embryo or animal composed of cells from two or more different origins. The Polish embryologist Andrezej Tarkowski reported the rst mouse chimeras in 1961. To construct them, he removed the zona pellucida from two cleavage-stage mouse embryos, obtaining denuded cell masses that are naturally sticky; he then pushed the sticky denuded embryos up against each other. Denuded embryos pressed together in this way form a single chimeric cell mass that is capable of undergoing normal development within the female reproductive tract. If the two embryos of a chimera come from different females mated to different males, the resulting individual is tetraparental, that is, has four parents. It is also possible to produce hexaparental animals derived from a combination of three embryos. Every organ and tissue in the adultincluding the germ linecan contain cells derived from all three original embryos. As we see later, the production of chimeric mice has been an essential compo-
Identical quadruplets
Embryo 2
Figure E.6
Early mammalian embryos are highly malleable in their development. There is no requirement for a one-to-one
correspondence between embryo and adult. (a) Creating identical quadruplets from a single fertilized egg. (b) Creating a single chimeric animal from the fusion of two embryos.
nent of the targeted mutagenesis technology that has revolutionized the use of the mouse as a model organism for studying human diseases. A comparison of the early developmental program of placental mammals with that of other animals, including C. elegans and D. melanogaster, shows how different these programs can be. In nematodes, embryonic cells are highly restricted in their developmental potential, or fate, beginning at the two-cell stage; and in fruit ies, polarization of the egg before fertilization generates distinct cytoplasmic regions dedicated to supporting different developmental programs within the nuclei that end up in these locations. Consequently, half a nematode embryo or half a y embryo can never give rise to a whole animal.
Events Restricting the Developmental Potency of Individual Cells Occur Near the End of the Preimplantation Stage
The rst differentiation events of mouse embryogenesis occur in the 16-cell embryo (see Fig. E.5). The cells on the outside of the embryo turn into a trophectoderm layer that
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will eventually take part in the formation of the placenta. Soon thereafter, the cells on the inside of the embryo compact into a small clump called the inner cell mass, or ICM, that remains attached to one spot along the inside of the hollow trophectoderm sphere. The entire fetus and animal are derived entirely from the cells of the ICM. As Figure E.5 shows, compaction of the ICM causes the appearance of a uid-lled space that is devoid of cellular material and surrounded by trophectoderm. This space is called the blastocoel cavity. The embryo is now called a blastocyst. Two more rounds of cell division occur during the blastocyst stage, producing the 64-cell embryo that implants. Throughout normal preimplantation development, the embryo remains protected within the inert zona pellucida. As a result, there is no difference in size between the 1-cell zygote and the 64-cell embryo. To accomplish implantation, the embryo must rst hatch from the zona pellucida so that it can make direct membrane-to-membrane contact with cells in the uterine wall. Embryonic and fetal development within a uterus inside the body of a female is a characteristic unique to all mammals except the primitive egg-laying platypus.
After Implantation, the Placenta Develops, the Embryo Grows, and the Tissues and Organs Emerge
Implantation initiates development of the placenta, a mix of embryonic and maternal tissues that mediates the ow of nutrients entering the embryo from the maternal blood supply and the ow of waste products exiting the embryo to the maternal circulation. The placenta maintains this intimate connection between mother and embryo, and later between mother and fetus, until the time of birth. Development of the placenta enables a period of rapid embryonic growth. Cells from the ICM differentiate into the three germ layers of endoderm, ectoderm, and mesoderm during a stage known as gastrulation. The foundation of the spinal cord is put into place, and the development of the various adult tissues and organs begins. With the appearance of organs, the embryo becomes a fetus, which continues to grow rapidly. Birth occurs at about 21 days after conception. Newborn animals remain dependent on their mothers during a suckling period that lasts 1825 days. By ve to eight weeks after birth, mice reach adulthood and are ready to begin the next reproductive cycle.
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Embryos transferred to a depression slide containing culture medium Culture medium Oil
Holding pipette
Pronucleus DNA to be injected Injection pipette Several injected embryos are placed into oviduct of receptive female.
Figure E.7 How transgenic mice are created. About 12 hours after conception, the female mouse is sacriced and the onecell embryos recovered. They are transferred to a depression on a specialized microscope slide containing a drop of culture medium under oil to prevent evaporation. The slide is placed on the stage of an inverted microscope (as the name implies, the objective lens is beneath the stage rather than above it, as in a typical microscope). This arrangement gives the researcher space to manipulate the embryos from above. (In the photo used here, only one of the two pronuclei is visible.) Suction holds the embryo in place on the blunt end of a special holding pipette. A second type of pipette with a very narrow bore (the injection pipette) is used to inject transgenic DNA through the plasma membrane and into the pronucleus, where the foreign DNA is released. The altered embryos are then placed into the oviduct of a physiologically receptive female.
the blastocyst stage into culture such that the embryonic cells from the ICM continue to divide without differentiating. Cultured cells that behave in this way are called embryonic stem cells, or ES cells for short. ES cells appear to be similar in their state of differentiation to cells from the ICM. It is possible to grow cultures containing many millions of ES cells from a single embryo and then recover a handful of cells from this culture for injection back into the blastocoel cavity of a normal embryo. Once inside the cavity, the ES cells can become incorporated into the ICM, and they can contribute to all of the tissues in the mouse that develops from the embryo. Most importantly for geneticists, the ES cells even contribute to the germ lines of these chimeric mice so that reproducing adults can transmit mutated genes present in the ES cells to future generations. The second critical advance that provided a foundation for targeted mutagenesis was development of a protocol for homologous recombination in ES cells. The transformation of mammalian cells is known as transfection. During the transfection of mouse cells with mouse-derived DNA, the foreign mouse DNA almost always integrates at random into a chromosome at a site other than its point of origin. Occasionally, however, the added DNA will nd and replace its homolog by homologous recombination. The frequency of homologous recombination events as a fraction of the total number of integrations is on the order of 103105. If researchers transfect mouse ES cells with unaltered, cloned fragments of mouse DNA, homologous recombination events do not cause genomic changes. But with the recombinant DNA technology described in Chapter 9 of the main textbook, investigators can modify cloned genes so that they no longer function; cloned genes modied in this way are known as knockout constructs. When homologous recombination occurs with a knockout construct, the nonfunctional knockout allele replaces the endogenous wild-type allele (Fig. E.8). To construct mice in which homologous recombination has knocked out specic genes, researchers developed protocols for identifying and recovering the very rare ES cells in which homologous recombination occurs.
Targeted Mutagenesis, a Second Transgenic Technology, Makes It Possible to Remove, or Knock out, Specic Sequences from the Mouse Genome
In addition to knocking out the function of a sequence altogether, targeted mutagenesis can produce an allele with an altered function. The emergence of targeted mutagenesis, which is technically more demanding and more complex than the nuclear injection technology just described, depended on two advances in cell culture techniques that occurred during the 1980s. The rst advance was the establishment of in vitro conditions that enable researchers to place mouse embryos at
E.2
How Biologists Use Transgenic Tools to Study Mice and Create a Mouse Model for Human Disease
Both add-on and knockout transgenic technologies have tremendous value in genetic research. The protocols enable researchers to determine the function of gene
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(a) Construction of a knockout allele in ES cells Early blastocyst ( 10,000) Clone containing gene of interest 5' Build knockout construct by adding in selectable marker. 5' Culture into millions of embryonic-like (ES) cells. 3' Marker disrupts transcription unit. Add cloned DNA to culture of cells. 3'
Finding the cell with the knockout allele. Subject culture to drug that kills all cells that do not contain selectable marker.
Survivor cells have knockout allele (1% or less). Begin new culture with survivor cells.
(b)
Figure E.8
Knocking out a mouse gene in ES cells. (a) An early mouse blastocyst can be grown in culture under conditions that allow the cells of the ICM (inner cell mass) to remain undifferentiated as ES cells. A DNA clone, containing the gene of interest, can be modied in the laboratory into a disrupted allele with a selectable marker. This knockout construct is added to the ES cell culture, where homologous recombination will occur. (b) A chimeric mouse composed of cells derived from a normal embryo (albino) and ones derived from the mutated ES cell (dark agouti).
products, characterize genetic regulatory regions, establish links between mutant phenotypes and particular transcriptional units as an aid to verifying the identication of a cloned gene, and create mouse models of human genetic diseases.
Figure E.9
The transgenic mouse protocol proves that SRY is the testis-determining locus responsible for the production of maleness during embryogenesis. DNA fragments con-
taining the mouse SRY gene and its regulatory sequence were injected into a series of embryos that were allowed to develop into live animals. Normal XX animals without a transgene develop as females. But the presence of the SRY transgene in an XX embryo induces development as a male.
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a mouse gene of interest with regulatory regions from other mouse genes, they can cause transgenic mice to express the natural transgene product in an unnatural manner: at a higher than normal level, in an alternative tissue, or at an alternative developmental stage. They can then use the aberrant level, time, or place of expression to elucidate the normal function of the wild-type gene. Experiments analyzing the myc gene demonstrate the power of transgenic technology for uncovering the function of genes in ectopic expression (that is, expression at an abnormal place, time, or level). Investigators originally discovered the myc gene in the genome of the myelocytomatosis chicken retrovirus; exposure to this virus caused cultured chicken cells to become tumorigenic. Hybridization studies demonstrated the existence of a myc gene homolog in the genomes of mice and other vertebrates, including humans (where it resides on the long arm of chromosome 8) but not in the genomes of nonvertebrate model organisms, such as Drosophila. Moreover, in human cancer cells obtained from patients with Burkitts lymphoma (a cancer of the immune systems B cells), the myc gene often appears close to one of the breakpoints of a reciprocal translocation characteristic of these cancer cells between the long arms of chromosomes 8 and 14; in this translocated position, the gene is usually expressed at a higher than normal level. Noncancerous animal cells have a very low level of myc gene expression. These ndings constitute circumstantial evidence that abnormally high levels of myc expression might help transform a cell to a cancerous state. (The biochemical mechanism by which the myc gene product functions is described in Chapter 18 of the main textbook.) The results of experiments using cells grown in culture support this hypothesis. In almost every case studied, however, cultured mammalian cells display programs of gene expression that do not correspond with those of the cells they are supposed to model. Thus, the results of cell culture studies do not necessarily reect how cells in vivo (that is, within the body) behave in response to a change in gene activity. In addition to differences in gene activity, there are differences in chromatin structure and patterns of DNA methylation between cells growing in vitro and in vivo. These discrepancies are not surprising since cells in the body, unlike those in culture, exist in a complex environment that includes constant exposure to molecular signals released by other body cells. The living organism also has a pervasive immune system that is impossible to imitate in vitro. It is therefore possible that a phenotype observed in response to the abnormal expression of a gene in cultured cells might be a consequence not of one genes abnormal expression but of interactions with other genes that are expressed differently in cultured cells than in cells in vivo. For these reasons, it is not possible to rely on cell culture results for an explanation of the true function of a gene; rather it is
(a) (1) The myc locus found in the mouse genome. Promoters 5' 1 kb (2) Hybrid DNA construct containing the myc coding region regulated by an inducible promoter 5' Inducible promoter (b) 3' Exons 3'
Figure E.10 Transgenic expression of the myc gene provides information on the genes role in tumor formation.
(a) Construction of a transgene containing the myc gene under the control of an inducible promoter: (1) structure of the endogenous myc gene and (2) transgene construct with the MTV (dexamethasone-inducible) promoter attached to a portion of the myc gene that contains the coding region. (b) Northern blot showing induction of transgene expression in a range of adult tissues.
necessary to examine the effects of aberrant gene expression in vivo. To learn whether increased expression of the myc gene affects tumor formation in various tissues of the mouse, researchers used transgenic technology (Fig. E.10). In one experiment, they attached the immunoglobulin gene promoter to the myc coding sequence to produce a transgenic mouse line that expressed myc at high levels in the precursors to immunoglobulin-producing B cells. In another experiment, they attached tissue-specic promoters, including one for mammary gland expression, to the myc coding region. And in yet another experiment, they attached to the myc coding region a promoter that is recognized in all tissues but only after the embryos or animals exposure to dexamethasone, a glucocorticoid hormone. The results of all these experiments showed that overexpression of the myc gene does not have any effect on normal developmental processes. Even when the gene was expressed at high levels in many developing tissues, normal animals were born. Moreover, even in the adult, most cells that overexpress the myc gene never display an aberrant phenotype. However, the rate of tumor formation increased signicantly in most, but not all, types of tissue. The conclusion was that aberrant expression of the myc gene alone does not cause cells to become cancerous; but it can operate with other somatic mutational events to
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transform a cell to the cancerous state. These observations support the hypothesis that the cancer phenotype results from the accumulation of multiple mutations in the clonal progeny of one cell (see Chapter 19 of the main textbook). Biochemical and genetic studies indicate that the protein product of the wild-type myc gene is a component of a transcription factor that helps control the growth of cell populations in almost every tissue of the body (as described in Chapter 18 of the main textbook).
Mouse genomic clone Coding region 5' Regulatory region 3' 5'
E. coli Mouse Inject transgene construct into one pronucleus of one-cell mouse embryos. Place embryos into oviduct of receptive female.
Mate transgenic animal to establish pregnancy. Recover and stain fetuses to detect -gal activity.
Figure E.11 Transgenic technology can be used to analyze cis-acting regulatory regions. A DNA construct containing the mouse regulatory region of interest is attached to the E. coli reporter gene. The function of the regulatory region can be ascertained by observing -gal expression in transgene fetuses. In the example shown here, expression is observed in the developing forelimb (blue) of three independently derived transgenic mice containing the complete regulatory region of the mouse Tbx5 gene, which is known to play a specic role in forelimb development of all vertebrates. John Schiementi, The Jackson Laboratory. Reproduced from Promoter Mapping of the Mouse TcP-10bt Gene I Transgenic Mice Identies Essential Male Germ Cell Regulatory Sequences, Ewulonu et al., Molecular Reproduction and Development 43:290297, 1996. Reproduced by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.
substrate known as X-Gal into a colored product that can be visualized under the microscope. To characterize the regulatory region associated with a gene, you rst create a series of different transgene constructs, each formed by splicing together different fragments or mutated forms of the putative regulatory region with the -galactosidase reporter gene. You next establish a series of transgenic lines by injecting the transgene constructs into different mouse embryos. Then, after setting up timed matings within each of these lines, you recover
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embryos at the developmental stage or stages during which the mouse gene normally undergoes expression. By examining the distribution of the colored -galactosidase product at each embryonic stage and comparing it to the distribution of the natural gene product, it is possible to map the extent of the cis-acting regulatory region. The distribution of the natural gene product can be tracked with specic antibodies labeled for examination by immunohistochemistry. If the antibody label is a different color than the -galactosidase label, the natural gene product and the -galactosidase enzyme can be simultaneously observed and distinguished under the microscope. Further studies of the effects of individual base-pair substitutions or small deletions on details of the expression pattern can lead to a highly sophisticated understanding of the various cis-control elements. The collective behavior of these cisacting regulatory elements helps determine the complex patterns of spatial and temporal expression of genes that play a role in development. An example of the use of transgenic mice to study gene regulation is the analysis of a gene called T complex protein10bt (Tcp10bt). Only differentiating male germ cells in the process of maturing into spermatozoa express Tcp10bt. Cells in the seminiferous tubules, called sertoli cells, mediate sperm differentiation. When investigators disrupt the testes and place sperm cells in culture, they cannot simulate natural conditions completely; as a result, germ cell differentiation continues in vitro for only a brief time. But differentiation from stem cell to mature spermatozoa takes six weeks in mice. Sperm differentiation is of interest to cell biologists because the mechanisms that regulate it may differ signicantly from those that control the differentiation of other cells. This is, in part, because the transformations of differentiating sperm cells are much more dramatic than those experienced by other types of cells. Not only do the spermatogenic cells change in shape and size from large round stem cells to tiny, sleek spermatozoa almost without cytoplasm, they also drastically change their genetic program: Stem cells have a normal program of gene expression as well as chromosomes with a normal chromatin structure; in contrast, the chromosomes of differentiated sperm cells have a unique chromatin structure with no histones attached, and they exhibit no gene activity. To understand these differences, mouse geneticists have tried to characterize the regulatory regions associated with gene activity in spermatogenic cells. To analyze the regulatory region associated with the Tcp10bt gene, geneticists rst made a series of transgene constructs carrying different lengths of DNA from the 5 anking region of the Tcp10bt gene, ligated to the lacZ coding sequence. The result was six DNA constructs containing from 0.61.6 kb of DNA from the putative Tcp10bt regulatory region. The researchers injected copies of each DNA construct into multiple mouse embryos, obtaining at least four independent transgenic
-galactosidase 1.3 kb 1.16 kb 0.97 kb 0.75 kb 0.6 kb Bg 0.97 1.16 H Bsp Bam Sfa X Bam 1.3 1.6 H Nhel Bam
(b)
Figure E.12 An example of the use of transgenic technology to map the cis-acting regulatory region associated with the Tcp10bt gene. (a) Different hybrid DNA constructs were
made with varying lengths of the 5 anking sequence adjacent to the Tcp10bt gene fused to the E. coli -galactosidase gene (which acts as a reporter). (b) Testicular RNA was obtained from transgenic mice containing the various lengths of 5 anking region (shown in kilobases). With 0.75 kb of anking region, no transcription of the reporter gene was observed, but with all larger anking regions, transcription did occur. Source E.12b: John Schiementi, The Jackson Laboratory. Reproduced from Promoter Mapping of the Mouse TcP-10bt Gene I Transgenic Mice Identies Essential Male Germ Cell Regulatory Sequences, Ewulonu et al., Molecular Reproduction and Development 43: 290297, 1996. Reproduced by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.
lines of mice for each construct. (It is important to use multiple transgenic lines in an experiment of this type to verify that sequences in the transgene construct itself, rather than sequences that coincidentally ank the transgene insertion site, are responsible for a particular phenotype.) Figure E.12 depicts how it was possible to map the regulatory region associated with Tcp10bt by simply testing for the presence of lacZ transcripts in Northern blots of testicular RNA obtained from each transgenic line of mice. As the gure shows, there was no detectable transcription of the lacZ gene in testes from transgenic mice that carried 0.75 kb or less of anking sequence 5 to the Tcp10bt gene; but with 0.97 kb or more of the anking sequence, high levels of transcription occurred in the testes, but in no other tissue, of all mouse lines. These observations located a critical testes-specic, cisregulatory sequence within a 227 bp region between 746 and 973 bases upstream of the Tcp10bt gene. With this information, it became possible to design additional experiments to examine the regulatory region in more detail and identify
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transacting proteins that bind to this region. Through these additional experiments, researchers identied specic testicular proteins that activate the Tcp10bt gene.
cloning of the mouse homolog. Development of the mouse model also required fabrication of a CFTR knockout construct, derivation of an ES cell culture from a mouse blastocyst, transfection of the ES cells with the CFTR knockout construct, selection of cells in which homologous recombination had replaced the wild-type CFTR gene with the mutant knockout allele, and nally, the production and analysis of chimeric mice and their offspring. Animals homozygous for the CFTR knockout allele display a mutant phenotype that is very similar to that expressed by humans suffering from cystic brosis. Thus, in developing drugs to alleviate CF symptoms in humans, pharmaceutical researchers can rst test new products in mice to determine their efcacy.
Figure E.13 Transgenic technology can be used to identify the locus responsible for a mutant phenotype. A dominant deletion mutation at the T locus causes a short tail. A transgenic animal containing the pme75 transgene is mated with a mutant animal to create animals containing both the deletion and the transgene. A normal phenotype demonstrates that the deletion of the pme75 gene is responsible for the short-tail phenotype.
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(a) Plasmid clone containing portion of mouse CFTR locus with first exon 3' exon 1 (b) Develop DNA construct by adding selectable marker (neo ) and TK gene to CFTR restriction fragments 5'
(c) Early blastocyst recovered from mating between two agouti parents of the 129/ SvJ strain
neo exon 1
TK
neo
TK
Develop ES cell culture by placing blastocysts in petri dish to undergo many cell divisions without differentiation
ES culture
disrupted CFTR exon 1 neo neo Expose ES culture to neomycin. Remaining cells contain CFTR construct integrated either randomly or homologously TK
Expose colonies to ganciclover. TK-containing cells eliminated Transfer remaining colony to plate. Begin new culture.
Figure E.14
E.2 How Biologists Use Transgenic Tools to Study Mice and Create a Mouse Model for Human Disease
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(f ) Mate B6 black mice. Embryos recovered from pregnant B6 female. (+/+) (+/+)
10 ES cells are placed in embryos which are returned to uterus of B6 foster mother.
(+/+) Chimera [agouti (+/)] and [black (+/+)] Mate chimera with B6 black mouse [agouti (+/)] and [black (+/+)] black (+/+)
(h)
Use DNA analysis to identify male and female agouti animals that are heterozygous for the knockout allele of CFTR (+/) and breed them toghether (+/) (+/)
Offspring homozygous for mutant allele serve as models for CF disease state. (+/+) (+/) (+/) (/)
Use DNA analysis to identify offspring homozygous for knockout allele to serve as models for cystic fibrosis disease state
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Anterior
Antennapedia locus
Posterior
lab
pb
(Zan)
Dfd
Scr
Antp
Ubx
Abd-A
Abd-B
5'
Hox A, chromosome 6
A1 A2 A3 A4 A5 A6 A7 A9 A10 A11 A13
Hox B, chromosome 11
B1 B2 B3 B4 B5 B6 B7 B8 B9
Hox C, chromosome 15
C4 C5 C6 C8 C9 C10 C11 C12 C13
Hox D, chromosome 2
D1 D3 D4 D8 D9 D10 D11 D12 D13
Figure E.15 The mouse Hox gene superfamily contains multiple homologs of each member of the Drosophila homeotic selector gene family. Genes within the four mouse Hox clusters are lined up according to their homology with each other and specic
Drosophila genes. Not all clusters have homologs of each Drosophila gene. From gene 9 and higher, there are multiple homologs of the Drosophila Abd-B gene in Hox clusters A, C, and D.
E.3
A particularly striking example of how mouse biologists used both nuclear injection and targeted mutagenesis technologies to decipher gene function comes from an analysis of the mouse Hox gene family. The Hox genes are homeotic selector genes, that is, genes that control the development of body segment characteristics. Members of the Hox family are distributed among four unlinked gene clusters that each contain 911 genes (Fig. E.15). Researchers discovered the mouse Hox gene family through cross-hybridization studies using the homeotic selector genes of D. melanogaster as probes. In fact, homeotic selector genes were rst identied on the basis of mutations that produced ies with four wings instead of two, ies whose mouthparts developed incorrectly as legs, and ies with legs instead of antenna growing out of their heads. The proteins encoded by these genes turned out to be transcription factors that act as on/off switches, instructing segments of the y to develop into one type of tissue or another. William Bateson, the same man who coined the term genetics, chose the designation of homeotic selector from the Greek word homoios, which describes a type of variation in which something has been changed into something else. Homeotic genes appear to control the development of each Drosophila body
segment (as discussed in the Drosophila portrait, Reference D on our website). The bizarre phenotypes just described result when expression of a particular homeotic gene does not occur at the proper time and place. Lack of appropriate expression flicks the binary switch, transforming the recipient body segment into a different type of tissue. Drosophila homeotic genes were rst cloned in the early 1980s. By the end of that decade, it had become clear from cross-hybridization and cloning studies that homologs of these genes are likely to exist in every species of multicellular animal, from C. elegans to Homo sapiens. In segmented animals such as ies, homeotic genes are active in the discrete segments that dene the body plan, where they determine the proper differentiation of tissues. But what do they do in mice and humans, organisms that do not have obvious body segments? To answer this question, researchers had to overcome a serious drawback: the lack of known mutations at any of the Hox loci. This problem was not unique to understanding the functions of mammalian Hox genes. Since the discovery of homeotic gene homologs in the mouse genome, it has become routine for developmental geneticists to use cross-hybridization protocols to look for mouse homologs of every Drosophila gene found to have a role in development. This strategy has led to the discovery of dozens of new mouse genes, most of which were not associated with any known mutant phenotypes.
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13
12
11
10
5'
HoxD13
Figure E.16
Spatial extent of expression of some representative Hox genes along the developing spine. The top of the
mature spine is shown to the right, bottom to the left, with vertebrae numbered and grouped by name.
How Scientists Determine the Function of a Gene in the Absence of Previously Characterized Mutations
Analyses of Expression Patterns in Developing Embryos Can Provide a Clue to the Time and Location of Gene Action
A clone of a gene can be a tool for analyzing the genes expression. One way to convert a clone into this type of tool is to label it, denature it, and use the resulting DNA strands as probes in in situ hybridization. To study development, investigators can perform in situ hybridization on the RNA present in xed tissue sections obtained from embryos at different stages of development. When developmental geneticists examined the expression patterns of the Hox genes, they discovered that each one is transcribed along a portion of the developing embryonic axis that extends from the same most posterior point to a specic anterior boundary (Fig. E.16). Analysis of the pooled data on Hox gene expression showed that the anterior boundary of expression corresponds with the position of each gene in its cluster. Genes at the 5 end of a cluster (for example, D13) have the
least extensive expression which is restricted to the posterior region of the embryonic axis. In contrast, genes at the 3 end of the cluster (for example, A1 and B1) have a broader range of expression that extends further to the anterior region of the embryonic axis. These data suggest that different Hox genes might be involved in controlling the development of different sections of the embryonic axis. Expression data alone, however, cannot provide conclusive evidence of function.
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Validating the Hypothesis That Expression of the 5 Gene in a Hox Cluster Is Epistatic to Expression of the More 3 Genes
The expression data show that some Hox genes are expressed across many segments of the embryo, even as those segments develop differently from each other. To account for this difference in the simplest way, researchers proposed that the only gene that counts in any particular embryonic segment is the most 5 gene in a Hox cluster. In other words, expression of the most 5 gene is epistatic to expression of the other, more 3 Hox genes. By examining Figures E.15 and E.16, you can see that this hypothesis could explain how each spinal segment develops in a different manner.
(a)
(b)
(c)
Figure E.17 Transgenic technology provides support for the mode of action of the Hox gene family. (a) A transgenic
construct is produced to missexpress HoxD4 in a more anterior region where HoxA1 is normally expressed (refer to Fig. E.16 for the normal extents of expression of each of these genes). In panel (b), the complete skeleton of a wild-type animal is shown on the left, and that of an animal expressing the transgene construct is shown on the right. A blowup of the cervical regions from both skeletons is shown in panel (c), again the wild type is on the left and the transgenic construct is on the right. In transgenic newborns, what would have been occipital bone (region E in wildtype animal) has been transformed into ectopic arches (E1) that look like cervical vertebrae.
Transgenic Studies Lead to an Understanding of the Developmental Role of Hox and Other Homeotic Genes
With the accumulation of transgene and knockout data for many of the mouse Hox genes, a general understanding of the developmental role played by this gene family, not only in mice but in other animals as well, has emerged. What the Hox genes and their homologs in other species apparently do is establish signals identifying the position of each region along the embryos anterior-posterior axis. The genes
Solved Problems
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do not determine the differentiation of any particular cell type or tissue. Rather, they provide positional information that other genes act on to promote the differentiation of particular tissues. The positional information as well as the genes that act on it vary from species to species. It is likely that the emergence of the Hox-like gene family in our most recent single-cell ancestor set the stage for the evolution of developmental complexity, with the consequent
appearance of metazoan organisms sometime between 1 billion and 600 million years before the present time. Thus, the analysis of the Hox gene family is an example of how detailed genetic studies in one model species can provide a general understanding of gene function across large segments of the animal kingdom as well as clues to the conserved mechanisms by which complex developmental processes are carried out.
Connections
Geneticists can now produce transgenic animals by combining the classic tools of mutagenesis with an understanding of molecular biology and embryology. Transgenic technology has become so sophisticated that, in theory, it is possible to make any genetic change imaginable to the mouse genome and determine its effect on the individual that emerges. With the ability to produce mice carrying add-ons and knockouts of Hox and other genes that play a role in development, mouse geneticists have begun to dissect the process by which the mouse embryo develops from the one-cell zygote, through gastrulation, and into the organbuilding stage. Remarkably, the development of all mammals is similar, especially in these early stages. For example, all the genes important to mouse development are conserved in the human genome. Thus, much of what we learn about the genetic basis for mouse development will apply to normal and abnormal human development.
Essential Concepts
1. The availability of hundreds of single-gene mutations and a short life cycle contribute to M. musculuss value as a model organism. 2. The mouse genome closely resembles the human genome in size, gene content, and syntenic loci. Researchers can thus use homology and conserved synteny analysis to identify, locate, and determine the function of genes in both species. 3. The mouse life cycle is representative of the mammalian life cycle, although the timing of events is unique to each species. The totipotency of preimplantation cells in mammals makes it possible to create chimeras. 4. Researchers can use the transgenic technology of adding genes to the mouse genome by nuclear injection to determine gene function, characterize regulatory regions, and correlate mutant phenotypes with specic transcription units. 5. Targeted mutagenesis is the basis for creating a mouse model of human diseases caused by a loss of gene function. 6. Studies using transgenic technology have revealed that the Hox genes generate signals that identify the position of each region along a mouse embryos anterior-posterior axis. Normal development of the embryo depends on this information. 7. Knowledge of how the Hox genes function in mice has elucidated the general role played by homeotic selector genes in the evolution of all metazoan organisms, including humans.
Solved Problems
I. Gain-of-function mutations can produce a novel phe-
a. b.
notype and act in a dominant fashion. In loss-offunction mutations no functional gene product is made; most, but not all, loss-of-function mutations are recessive.
Which of these types of mutations would you study using add-on transgenic technology? Which type of mutation would you have to study with the implementation of homologous recombination in ES cells? Why?
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Answer
This problem requires an understanding of add-on transgenic and homologous recombination techniques and their applications. a. Dominant alleles caused by gain-of-function mutations are expressed phenotypically when a single copy of the dominant allele is present. The effect of a dominant gain-of-function mutation can be characterized in transgenic mice where a transgene containing the dominant allele has been inserted into the genome and the original gene copy is still present. b. Loss-of-function mutations would have to be studied using homologous recombination, where a normal copy of the gene is replaced with a defective version. To observe a phenotype of a recessive loss-of-function mutation, there should not be any normal version of the gene present. After replacing one gene copy with a mutant version, heterozygous mice would be mated to obtain a mouse homozygous for the recessive allele and to observe the resulting phenotype.
II. You have isolated muscle cell RNA and made a muscle
bp
DNA in subclone 1. 2. 3. 4. 5. 6. 7.
Expression in bone + + +
Expression in muscle + + +
Answer
This problem requires familiarity with several molecular techniques including analysis of cDNAs and regulatory sequences in the promoter. a. The cDNA clone was derived from the mRNA and therefore it will probably not contain the regulatory region usually found upstream of 1 5 to) the transcription start site. b. The cDNA clone is a good source of a probe for nding the genomic clone in a genomic library. 1. Isolate a DNA fragment from the cDNA clone and label for use as a probe. 2. Grow clones (bacteriophages or cosmids) from a genomic library on plates. 3. Transfer clones from plates to lter paper. 4. Incubate the labeled probe with the lters to nd, through hybridization, a genomic clone containing the gene. c. To study developmental expression of this gene, the fusion construct would be transferred into the mouse genome, using transgenic techniques. At different stages in development the tissues of the mouse can be tested for the presence of the mRNA or protein using the lacZ DNA or antibody that recognizes the -galactosidase protein respectively. d. To examine the transcripts in these two cell types, you would prepare mRNA from isolated muscle and bone cells and use the cDNA clone to probe separated RNAs that have been transferred to a lter (Northern hybridization analysis). Transcripts may be different sizes because of different transcription start sites or different processing (splicing) events. e. The presence or absence of bone or muscle transcripts in mice that carry different transgene constructs provides a way to identify DNA fragments containing tissue-specific regulatory sequences. There are three constructs (4, 5, and 6) that express the transgene in bone tissue. The
cDNA library. You want to study the regulatory region of one of the genes you identied as being expressed in muscle cells. a. You need to isolate a genomic clone of this gene. Why is the cDNA clone not sufcient for your studies? b. Outline the steps to isolate the genomic clone of this gene. c. You isolated a genomic clone containing the entire coding region and 1.2 kb upstream of the coding region. A map of the genomic clone is shown here. To make a fusion construct of the presumed regulatory region attached to the reporter gene lacZ, you cloned the 1.3 kb BamHI fragment into a vector containing the lacZ gene. The fusion construct, when injected into cultured muscle cells, expressed -galactosidase protein. What experiment would you do next to determine if the fusion construct is developmentally regulated? d. Using an antibody against the -galactosidase portion of the fusion protein you analyzed the tissues of a transgenic mouse and found that the protein encoded by this gene is also present in bone cells. How could you determine if the same transcript was expressed in both cell types? e. You decided to analyze the promoter region of the gene to identify sequences responsible for the expression in the two different cell types. The results obtained with constructs containing different fragments from the 5 region flanking the gene to the lacZ coding region are shown here. What region(s) are important for expression in muscle? in bone?
Problems
129
only portion of the regulatory region that they all have in common is a 50 bp Sau3A fragment. The bone-specic regulatory element must be within this fragment. For muscle expression, there are three clones (5, 6, and 7) that express the transgene in muscle tissue and these share the adja-
cent 350 bp Sau3A fragment. This analysis shows that expression of the same gene is subject to different mechanisms of regulation in two tissue types that use different regulatory sites in the 5 region anking the gene.
Problems
E-1 Choose the matching phrase in the right column for E-4 A mouse gene was localized to a region of chromo-
some 4. From the synteny map, it appears this gene would localize to chromosome 8 in humans. a. How could you determine if the gene is in this region of human chromosome 8? (Do not use the approach of cloning the gene here.) b. Briey outline how you could obtain the human clone containing the homolog of this mouse gene.
E-5 Why do identical twins occur in mice (and other mam-
the C. elegans genome (~100 Mb). a. What challenges are there for a geneticist studying an organism that has a large genome size? b. Despite the large genome size, many geneticists choose to use the mouse as a model system? Why?
E-3 The CFTR gene, which is defective in humans with
cystic brosis, encodes a membrane protein that acts as a channel for the passage of Cl. Although one particular mutation (a deletion called D508) is predominant in Caucasians, more than 200 different mutations in the gene have been identied that cause the disease. Many Cl channel genes, including CFTR homologs, have been identied in other organisms such as C. elegans and yeast. For each of the following research questions, indicate whether you would be more likely to pursue an answer using yeast or mouse and why you would choose that organism. a. Do mutations in different regions of the gene have the same effect on Cl channel function? b. Do mutations in different regions of the gene affect channel function in all organs normally involved in cystic brosis? c. What portion of the protein receives a signal to open the channel? d. Are there drugs that can cause an opening of the channel?
pressed for several days during development of the embryonic heart and pituitary in mice. Expression continues in the pituitary in the adult and also is seen in the germ cells of the adult testis. a. You bred a male mouse carrying one normal CTF allele () and one disrupted CTF allele () with a female heterozygous for the same alleles and examined 30 of their offspring. (The disrupted allele contains an insertion within the gene.) Twentyone of the offspring were / and 9 were / . What would you conclude about the CTF gene in this case? b. Suppose instead that you obtained 30 offspring and 7 of the mice that had the ( / ) genotype were half the normal size. Of these, all 4 males were sterile, whereas the 3 females were fertile. What would you conclude about the CTF gene from these results? c. In the preceding experiments, what technique(s) were used to determine if the offspring mice were ( / ), ( / ), or ( / )?
E-8 Retinoic acid, which acts on cells via a protein recep-
tor, is thought to be important for limb development in mammals. a. Several different strains of mice that have recessive defects in limb development are available. You have tested each of these strains for the presence of the retinoic acid receptor (RAR protein), RAR mRNA,
130
and the RAR gene using Western (protein), Northern (RNA), and Southern (DNA) blots, respectively. Based on the following data, give a reasonable hypothesis regarding the defect in each mutant.
Strain RAR protein RAR mRNA RAR gene
A Normal* Normal Normal B Absent Normal Normal C Absent Short Normal D Absent Short Altered E Absent Absent Altered F Absent Absent Absent *Normal means that the protein, RNA, and DNA bands were normal in size and abundance; normal does not refer to function of the protein. One of the bands hybridizing with the RAR cDNA clone migrated to a different position in strains D and E.
b.
c.
The limb deformity in strain A could be due to a defect in some gene other than RAR. Suggest a simple genetic experiment that would enable you to test this possibility. If you wanted to test the developmental consequences of RAR gene expression in neurons, which do not normally make RAR, how would you do this?
ed that have an obese phenotype. The Ob gene, defective in one class of these mutants, was the rst gene involved in obesity to be cloned. The Ob gene product is made in fat cells and is transported to the brain, where it informs the brain that the animal is satiated (full). a. You made an antibody to the Ob protein and used this to test predictions of the hypothesis that Ob is a satiety factor. You isolated protein from mice that had been eating normally, from mice that had been starved, and from mice that had been force-fed a high-calorie diet. You did a Western analysis using your antibody as a probe against proteins from these animals. Results are shown here. Are these results consistent with the hypothesis for the role of the Ob protein? Why or why not?
Normal Starved Force- fed
the DNA can insert at random in any of the chromosomes. Subsequent matings produce animals homozygous for the transgene insertion. Sometimes an interesting mutant phenotype is generated by the insertion event. In one case, after injection of DNA containing the mouse mammary tumor virus (MMTV) promoter fused to the c-myc gene, investigators identied a recessive mutation that causes limb deformity. In this mouse, the distal bones were reduced and fused together; the mutation also caused kidney malfunction. a. The mutant phenotype could be due to insertion of the transgene in a particular region of the chromosome or a chance point mutation that arose in the mouse. How could you distinguish between these two possibilities? b. The mutation in this example was in fact caused by insertion of the transgene. How could you use this transgene insertion as a tag for cloning? c. The insertion mutation was mapped to chromosome 2 of mice in a region where a mutation called limb deformity (ld) had previously been identied. Mice carrying this mutation are available from a major mouse research laboratory. How could you tell if the ld mutation was in the same gene as the transgenic insertion mutation? d. Analysis of transcripts from the ld gene showed that many different transcripts (formed by alternate splicing) were present in both the embryo and adult. Is this consistent with a role of the ld gene product in limb development in the embryo?
How could you determine if the Ob gene is transcriptionally regulated? c. If the amount of mRNA was the same for all three types of mice, what type of regulation is involved? d. You isolated RNA from several different Ob mutants and analyzed the RNA using Northern analysis. What conclusions could you reach about each mutation based on the results shown here? b.
Wild type A B C D
Problems
131
e.
You have now decided to clone the receptor for the Ob protein and express the gene in mammalian cells. Which is the correct alternative for each of the following steps? 1. (a) Isolate brain mRNA. (b) Isolate fat mRNA. 2. (a) Clone cDNA into a plasmid vector next to a mouse metallothionein promoter. (b) Clone cDNA into a plasmid vector next to the yeast LEU2 promoter.
3. (a) Treat cells with zinc. (b) Deprive cells of leucine. f. You identied the Ob receptor clone using the screen outlined in part e and decided to make a knockout of the gene in mice. What phenotype would you predict for mice that lack the Ob receptor? (obese, slim, normal) g. If you inject Ob protein into Ob mutant mice, what effect do you predict there will be on the phenotype?