Mus musculus:Genetic Portrait of the House Mouse Referencelibrary01.forbiddenmousecity.com/L028.pdf · 110 Reference EMus musculus: Genetic Portrait of the House Mouse homology analysisto

The common house mouse, Mus musculus, has played a prominentrole in the study of genetics ever since Carl Correns, Hugo De Vries,and Erich Von Tschermak independently rediscovered Mendel’s lawsat the beginning of the twentieth century. Because these three scien-tists, as well as Mendel himself, performed their research entirely onplants, many in the scientific community questioned whetherMendel’s laws could explain the basis for inheritance in animals, espe-cially humans. The reason for this skepticism is easy to see. People, forexample, differ in the expression of many commonly inherited traits—such as skin color, eye color, curliness of hair, and height—that showno evidence of transmission according to Mendel’s laws. We nowknow that these traits result from the interaction of many genes withmultiple alleles that each segregate according to Mendel’s first laweven though the traits themselves do not. At the beginning of thetwentieth century, however, a demonstration of the applicability ofMendel’s laws to animal inheritance required the analysis of simpletraits controlled by single genes.

M. musculus has many features that enhance its value as a model organism forgenetic analysis, and foremost among these is the availability of hundreds of single-gene mutations. These mutations arose during the mouse’s long history of domesti-cation as a pet. Over the centuries, dealers in what became known as the “fancymouse” trade selected and bred mice with numerous coat colors and other visiblemutations, first in China and Japan, later in Europe (Fig. E.1a). In contrast to thevariation that occurs naturally in wild populations, new traits that appear suddenlyin captive-bred mice are almost always the result of single-gene mutations. Earlyanimal geneticists made note of this fact and used fancy mice to demonstrate thatMendel’s laws apply to mammals and, by extrapolation, to humans.

In addition to providing a ready source of single-gene mutations, the housemouse has several other features that make it the mammal of choice for geneticanalysis. Mice have a very short generation time of just eight to nine weeks. Theyare small enough so that thousands can live in relatively small rooms. They havelarge litters of eight or more pups. They breed readily in captivity. Fathers do notharm their young. And after centuries of artificial selection, domesticated mice aredocile and easy to handle (Fig. E.1b).

But why study a mammal at all when animals like fruit flies and nematodes areeven smaller and more amenable to genetic analysis? The answer is that a majorgoal of current biological research is the understanding of human beings. Andalthough many features of human biology, especially at the cellular and molecularlevels, are common to a broad spectrum of life-forms, the most advanced organism-level human characteristics appear in a limited subset of animals. In fact, manyaspects of human development and disease are common only to placenta-bearingmammals such as the mouse. Thus, the mouse provides a powerful model systemfor investigating the genetic basis of simple and complex human traits, especiallythose related to development and disease (Fig. E.2).

Two general themes emerge from our presentation of M. musculus. First, becauseof the many similarities between mouse and human genomes, researchers can use

Mus musculus: Genetic Portrait of the House Mouse ReferenceE

109

A member of the 129 strain ofinbred mice commonly used intargeted mutagenesis studies.

har06584_refE_109-132 11/06/2006 06:11 PM Page 109

110 Reference E Mus musculus: Genetic Portrait of the House Mouse

homology analysis to identify and locate the same genes in both species. In this con-text, homologs are genes or regulatory DNA sequences that are similar in differentspecies because of descent from a common ancestral sequence. Second, geneticanalysis in mice exemplifies the combined use of molecular (that is, recombinantDNA) and classical breeding techniques to identify and understand the function ofcomplex genetic systems.

Our genetic portrait of the house mouse describes:

• An overview of M. musculus in the laboratory, including a look at themouse genome, the mouse life cycle, and two powerful transgenic protocols:the addition of specific genes to the mouse genome by nuclear injectionand the removal of specific genes from the mouse genome by targetedmutagenesis.

• The uses of transgenic technology in determining the function of gene prod-ucts, characterizing regulatory regions, establishing links between mutantphenotypes and particular transcription units, and creating a mouse modelfor a human disease.

• The Hox genes: a comprehensive example.

(a)

(b)

(a)

(b)

Figure E.1 The mouse is a model system for humanbiology. (a) Examples of visible phenotypes caused by single-genemutations. (b) Mother mouse with her pups.

Figure E.2 Hirschsprung disease: A human developmentaldisease that causes deformities of the colon (a) and (b) a mousemodel of the disease with a classical mutation (piebald coat).

har06584_refE_109-132 11/06/2006 06:11 PM Page 110

E.1 An Overview of Mus musculus in the Laboratory

The Mouse GenomeThe most important feature of the mouse genome for con-temporary geneticists is its close resemblance to the humangenome (Table E.1). The haploid genomes of humans andmice (and other placental mammals as well) contain ap-proximately 3 billion base pairs of DNA. Nearly every hu-man gene has a homolog in the mouse genome. This doesnot mean that the two genomes are equivalent in content.Nearly all differences, however, appear to result fromspecies-specific additions to gene families that alreadyexisted in the common ancestor of mice and humans.

The human genome is distributed among 22 autosomesand 2 sex chromosomes, while the mouse genome is con-tained within 19 autosomes and 2 sex chromosomes. As wesaw at the beginning of Chapter 14 of the main textbook,examinations of mouse and human karyotypes under themicroscope reveal no evidence of chromosome bandingsimilarities between the two species. With the mapping ofthousands of homologous genes in both species, however, aremarkable pattern has emerged. Genes that are closelylinked in one species are usually closely linked in the other.When two or more loci are found to be linked in onespecies, they are said to be syntenic (meaning “on the samethread,” or chromosome). When the same set of loci arealso found to be linked in a second species, they are said toexist in a state of conserved synteny. A comparison of ge-netic maps of the whole mouse genome with genetic mapsof the whole human genome shows that regions of con-served synteny extend across nearly the complete length ofboth. The average size of each conserved syntenic region isroughly 17.6 Mb. The implication of this finding is that

during the 75 million years that mice and humans havebeen evolving apart from a common ancestor, theirgenomes have broken apart and rearranged some 170 times(17.6 Mb � 170 � about 3000 Mb � the size of the mam-malian genome). Conversely, if the proper genome-scalescissors and glue were available, one could break themouse genome into about 170 pieces and reassemble thosepieces—like a puzzle—in 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 orshe can look at a homology map and immediately identifyits likely map position in the other species. Of course, forgenes that have already been cloned, it is possible to useDNA-DNA hybridization, or computer analysis of a whole-genome sequence, to pick out gene homologs from theother species. But for loci characterized only by their phe-notypic expression, conserved synteny enables geneticiststo move back and forth between the analysis of a trait in hu-mans and the analysis of a model for that trait in mice.

The discovery of a locus that predisposes female miceto excessive consumption of 10% ethanol (the concentra-tion of alcohol found in many wines) provides an exampleof the use of conserved synteny for locating human ho-mologs of mouse genes. Mouse geneticists used DNAmarkers (as described in Chapter 11 of the main textbook)to map the Alcohol-preference-2 (Alcp2) locus to the mid-dle of mouse chromosome 11. Now that the whole mousegenome has been cloned and sequenced, the genes as tran-scription units in Alcpz region have been identified, butwhich one is actually Alcpz is not yet known. However,even though researchers have not yet identified the specificgene, scrutiny of a conserved synteny homology mapshows that the most likely location for the human homologof Alcp2 is on the short arm of human chromosome 17,close to the centromere (Fig. E.3). With this informationabout the likely location of an alcohol-preference locus,

E.1 An Overview of Mus musculus in the Laboratory 111

TABLE E.1 Comparison of Mice and Humans

Trait Mice Humans

Average weight 30 g 77,000 g (170 lb)

Average length 10 cm (without tail) 175 cm

Genome size ~3,000,000,000 bp ~3,000,000,000 bp

Haploid gene number ~25,000 ~25,000

Number of chromosomes 19 autosomes � X and Y 22 autosomes � X and Y

Gestation period 3 weeks Average, 38 weeks (8.9 months)

Age at puberty 5–6 weeks Average, 624–728 weeks (12–14 years)

Estrus cycle 4 days Average, 28 days

Life span 2 years Average, 78 years

har06584_refE_109-132 11/06/2006 06:11 PM Page 111

human geneticists can now look for linkage between DNAmarkers on human chromosome 17 and a phenotypicexpression of a predisposition to alcohol abuse.

The Mammalian Life CycleThe mouse’s life cycle is similar to that of humans and allother placental mammals, although the timing of events isunique to each species (see Table E.1). For example, formice, the average life span is just 2 years; for humans it av-erages 78 years. Gestation—the period from fertilization tobirth—lasts only 21 days in mice but 8.9 months in humans.After birth, mice reach puberty in just 5–6 weeks, 100 timessooner than humans, who mature for roughly 12 years beforethey attain puberty and the ability to conceive children oftheir own (although in most societies, they wait 5–10 yearslonger). It is thus possible to go from the birth of one mouseto the birth of its offspring in just 8 weeks, whereas with hu-mans, completion of the same cycle would take 13–25 years.

Even with significant differences in timing, however,the details of each stage of development, both before andafter birth, are remarkably similar in all mammalianspecies. As a result, the overview presented here appliesequally to mice and humans.

The mammalian life cycle, like that of all sexually re-producing species, can be visualized as continuous circles(Fig. E.4), with any point along the circumferences mark-ing the start. For ease of presentation, we begin with thehaploid phase in both males and females.

Male Germ Cell Development

As we saw in Chapter 4 of the main textbook, once a malemammal has reached puberty, he continuously produces alarge number of haploid germ cells for the rest of his life.The mature haploid cell is a sperm cell, or spermatozoa,and the process by which it arises is spermatogenesis (re-view Fig. 4.19 in the main textbook). Mature spermatozoareleased into the lumen at the center of the seminiferoustubule join millions of other sperm cells, which togetherpass through countless passageways to reach the epididymis;there they mature further and then continue on to the vasdeferens, where they await ejaculation during copulation.

Female Germ Cell Development

Unlike males, females are born with all the haploid cellsthey will ever have (~50,000 in the mouse; 1 million inwomen). When mature, these haploid cells are known aseggs, or oocytes, and the process by which they arise isoogenesis (review Fig. 4.18 in the main textbook). As wesaw in Chapter 4, oogenesis begins inside the newlyformed ovaries of the developing fetus. Long before birth,primordial germ cells differentiate into oogonia and entermeiosis, but they stop at the diplotene stage of the firstmeiotic prophase. These primary oocytes remain arrestedin suspended animation—for weeks in mice and manyyears in humans—until after puberty.


Boxes showregions ofconservedsynteny inthe human genome

Mousechromosome

Mouseloci

Humanhomologs

Humanmap positions

Alcp2

Figure E.3 Conserved synteny: An example with mousechromosome 11. The mouse chromosome, on the left, showspositions of loci with mapped homologs in the human genome.The map locations of human homologs are on the right.

Male Female

Ovary

Blastocyst

ImplantationFetus

Zygote

TestesOogonia

Primaryoocytes

Birth

Spermatozoa

Spermatogonia

Birth

Sperm EggFertilization

OvulationMaturesecondaryoocyte

Diploid phase

Haploid phase

Figure E.4 The mammalian life cycle. The life cycles of malesand females are shown separately in the traditional format. Noticethat primary germ cells are formed in the female before birthbut in the male after birth. Notice also that male germ cells areself-renewing, while in females, no new germ cells are formedafter birth.

har06584_refE_109-132 11/06/2006 06:11 PM Page 112

From this time on, the female progresses through anestrus cycle that lasts about 4 days in mice and about 28days in humans. During each cycle, primary oocytes (8–10in mice, usually only 1 in women) are stimulated to com-plete the first meiotic division and extrude the first polarbody at the end of this division; the resulting secondaryoocyte begins the second meiotic division but stops atmetaphase and is released from the ovary in a processknown as ovulation. Following ovulation, the secondaryoocyte passes into an oviduct (called a fallopian tube in hu-mans), where for a brief time, known as estrus, it remainsalive and receptive to fertilization.

In nature, most mammals die while they still have theability to reproduce. Many human females, however, livelong enough to pass through menopause, during whichthey stop cycling through estrus, no longer ovulate, andthus lose the ability to reproduce.

Fertilization

Just before and during the estrus phase of the estrus cycle,female mammals of nonhuman species release species-specific chemical signals, or pheromones. In a behavioralresponse to these pheromones, a male will copulate with afemale and ejaculate semen containing millions of sperminto her reproductive tract. The sperm swim from thevagina into the uterus and thence up the oviducts. Only 100or 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 pellucida—the thicksolid shell composed of glycoproteins that surrounds the eggproper. The act of binding induces each sperm to release spe-cial 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, usuallyonly one fuses with the egg. This fusion causes rapid electro-chemical changes in the egg membrane that prevent the entryof additional sperm and activate the newly fertilized egg toenter the pathway of animal development.

After fusion, the fertilized egg, or zygote, contains twohaploid pronuclei. The two pronuclei never merge; instead,replication occurs within both of the pronuclei. The one-cell embryo carries two replicated pronuclei right up to themoment of the first mitosis, at which time the membranesof the two pronuclei break down, and the two sets of chro-mosomes, one from the paternal pronucleus, the other fromthe maternal pronucleus, align along the midplane of thefertilized egg and thence segregate chromatids into the twodaughter cells.

For the purposes of analysis, scientists divide mousedevelopment into two distinct stages of unequal length,separated by the process of embryonic implantation intothe uterus: a preimplantation stage that lasts 4–5 days inmice, and a postimplantation stage that lasts about 16.5days in mice. During the preimplantation phase, the em-bryo is a free-floating object within the female’s body. It iseasy to remove this naturally free-floating preimplantationembryo from the animal, culture it in a petri plate, andexpose it to genetic manipulation before placing it backin the reproductive tract of an adult female for developmentto a newborn animal. After implantation, however, suchmanipulation is no longer possible because the embryo, ifremoved from the adult’s body, cannot be returned. Theaccessibility of the preimplantation embryo provides thebasis for many of the genetic manipulations researchersuse to study mammalian development.


Ovulation expands

sperm head

First cleavage

Second cleavage

Thirdcleavage

8-cell embryo

Fourthcleavage

outside cells differentiateinto trophectoderm

Blastocystformation

Blastocyst

hatching, and implantation

Zona pellucida

SpermOne sperm penetrates

Maternal pronucleus

Paternal pronucleus

TrophectodermInner cell mass(ICM)

Blastocoele cavity

Uterine wall

Fertilized egg = zygote = 1-cell embryo

Fertilization2-cellembryo

4-cellembryo

16-cell embryo:

Figure E.5 Early development of mammals from fertilization to implantation.

har06584_refE_109-132 11/06/2006 06:11 PM Page 113

For Most of the Preimplantation Stage, theEmbryonic Cells Remain Undifferentiated

The preimplantation stage starts with the zygote (Fig. E.5).Development proceeds slowly in the beginning, with thefirst 22 hours devoted to the expansion of the highly com-pacted sperm head into a paternal pronucleus that matchesthe size of the egg’s maternal pronucleus. After the paternalpronucleus has completed its expansion and replicated itschromosomes and the maternal pronucleus has replicatedits chromosomes, the embryo undergoes the first of fourequal divisions, or cleavages, that increase the number ofcells from 1 to 16 over 60 hours.

The period of these four equal divisions is called thecleavage stage. During this stage, all the cells in the develop-ing embryo are equivalent and totipotent, that is, they havenot yet differentiated and each one retains the ability, orpotency, to produce every type of cell found in the developingembryo and adult animal. This is very different from the de-velopmental patterns found in most nonmammalian animalspecies, including Caenorhabditis elegans, where totipo-tency disappears as early as the two-cell stage. Because of themouse cells’ totipotency, cleavage-stage embryos can bedivided into smaller groups of cells that each have thepotential to develop into a normal individual. Identical hu-man twins, or more rarely, identical triplets or quadruplets,are examples of the outcome of this process. (Twinning is im-possible in C. elegans or Drosophila melanogaster.) In thelaboratory, scientists have obtained completely normal micefrom individual cells that they dissected out of the four-cell-stage mouse embryo and placed back into the femalereproductive tract (Fig. E.6a). This experimental feat demon-strates the theoretical possibility of obtaining four identicalclones from a single embryo of any mammalian species.

Another more bizarre consequence of the equivalencyof cleavage-stage cells is the formation of chimeras, whichare the opposite of clones (Fig. E.6b). The term “chimera”comes from the Greek word for a mythological beast that ispart lion, part goat, and part serpent. Geneticists use the termto designate an embryo or animal composed of cells fromtwo or more different origins. The Polish embryologist An-drezej Tarkowski reported the first mouse chimeras in 1961.To construct them, he removed the zona pellucida from twocleavage-stage mouse embryos, obtaining denuded cellmasses that are naturally sticky; he then pushed the stickydenuded embryos up against each other. Denuded embryospressed together in this way form a single chimeric cell massthat is capable of undergoing normal development within thefemale reproductive tract. If the two embryos of a chimeracome from different females mated to different males, the re-sulting individual is tetraparental, that is, has four parents. Itis also possible to produce hexaparental animals derivedfrom a combination of three embryos. Every organ and tis-sue in the adult—including the germ line—can contain cellsderived from all three original embryos. As we see later, theproduction of chimeric mice has been an essential compo-

nent of the targeted mutagenesis technology that has revolu-tionized the use of the mouse as a model organism for study-ing human diseases.

A comparison of the early developmental program ofplacental mammals with that of other animals, includingC. elegans and D. melanogaster, shows how different theseprograms can be. In nematodes, embryonic cells are highlyrestricted in their developmental potential, or fate, begin-ning at the two-cell stage; and in fruit flies, polarization ofthe egg before fertilization generates distinct cytoplasmicregions dedicated to supporting different developmentalprograms within the nuclei that end up in these locations.Consequently, half a nematode embryo or half a fly embryocan never give rise to a whole animal.

Events Restricting the Developmental Potency of Individual Cells Occur Near the End of the Preimplantation Stage

The first differentiation events of mouse embryogenesisoccur in the 16-cell embryo (see Fig. E.5). The cells on theoutside of the embryo turn into a trophectoderm layer that


(a)

(b)

Four-cell embryo

Two four-cell embryos

Embryo 1

Chimeric embryo

Chimeric mouse

Embryo 2

Identical quadruplets

Figure E.6 Early mammalian embryos are highly malleablein their development. There is no requirement for a one-to-onecorrespondence between embryo and adult. (a) Creating identicalquadruplets from a single fertilized egg. (b) Creating a singlechimeric animal from the fusion of two embryos.

har06584_refE_109-132 11/06/2006 06:11 PM Page 114

will eventually take part in the formation of the placenta.Soon thereafter, the cells on the inside of the embryo com-pact into a small clump called the inner cell mass, or ICM,that remains attached to one spot along the inside of thehollow trophectoderm sphere. The entire fetus and animalare derived entirely from the cells of the ICM. As Figure E.5shows, compaction of the ICM causes the appearance of afluid-filled space that is devoid of cellular material and sur-rounded by trophectoderm. This space is called the blasto-coel cavity. The embryo is now called a blastocyst. Twomore rounds of cell division occur during the blastocyststage, producing the 64-cell embryo that implants.

Throughout normal preimplantation development, theembryo remains protected within the inert zona pellucida.As a result, there is no difference in size between the 1-cellzygote and the 64-cell embryo. To accomplish implanta-tion, the embryo must first “hatch” from the zona pellucidaso that it can make direct membrane-to-membrane contactwith cells in the uterine wall. Embryonic and fetal develop-ment within a uterus inside the body of a female is a char-acteristic unique to all mammals except the primitiveegg-laying platypus.

After Implantation, the Placenta Develops, the Embryo Grows, and the Tissues and Organs Emerge

Implantation initiates development of the placenta, a mixof embryonic and maternal tissues that mediates the flow ofnutrients entering the embryo from the maternal blood sup-ply and the flow of waste products exiting the embryo tothe maternal circulation. The placenta maintains this inti-mate connection between mother and embryo, and later be-tween mother and fetus, until the time of birth.Development of the placenta enables a period of rapid em-bryonic growth. Cells from the ICM differentiate into thethree germ layers of endoderm, ectoderm, and mesodermduring a stage known as gastrulation. The foundation of thespinal cord is put into place, and the development of thevarious adult tissues and organs begins. With the appear-ance of organs, the embryo becomes a fetus, which contin-ues to grow rapidly. Birth occurs at about 21 days afterconception. Newborn animals remain dependent on theirmothers during a suckling period that lasts 18–25 days. Byfive to eight weeks after birth, mice reach adulthood andare ready to begin the next reproductive cycle.

Two Powerful Transgenic Techniques for Analyzing the Mouse GenomeGeneticists have capitalized on certain features of themouse genome and the mouse life cycle to develop proto-cols that make it possible to add and remove specific genesfrom embryonic or germ cells.

The Addition of Genes to the Mouse Genome by Nuclear Injection

The 1981 development of a method for inserting foreignDNA into the germ line of mice thrust a primarily obser-vational discipline into the realm of genetic engineeringwith all its implications. Yet the incredibly powerful trans-genic technology is based on a very simple process. Recallthat a transgene is any piece of foreign DNA thatresearchers have inserted into the genome of a complexorganism, such as a mouse or a pea plant, through experi-mental manipulation of early stage embryos or germ cells;any individual carrying a transgene is known as a transgenicanimal or plant.

To create a transgenic mouse carrying a foreign DNAsequence integrated into one of its chromosomes, a re-searcher simply injects foreign DNA into a pronucleus of afertilized egg and then places the injected one-cell embryoback into a female oviduct, where it can continue its devel-opment. Roughly 25% to 50% of the time, for a skilledinvestigator, the injected DNA will integrate at random intoa chromosomal location. Integration can occur while theembryo is still in the one-cell stage, in which case the trans-gene will appear in every cell of the adult body. Or integra-tion may occur somewhat later, after the embryo hascompleted one or two cell divisions; in this case, the mousewill be a mosaic of cells, some with the transgene and somewithout it. The relatively high rate of integration appears tobe a consequence of naturally occurring DNA repair en-zymes present in all eukaryotic cells. During evolution,these enzymes acquired the ability to seek out and ligate to-gether open-ended DNA molecules, which can result natu-rally from mutagenesis. Figure E.7 illustrates the details ofthe transgenic procedure.

Up to 50% of the mice born from injected embryoshave the foreign DNA stably integrated into their genomes.They will thus transmit this DNA to their offspring. Thereare no limits to the type of DNA that can be incorporated.It can come from any natural source—animal, plant, ormicrobial—or directly from a DNA synthesizer. It is verycommon for investigators to construct DNA molecules(called “DNA constructs”) composed of genetic elementsfrom different sources. For example, a DNA constructmight have a coding region that is a composite of humanand Escherichia coli sequences flanked by an upstreamregulatory region that is a composite of mouse and syntheticsequences.

Although embryonic nuclear injection is a powerfultransgenic tool, it has two significant limitations. First, itcan only add—not subtract—genetic material. Second,experimenters cannot target the insertion of foreign DNAto specific genomic locations. Consequently, transgenicmice produced by embryonic nuclear injection are usefulonly for the analysis of dominant phenotypes. By 1989,geneticists had developed a way to circumvent theselimitations.


har06584_refE_109-132 11/06/2006 06:11 PM Page 115

Targeted Mutagenesis, a Second Transgenic Technology, Makes It Possible to Remove, or Knock out, SpecificSequences from the Mouse Genome

In addition to knocking out the function of a sequence alto-gether, targeted mutagenesis can produce an allele with analtered function. The emergence of targeted mutagenesis,which is technically more demanding and more complexthan the nuclear injection technology just described, de-pended on two advances in cell culture techniques thatoccurred during the 1980s.

The first advance was the establishment of in vitro con-ditions that enable researchers to place mouse embryos at

the blastocyst stage into culture such that the embryoniccells from the ICM continue to divide without differenti-ating. Cultured cells that behave in this way are calledembryonic stem cells, or ES cells for short. ES cells ap-pear to be similar in their state of differentiation to cellsfrom the ICM. It is possible to grow cultures containingmany millions of ES cells from a single embryo and thenrecover a handful of cells from this culture for injectionback into the blastocoel cavity of a normal embryo. Onceinside the cavity, the ES cells can become incorporatedinto the ICM, and they can contribute to all of the tissuesin the mouse that develops from the embryo. Most impor-tantly for geneticists, the ES cells even contribute to thegerm lines of these chimeric mice so that reproducingadults can transmit mutated genes present in the ES cellsto future generations.

The second critical advance that provided a foundationfor targeted mutagenesis was development of a protocol forhomologous recombination in ES cells. The transformationof mammalian cells is known as transfection. During thetransfection of mouse cells with mouse-derived DNA, theforeign mouse DNA almost always integrates at randominto a chromosome at a site other than its point of origin.Occasionally, however, the added DNA will “find” and re-place its homolog by homologous recombination. The fre-quency of homologous recombination events as a fractionof the total number of integrations is on the order of10�3–10�5.

If researchers transfect mouse ES cells with unaltered,cloned fragments of mouse DNA, homologous recombina-tion events do not cause genomic changes. But with therecombinant DNA technology described in Chapter 9 ofthe main textbook, investigators can modify cloned genesso that they no longer function; cloned genes modified inthis way are known as knockout constructs. Whenhomologous recombination occurs with a knockoutconstruct, the nonfunctional knockout allele replaces theendogenous wild-type allele (Fig. E.8). To construct micein which homologous recombination has knocked outspecific genes, researchers developed protocols for identi-fying and recovering the very rare ES cells in whichhomologous recombination occurs.

E.2 How Biologists Use Transgenic Tools to Study Mice and Create a MouseModel for Human Disease

Both add-on and knockout transgenic technologies havetremendous value in genetic research. The protocolsenable researchers to determine the function of gene


Several embryos recovered from sacrificed female

Embryos transferred to a depression slide containing culture medium

As embryo is held in place, DNAis injected into pronucleus.

Several injected embryos are placed into oviduct of receptive female.

Culture mediumOil

Holding pipette

Injection pipette

DNA to be injected

Pronucleus

Figure E.7 How transgenic mice are created. About 12hours after conception, the female mouse is sacrificed and the one-cell embryos recovered. They are transferred to a depression on aspecialized microscope slide containing a drop of culture mediumunder oil to prevent evaporation. The slide is placed on the stage ofan inverted microscope (as the name implies, the objective lens isbeneath the stage rather than above it, as in a typical microscope).This arrangement gives the researcher space to manipulate the em-bryos from above. (In the photo used here, only one of the twopronuclei is visible.) Suction holds the embryo in place on the bluntend of a special “holding pipette.” A second type of pipette with avery narrow bore (the injection pipette) is used to inject transgenicDNA through the plasma membrane and into the pronucleus,where the foreign DNA is released. The altered embryos are thenplaced into the oviduct of a physiologically receptive female.

har06584_refE_109-132 11/06/2006 06:11 PM Page 116

products, characterize genetic regulatory regions, estab-lish links between mutant phenotypes and particulartranscriptional units as an aid to verifying the identifica-tion of a cloned gene, and create mouse models of humangenetic diseases.

Using Transgenic Technology to Determine Gene FunctionIn Chapter 11 of the main textbook we saw how geneticistsused transgenic technology to demonstrate that the clonedSRY (for sex-determining region on the Y chromosome)gene could confer maleness on an animal without aY chromosome. Incorporation of the SRY gene’s codingregion and regulatory sequences into a mouse embryowith two X chromosomes produced an animal with malegenitalia and testes. This result demonstrated that theproduct of a single gene on the Y chromosome is all that isneeded to switch the developmental pathway of the fetusfrom female to male (Fig. E. 9).

Biologists have used this same transgenic technology toexamine the functions of many other genes. By combining

E.2 How Biologists Use Transgenic Tools to Study Mice and Create a Mouse Model for Human Disease 117

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.

(a) Construction of a knockout allele in ES cells

(b)

Early blastocyst( 10,000)

Culture into millionsof embryonic-like(ES) cells.

Homologous recombination inside ES cell nucleus

ES cell chromosome with wild-type allele

ES cell chromosome with knockout allele

Knockout construct

Clone containing gene of interest

5'Build knockout construct byadding in selectable marker.

3'

5'Marker disrupts transcriptionunit.

3'

5' 3'

Add cloned DNA toculture of cells.

Figure E.8 Knocking out a mouse gene in ES cells. (a) An early mouse blastocyst can be grown in culture under conditions that al-low the cells of the ICM (inner cell mass) to remain undifferentiated as ES cells. A DNA clone, containing the gene of interest, can bemodified 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 onesderived from the mutated ES cell (dark agouti).

Two one-cell female mouse embryos (with two X chromosomes)

No injection

Pronuclei

Inject SRY DNA

FEMALE MALE

19 pairs autosomes,two X chromosomes

19 pairs autosomes,two X chromosomes, and SRY transgene

Figure E.9 The transgenic mouse protocol proves that SRYis the “testis-determining” locus responsible for the produc-tion of maleness during embryogenesis. DNA fragments con-taining the mouse SRY gene and its regulatory sequence wereinjected into a series of embryos that were allowed to developinto live animals. Normal XX animals without a transgene de-velop as females. But the presence of the SRY transgene in an XXembryo induces development as a male.

har06584_refE_109-132 11/06/2006 06:11 PM Page 117

a mouse gene of interest with regulatory regions from othermouse genes, they can cause transgenic mice to express thenatural transgene product in an unnatural manner: at ahigher than normal level, in an alternative tissue, or at an al-ternative developmental stage. They can then use the aber-rant level, time, or place of expression to elucidate thenormal function of the wild-type gene.

Experiments analyzing the myc gene demonstrate thepower of transgenic technology for uncovering the func-tion of genes in ectopic expression (that is, expression atan abnormal place, time, or level). Investigators originallydiscovered the myc gene in the genome of the myelocy-tomatosis chicken retrovirus; exposure to this viruscaused cultured chicken cells to become tumorigenic. Hy-bridization studies demonstrated the existence of a mycgene homolog in the genomes of mice and other verte-brates, including humans (where it resides on the longarm of chromosome 8) but not in the genomes of nonver-tebrate model organisms, such as Drosophila. Moreover,in human cancer cells obtained from patients withBurkitts lymphoma (a cancer of the immune system’s Bcells), the myc gene often appears close to one of thebreakpoints of a reciprocal translocation characteristic ofthese cancer cells between the long arms of chromosomes8 and 14; in this translocated position, the gene is usuallyexpressed at a higher than normal level. Noncancerousanimal cells have a very low level of myc gene expression.These findings constitute circumstantial evidence that ab-normally high levels of myc expression might help trans-form a cell to a cancerous state. (The biochemicalmechanism by which the myc gene product functions isdescribed in Chapter 18 of the main textbook.)

The results of experiments using cells grown in cul-ture support this hypothesis. In almost every case stud-ied, however, cultured mammalian cells displayprograms of gene expression that do not correspond withthose of the cells they are supposed to model. Thus, theresults of cell culture studies do not necessarily reflecthow cells in vivo (that is, within the body) behave in re-sponse to a change in gene activity. In addition to differ-ences in gene activity, there are differences in chromatinstructure and patterns of DNA methylation between cellsgrowing in vitro and in vivo. These discrepancies are notsurprising since cells in the body, unlike those in culture,exist in a complex environment that includes constant ex-posure to molecular signals released by other body cells.The living organism also has a pervasive immune systemthat is impossible to imitate in vitro. It is therefore possi-ble that a phenotype observed in response to the abnor-mal expression of a gene in cultured cells might be aconsequence not of one gene’s abnormal expression butof interactions with other genes that are expressed differ-ently in cultured cells than in cells in vivo. For these rea-sons, it is not possible to rely on cell culture results foran explanation of the true function of a gene; rather it is

necessary to examine the effects of aberrant gene expres-sion in vivo.

To learn whether increased expression of the mycgene affects tumor formation in various tissues of themouse, researchers used transgenic technology (Fig. E.10).In one experiment, they attached the immunoglobulin genepromoter to the myc coding sequence to produce a trans-genic mouse line that expressed myc at high levels in theprecursors to immunoglobulin-producing B cells. In an-other experiment, they attached tissue-specific promot-ers, including one for mammary gland expression, to themyc coding region. And in yet another experiment, theyattached to the myc coding region a promoter that isrecognized in all tissues but only after the embryo’s oranimal’s exposure to dexamethasone, a glucocorticoidhormone.

The results of all these experiments showed that over-expression of the myc gene does not have any effect on nor-mal developmental processes. Even when the gene wasexpressed at high levels in many developing tissues, nor-mal animals were born. Moreover, even in the adult, mostcells that overexpress the myc gene never display an aber-rant phenotype. However, the rate of tumor formation in-creased significantly in most, but not all, types of tissue.The conclusion was that aberrant expression of the mycgene alone does not cause cells to become cancerous; butit can operate with other somatic mutational events to


(1) The myc locus found in the mouse genome.Promoters Exons

5'

5'

3'

3'

(2) Hybrid DNA construct containing the myc coding region regulated by an inducible promoter

Induciblepromoter

1 kb

(a)

(b)

Figure E.10 Transgenic expression of the myc gene pro-vides information on the gene’s role in tumor formation.(a) Construction of a transgene containing the myc gene underthe control of an inducible promoter: (1) structure of the en-dogenous myc gene and (2) transgene construct with the MTV(dexamethasone-inducible) promoter attached to a portion ofthe myc gene that contains the coding region. (b) Northernblot showing induction of transgene expression in a range ofadult tissues.

har06584_refE_109-132 11/06/2006 06:11 PM Page 118

transform a cell to the cancerous state. These observationssupport the hypothesis that the cancer phenotype resultsfrom the accumulation of multiple mutations in the clonalprogeny of one cell (see Chapter 19 of the main textbook).

Biochemical and genetic studies indicate that the pro-tein product of the wild-type myc gene is a component of atranscription factor that helps control the growth of cellpopulations in almost every tissue of the body (as describedin Chapter 18 of the main textbook).

Using Transgenic Technology toCharacterize Regulatory RegionsTo understand embryonic development, it is essential tolearn not only how the proteins critical to this complexprocess function but also how the genes that encode theseproteins are regulated. The pattern of gene expression in anormally differentiating cell is finely tuned, with each geneexpressed at exactly the right level and each gene turned onand off at exactly the right times. Geneticists know that cis-control elements (that is, regulatory regions on the samechromosome) that are closely linked to each gene regulatethe timing and intensity of each gene’s expression. Trans-genic technology can help determine the location andsequence of such regions.

Researchers can characterize the cis-control elementsof single-cell organisms, such as bacteria and yeast, byrandom mutagenesis experiments. In these experiments,they select from cultures of millions of mutagenized cellsthose cells with single base changes or small deletions ingenetic regions that influence the cis-regulation of a geneof interest. They can then use the regulatory mutations toidentify the base pairs necessary for proper gene function.Because of the large number of organisms required, it isnot possible to use a similar approach to study gene regu-lation in mice. Instead, mouse geneticists must turn totransgenics.

Once they have obtained a genomic clone of a mousegene of interest, researchers can subclone flanking se-quences that are likely to contain its regulatory region. Inmost cases studied to date, the regulatory regions have beenconfined to the 5–10 kb of DNA just upstream (to the side) of the coding sequence; thus, this is the first region aninvestigator examines at the start of a project. Although itmight seem counterintuitive at first, the researcher has nouse for the actual coding region of a gene when the aim isto study the gene’s regulatory region. In fact, it is best toreplace the true coding region with that of a “reporter gene”whose protein product is easy to detect. The most com-monly used reporter gene is the (lacZ)gene from E. coli bacteria (Fig. E.11). As described inChapter 9 of the main textbook, the enzyme encoded by this gene will convert a special

�-galactosidase

�-galactosidase

5¿

substrate known as X-Gal into a colored product that canbe visualized under the microscope.

To characterize the regulatory region associated witha gene, you first create a series of different transgene con-structs, each formed by splicing together different frag-ments or mutated forms of the putative regulatory regionwith the reporter gene. You next establisha series of transgenic lines by injecting the transgene con-structs into different mouse embryos. Then, after settingup timed matings within each of these lines, you recover

�-galactosidase


Mouse genomic cloneCoding region

Regulatoryregion

Inject transgene construct into one pronucleus of one-cell mouseembryos. Place embryos into oviduct of receptive female.

E. coli β-galactosidase gene

Recover and stain fetuses to detect β-gal activity.

5' 3' 5' 3'

Mouse E. coli

Mate transgenic animal to establish pregnancy.

Figure E.11 Transgenic technology can be used to ana-lyze cis-acting regulatory regions. A DNA construct contain-ing the mouse regulatory region of interest is attached to theE. coli reporter gene. The function of the regulatory region canbe ascertained by observing �-gal expression in transgene fe-tuses. In the example shown here, expression is observed in thedeveloping forelimb (blue) of three independently derivedtransgenic mice containing the complete regulatory region ofthe mouse Tbx5 gene, which is known to play a specific role inforelimb development of all vertebrates. © John Schiementi,The Jackson Laboratory. Reproduced from “Promoter Mappingof the Mouse TcP-10bt Gene I Transgenic Mice Identifies Essen-tial Male Germ Cell Regulatory Sequences,” Ewulonu et al.,Molecular Reproduction and Development 43:290–297, © 1996.Reproduced by permission of Wiley-Liss, Inc., a subsidiary ofJohn Wiley & Sons, Inc.

har06584_refE_109-132 11/06/2006 06:11 PM Page 119

embryos at the developmental stage or stages duringwhich the mouse gene normally undergoes expression. Byexamining the distribution of the colored product at each embryonic stage and comparing it to thedistribution of the natural gene product, it is possible tomap the extent of the cis-acting regulatory region. The dis-tribution of the natural gene product can be tracked withspecific antibodies labeled for examination by immuno-histochemistry. If the antibody label is a different colorthan the label, the natural gene productand the enzyme can be simultaneously ob-served and distinguished under the microscope. Furtherstudies of the effects of individual base-pair substitutionsor small deletions on details of the expression pattern canlead to a highly sophisticated understanding of the variouscis-control elements. The collective behavior of these cis-acting regulatory elements helps determine the complexpatterns of spatial and temporal expression of genes thatplay a role in development.

An example of the use of transgenic mice to study generegulation is the analysis of a gene called T complex protein-10bt (Tcp10bt). Only differentiating male germ cells in theprocess of maturing into spermatozoa express Tcp10bt.Cells in the seminiferous tubules, called sertoli cells, medi-ate sperm differentiation. When investigators disrupt thetestes and place sperm cells in culture, they cannot simulatenatural conditions completely; as a result, germ cell differ-entiation continues in vitro for only a brief time. But differ-entiation from stem cell to mature spermatozoa takes sixweeks in mice.

Sperm differentiation is of interest to cell biologistsbecause the mechanisms that regulate it may differ signifi-cantly from those that control the differentiation of othercells. This is, in part, because the transformations of differ-entiating sperm cells are much more dramatic than thoseexperienced by other types of cells. Not only do the sper-matogenic cells change in shape and size from large roundstem cells to tiny, sleek spermatozoa almost without cyto-plasm, they also drastically change their genetic program:Stem cells have a normal program of gene expression aswell as chromosomes with a normal chromatin structure; incontrast, the chromosomes of differentiated sperm cellshave a unique chromatin structure with no histones at-tached, and they exhibit no gene activity. To understandthese differences, mouse geneticists have tried to character-ize the regulatory regions associated with gene activity inspermatogenic cells.

To analyze the regulatory region associated with theTcp10bt gene, geneticists first made a series of transgeneconstructs carrying different lengths of DNA from the flanking region of the Tcp10bt gene, ligated to the lacZcoding sequence. The result was six DNA constructscontaining from 0.6–1.6 kb of DNA from the putativeTcp10bt regulatory region. The researchers injectedcopies of each DNA construct into multiple mouse em-bryos, obtaining at least four independent transgenic

5¿

�-galactosidase�-galactosidase

�-galactosidase

lines of mice for each construct. (It is important to usemultiple transgenic lines in an experiment of this typeto verify that sequences in the transgene constructitself, rather than sequences that coincidentally flank thetransgene insertion site, are responsible for a particularphenotype.)

Figure E.12 depicts how it was possible to map theregulatory region associated with Tcp10bt by simply test-ing for the presence of lacZ transcripts in Northern blots oftesticular RNA obtained from each transgenic line of mice.As the figure shows, there was no detectable transcriptionof the lacZ gene in testes from transgenic mice that carried0.75 kb or less of flanking sequence to the Tcp10bt gene;but with 0.97 kb or more of the flanking sequence, highlevels of transcription occurred in the testes, but in no othertissue, of all mouse lines.

These observations located a critical testes-specific, cis-regulatory sequence within a 227 bp region between 746 and973 bases upstream of the Tcp10bt gene. With this informa-tion, it became possible to design additional experiments toexamine the regulatory region in more detail and identify

5¿


R Bg H Bsp Bam Sfa X Bam H Nhel Bam

1.6 kb1.3 kb

1.16 kb0.97 kb

0.75 kb0.6 kb

1 2 3 4 5 6

β-galactosidase(a)

T T T T T

0.75

0.97

1.16

1.3

1.6(b)

Figure E.12 An example of the use of transgenic technol-ogy to map the cis-acting regulatory region associated withthe Tcp10bt gene. (a) Different hybrid DNA constructs weremade with varying lengths of the flanking sequence adjacent tothe Tcp10bt gene fused to the E. coli gene (whichacts as a reporter). (b) Testicular RNA was obtained from transgenicmice containing the various lengths of flanking region (shown inkilobases). With 0.75 kb of flanking region, no transcription of thereporter gene was observed, but with all larger flanking regions,transcription did occur. Source E.12b: © John Schiementi, TheJackson Laboratory. Reproduced from “Promoter Mapping of theMouse TcP-10bt Gene I Transgenic Mice Identifies Essential MaleGerm Cell Regulatory Sequences,” Ewulonu et al., MolecularReproduction and Development 43: 290–297, © 1996. Reproducedby permission of Wiley-Liss, Inc., a subsidiary of John Wiley &Sons, Inc.

5¿

�-galactosidase5¿

har06584_refE_109-132 11/06/2006 06:11 PM Page 120

transacting proteins that bind to this region. Through theseadditional experiments, researchers identified specific testic-ular proteins that activate the Tcp10bt gene.

Using Transgenic Technology to Link Mutant Phenotypes to Specific Transcription UnitsA third significant use of transgenic tools is in establishingwhether a cloned gene corresponds to a locus previouslydefined by a mutant phenotype. Consider, for example, themouse Brachyury locus, symbolized by a T. This locus isdefined genetically by a dominant, X-ray-induced mutationthat causes heterozygous T /� animals to develop shorttails. Through high-resolution linkage analysis and posi-tional cloning (described in Chapter 11), researchersshowed that the T locus mutation is associated with a 200 kbdeletion on chromosome 17. They also identified a transcrip-tion unit called pme75 that is located in this same 200 kbdeletion; normally, pme75 is expressed in the embryonictissue that develops into the tail. While highly suggestive,these data do not prove that the absence of the pme75 genecauses the short-tail phenotype. It is very likely that the200 kb deletion also removes other genes, and on the basisof the genetic data alone, you cannot rule out the possibil-ity that the absence of one of these undiscovered genes pro-duces the mutant phenotype.

You can resolve this impasse with the transgenic pro-tocol illustrated in Fig. E.13. The first step is to make atransgene construct containing the complete pme75 codingregion together with its regulatory sequences. You next injectthe construct into wild-type mouse embryos to create atransgenic line. By breeding this transgenic line to animalswith the T locus mutation, you can obtain offspring thathave the 200 kb deletion over the T locus as well as a func-tional pme75 gene on a different chromosome (at the locuswhere the transgene construct integrated at random). Theseanimals sport a tail of normal length. By demonstratingthat the transgene can correct the mutant phenotype, thisresult proves that the T locus is the equivalent of the pme75gene.

Using Targeted Mutagenesis to Create a Mouse Model for Human DiseaseResearchers can use targeted mutagenesis, in combinationwith other protocols, to create a mouse model for humandiseases that result from a loss of gene function. As an ex-ample, Figure E.14 illustrates the step-by-step creation ofa mouse model for cystic fibrosis. With the identificationand cloning of the human cystic fibrosis gene (designatedCFTR), the first step toward a mouse model was the

cloning of the mouse homolog. Development of the mousemodel also required fabrication of a CFTR knockout con-struct, derivation of an ES cell culture from a mouse blas-tocyst, transfection of the ES cells with the CFTR knockoutconstruct, selection of cells in which homologous recom-bination had replaced the wild-type CFTR gene with themutant knockout allele, and finally, the production andanalysis of chimeric mice and their offspring. Animalshomozygous for the CFTR knockout allele display a mutantphenotype that is very similar to that expressed by humanssuffering from cystic fibrosis. Thus, in developing drugsto alleviate CF symptoms in humans, pharmaceuticalresearchers can first test new products in mice to determinetheir efficacy.


Transgene constructcontains pme75 gene withits regulatory sequences

Inject into pronucleus of wild-type one-cell mouse embryos

Create transgenic line

Transgenic animal withTg (pme75)Normal phenotype

Normal tail

Offspring: T /+ genotypeTg (pme75)Normal phenotype

T /+ genotypeShort-tail phenotype

X

Figure E.13 Transgenic technology can be used to iden-tify the locus responsible for a mutant phenotype. A domi-nant deletion mutation at the T locus causes a short tail. Atransgenic animal containing the pme75 transgene is matedwith a mutant animal to create animals containing both thedeletion and the transgene. A normal phenotype demonstratesthat the deletion of the pme75 gene is responsible for theshort-tail phenotype.

har06584_refE_109-132 11/06/2006 06:11 PM Page 121


ES culture

Plasmid clone containing portion ofmouse CFTR locus with first exon

Early blastocyst recovered from mating between two agouti parentsof the 129/ SvJ strain

Develop ES cell culture by placing blastocysts in petri dish to undergo many cell divisions without differentiation

Develop DNA constructby adding selectable marker (neo )and TK gene to CFTR restrictionfragments

Add cloned DNA knockoutconstruct to cultureof ES cells

DNA construct canintegrate through

two different mechanisms

Expose colonies toganciclover. TK-containingcells eliminated

Expose ES culture to neomycin.Remaining cells contain CFTR constructintegrated either randomly or homologously

Cell contains disrupted copy of CFTR exon 1

Transfer remainingcolony to plate. Begin new culture.

HOMOLOGOUS RECOMBINATION

disrupted CFTR exon 1

INTEGRATION INTO RANDOM LOCUS

neo

exon 1

exon 1

exon 1

endogeneousrandom locus

5' 3'

neo TK

neo TK

neo TK

(a) (c)

(b)

(d)

(e)

neo

neo TK

TK

Figure E.14 Creating a mouse model for cystic fibrosis.

har06584_refE_109-132 11/06/2006 06:11 PM Page 122


Mate B6 black mice. Embryos recoveredfrom pregnant B6 female.

Three types of offspring

Use DNA analysis toidentify male and female agouti animals that are heterozygousfor the knockout allele of CFTR (+/–) and breed them toghether

Use DNA analysis to identifyoffspring homozygous forknockout allele to serve asmodels for cystic fibrosisdisease state

Offspring homozygousfor mutant allele serveas models for CF diseasestate.

10 ES cells are placed in embryos whichare returned to uterus of B6 foster mother.

Colony of ES cells heterozygousfor a knockout of the CFTR locus (+/–)

Embryos develop into live-born mice

Mate chimera with B6 black mouse

Chimera

(+/+) (+/+)

(+/+)

black (+/+)agouti (+/+)agouti (+/–)

(+/+) (–/–)(+/–) (+/–)

(+/–) (+/–)

[agouti (+/–)] and [black (+/+)]

[agouti (+/–)] and [black (+/+)] black (+/+)

(f )

(g)

(h)

har06584_refE_109-132 11/06/2006 06:11 PM Page 123

E.3 The Hox Genes:AComprehensive Example

A particularly striking example of how mouse biologistsused both nuclear injection and targeted mutagenesis tech-nologies to decipher gene function comes from an analysisof the mouse Hox gene family. The Hox genes arehomeotic selector genes, that is, genes that control the de-velopment of body segment characteristics. Members ofthe Hox family are distributed among four unlinked geneclusters that each contain 9–11 genes (Fig. E.15).

Researchers discovered the mouse Hox gene familythrough cross-hybridization studies using the homeoticselector genes of D. melanogaster as probes. In fact,homeotic selector genes were first identified on the basisof mutations that produced flies with four wings insteadof two, flies whose mouthparts developed incorrectly aslegs, and flies with legs instead of antenna growing out oftheir heads. The proteins encoded by these genes turnedout to be transcription factors that act as on/off switches,instructing segments of the fly to develop into one type oftissue or another. William Bateson, the same man whocoined the term “genetics,” chose the designation of“homeotic selector” from the Greek word homoios, whichdescribes a type of variation in which “something hasbeen changed into something else.” Homeotic genes ap-pear to control the development of each Drosophila body

segment (as discussed in the Drosophila portrait, Refer-ence D on our website). The bizarre phenotypes justdescribed result when expression of a particular homeoticgene does not occur at the proper time and place. Lack ofappropriate expression flicks the binary switch, trans-forming the recipient body segment into a different typeof tissue.

Drosophila homeotic genes were first cloned in theearly 1980s. By the end of that decade, it had becomeclear from cross-hybridization and cloning studies thathomologs of these genes are likely to exist in everyspecies of multicellular animal, from C. elegans to Homosapiens.

In segmented animals such as flies, homeotic genes areactive in the discrete segments that define the body plan,where they determine the proper differentiation of tissues.But what do they do in mice and humans, organisms that donot have obvious body segments? To answer this question,researchers had to overcome a serious drawback: the lackof known mutations at any of the Hox loci. This problemwas not unique to understanding the functions of mam-malian Hox genes. Since the discovery of homeotic genehomologs in the mouse genome, it has become routine fordevelopmental geneticists to use cross-hybridization proto-cols to look for mouse homologs of every Drosophilagene found to have a role in development. This strategyhas led to the discovery of dozens of new mouse genes,most of which were not associated with any known mutantphenotypes.


Anterior Posterior

Drosophila

MouseHoxA, chromosome 6

HoxB, chromosome 11

HoxC, chromosome 15

HoxD, chromosome 2

Embryonic axis

Bithorax locusAntennapedia locus

lab pb (Zan) Dfd Scr Antp Ubx Abd-A Abd-B

A1

B1

D1 D3 D4 D8 D9 D11 D12 D13

A2

B2

A3

B3

A4

B4

C4 C5 C6 C8 C9 C10 C11 C12 C13

A5 A6

B5 B6 B7 B8 B9

A7 A9 A13A11A10

Direction of transcription of mouse genes

D10

Drosophila homeotic selector gene3' 5'

Figure E.15 The mouse Hox gene superfamily contains multiple homologs of each member of the Drosophila homeoticselector gene family. Genes within the four mouse Hox clusters are lined up according to their homology with each other and specificDrosophila genes. Not all clusters have homologs of each Drosophila gene. From gene 9 and higher, there are multiple homologs of theDrosophila Abd-B gene in Hox clusters A, C, and D.

har06584_refE_109-132 11/06/2006 06:11 PM Page 124

How Scientists Determine the Function of a Gene in the Absence of Previously Characterized MutationsAnalyses of Expression Patterns in DevelopingEmbryos Can Provide a Clue to the Time andLocation of Gene Action

A clone of a gene can be a tool for analyzing the gene’s ex-pression. One way to convert a clone into this type of toolis to label it, denature it, and use the resulting DNA strandsas probes in in situ hybridization. To study development,investigators can perform in situ hybridization on the RNApresent in fixed tissue sections obtained from embryos atdifferent stages of development. When developmental ge-neticists examined the expression patterns of the Hoxgenes, they discovered that each one is transcribed along aportion of the developing embryonic axis that extends fromthe same most posterior point to a specific anterior bound-ary (Fig. E.16). Analysis of the pooled data on Hox geneexpression showed that the anterior boundary of expressioncorresponds with the position of each gene in its cluster.Genes at the end of a cluster (for example, D13) have the5¿

least extensive expression which is restricted to the poste-rior region of the embryonic axis. In contrast, genes at the

end of the cluster (for example, A1 and B1) have abroader range of expression that extends further to the an-terior region of the embryonic axis. These data suggest thatdifferent Hox genes might be involved in controlling thedevelopment of different sections of the embryonic axis.Expression data alone, however, cannot provide conclusiveevidence of function.

Ultimately, Only Genetic Tests Can Determine Gene Function

To understand what role a particular Hox gene plays indevelopment, a scientist must be able to examine em-bryos that do not express that gene at all, or express itoutside its normal time or place. Examining the changesin development that arise as a result of these geneticchanges makes it possible to decipher the normal role ofthe gene and, in the case of the Hox family, test generalhypotheses concerning the functional interactions of dif-ferent Hox genes. We now present two examples of thisapproach.

3¿

E.3 The Hox Genes: A Comprehensive Example 125

AnteriorPosterior

Caudal Sacral Lumbar Thoracic Cervical Occipital

Extent of expression12345612341234 56 7 1234567812345678 910111213

atlas

axis

Vertebrae

HoxA1

HoxB1

HoxA3

HoxD4

HoxA4

HoxB4

HoxA5

HoxB5

HoxA6

HoxA7

HoxB9

HoxB7

HoxC9

HoxD8

HoxD9

HoxD10

HoxD11

HoxD12

HoxD13

3'

5'

Genes

Figure E.16 Spatial extent of expression of some representative Hox genes along the developing spine. The top of themature spine is shown to the right, bottom to the left, with vertebrae numbered and grouped by name.

har06584_refE_109-132 11/06/2006 06:11 PM Page 125

Validating the Hypothesis That Expressionof the Gene in a Hox Cluster Is Epistaticto Expression of the More GenesThe expression data show that some Hox genes are expressedacross many segments of the embryo, even as those segmentsdevelop differently from each other. To account for this dif-ference in the simplest way, researchers proposed that theonly gene that counts in any particular embryonic segment isthe most gene in a Hox cluster. In other words, expressionof the most gene is epistatic to expression of the other,more Hox genes. By examining Figures E.15 and E.16,you can see that this hypothesis could explain how eachspinal segment develops in a different manner.

A Transgene Test Confirmed the Prediction of a Homeotic Transformation

As one test of this hypothesis, investigators made a trans-gene construct with a HoxA1 regulatory region attached toa HoxD4 coding sequence (Fig. E.17a). According to thehypothesis, HoxD4 normally controls the development ofthe C1 and C2 vertebrae in the cervical region of the spinalcolumn, while HoxA1 normally controls the developmentof the occipital bone at the base of the skull. In a transgenicfetus, however, the presence of the transgene constructcauses expression of the HoxD4 gene in the occipital re-gion along with HoxA1; and as predicted by the hypothesis,a homeotic transformation converts the occipital bone intocervical vertebrae (Fig. E.17b and c).

Knockout Studies Confirmed Predictions of Aberrant Phenotypes

The hypothesis of epistasis also leads to the predictionthat aberrant phenotypes will arise when different Hox genesare knocked out by homologous recombination. With eachknockout, one would expect a particular embryonic segmentto become transformed to a more anterior-like structure. Thedata obtained from knocking out various Hox genes supportthe hypothesis of epistasis. For example, a knockout ofHoxB4 produces a partial homeotic transformation of thesecond cervical vertebra from axis to atlas (see Fig. E.16).

Transgenic Studies Lead to anUnderstanding of the Developmental Role of Hox and Other Homeotic GenesWith the accumulation of transgene and knockout data formany of the mouse Hox genes, a general understanding ofthe developmental role played by this gene family, not onlyin mice but in other animals as well, has emerged. What theHox genes and their homologs in other species apparentlydo is establish signals identifying the position of each re-gion along the embryo’s anterior-posterior axis. The genes

5¿

5¿

3¿5¿

5¿

3¿5¿


HoxA1 regulatory region HoxD4 coding sequence

(a)

(b)

(c)

Figure E.17 Transgenic technology provides support forthe mode of action of the Hox gene family. (a) A transgenicconstruct is produced to missexpress HoxD4 in a more anteriorregion where HoxA1 is normally expressed (refer to Fig. E.16 forthe normal extents of expression of each of these genes). In panel(b), the complete skeleton of a wild-type animal is shown on theleft, and that of an animal expressing the transgene construct isshown on the right. A blowup of the cervical regions from bothskeletons is shown in panel (c), again the wild type is on the leftand the transgenic construct is on the right. In transgenic new-borns, what would have been occipital bone (region E in wild-type animal) has been transformed into ectopic arches (E1) thatlook like cervical vertebrae.

har06584_refE_109-132 11/06/2006 06:11 PM Page 126

Solved Problems 127

Connections

Geneticists can now produce transgenic animals by com-bining the classic tools of mutagenesis with an understand-ing of molecular biology and embryology. Transgenictechnology has become so sophisticated that, in theory, it ispossible to make any genetic change imaginable to themouse genome and determine its effect on the individualthat emerges.

With the ability to produce mice carrying add-ons andknockouts of Hox and other genes that play a role in devel-

opment, mouse geneticists have begun to dissect theprocess by which the mouse embryo develops from theone-cell zygote, through gastrulation, and into the organ-building stage. Remarkably, the development of all mam-mals is similar, especially in these early stages. Forexample, all the genes important to mouse development areconserved in the human genome. Thus, much of what welearn about the genetic basis for mouse development willapply to normal and abnormal human development.

1. The availability of hundreds of single-gene mutationsand a short life cycle contribute to M. musculus’svalue as a model organism.

2. The mouse genome closely resembles the humangenome in size, gene content, and syntenic loci. Re-searchers can thus use homology and conserved syn-teny analysis to identify, locate, and determine thefunction of genes in both species.

3. The mouse life cycle is representative of the mam-malian life cycle, although the timing of events isunique to each species. The totipotency of preimplan-tation cells in mammals makes it possible to createchimeras.

4. Researchers can use the transgenic technology ofadding genes to the mouse genome by nuclear injec-

tion to determine gene function, characterize regula-tory regions, and correlate mutant phenotypes withspecific transcription units.

5. Targeted mutagenesis is the basis for creating amouse model of human diseases caused by a loss ofgene function.

6. Studies using transgenic technology have revealedthat the Hox genes generate signals that identify theposition of each region along a mouse embryo’santerior-posterior axis. Normal development of theembryo depends on this information.

7. Knowledge of how the Hox genes function in micehas elucidated the general role played by homeoticselector genes in the evolution of all metazoan organ-isms, including humans.

Essential Concepts

Solved Problems

I. Gain-of-function mutations can produce a novel phe-notype and act in a dominant fashion. In loss-of-function mutations no functional gene product ismade; most, but not all, loss-of-function mutationsare recessive.

a. Which of these types of mutations would youstudy using add-on transgenic technology?

b. Which type of mutation would you have to studywith the implementation of homologous recombi-nation in ES cells? Why?

do not determine the differentiation of any particular celltype or tissue. Rather, they provide positional informationthat other genes act on to promote the differentiation ofparticular tissues. The positional information as well as thegenes that act on it vary from species to species.

It is likely that the emergence of the Hox-like gene fam-ily in our most recent single-cell ancestor set the stage for theevolution of developmental complexity, with the consequent

appearance of metazoan organisms sometime between 1 bil-lion and 600 million years before the present time.

Thus, the analysis of the Hox gene family is an exampleof how detailed genetic studies in one model species can pro-vide a general understanding of gene function across largesegments of the animal kingdom as well as clues to the con-served mechanisms by which complex developmentalprocesses are carried out.

har06584_refE_109-132 11/06/2006 06:11 PM Page 127


Answer

This problem requires an understanding of add-ontransgenic and homologous recombination techniquesand their applications.a. Dominant alleles caused by gain-of-function muta-

tions are expressed phenotypically when a singlecopy of the dominant allele is present. The effect of adominant gain-of-function mutation can be charac-terized in transgenic mice where a transgene con-taining the dominant allele has been inserted into thegenome and the original gene copy is still present.

b. Loss-of-function mutations would have to be stud-ied using homologous recombination, where anormal copy of the gene is replaced with a defec-tive version. To observe a phenotype of a recessiveloss-of-function mutation, there should not be anynormal version of the gene present. After replac-ing one gene copy with a mutant version, het-erozygous mice would be mated to obtain a mousehomozygous for the recessive allele and to observethe resulting phenotype.

II. You have isolated muscle cell RNA and made a musclecDNA library. You want to study the regulatory regionof one of the genes you identified as being expressed inmuscle cells.a. You need to isolate a genomic clone of this gene.

Why is the cDNA clone not sufficient for yourstudies?

b. Outline the steps to isolate the genomic clone ofthis gene.

c. You isolated a genomic clone containing the entirecoding region and 1.2 kb upstream of the codingregion. A map of the genomic clone is shown here.To make a fusion construct of the presumed regu-latory region attached to the reporter gene lacZ,you cloned the 1.3 kb BamHI fragment into a vec-tor containing the lacZ gene. The fusion construct,when injected into cultured muscle cells, ex-pressed protein. What experimentwould you do next to determine if the fusion con-struct is developmentally regulated?

d. Using an antibody against the portion of the fusion protein you analyzed the tis-sues of a transgenic mouse and found that the pro-tein encoded by this gene is also present in bonecells. How could you determine if the same tran-script was expressed in both cell types?

e. You decided to analyze the promoter region ofthe gene to identify sequences responsible for theexpression in the two different cell types. The re-sults obtained with constructs containing differ-ent fragments from the region flanking thegene to the lacZ coding region are shown here.What region(s) are important for expression inmuscle? in bone?

5¿

�-galactosidase

�-galactosidase

Answer

This problem requires familiarity with several molecu-lar techniques including analysis of cDNAs and regu-latory sequences in the promoter.a. The cDNA clone was derived from the mRNA and

therefore it will probably not contain the regula-tory region usually found upstream of to) thetranscription start site.

b. The cDNA clone is a good source of a probe forfinding the genomic clone in a genomic library.

1. Isolate a DNA fragment from the cDNA cloneand label for use as a probe.

2. Grow clones (bacteriophages or cosmids)from a genomic library on plates.

3. Transfer clones from plates to filter paper.

4. Incubate the labeled probe with the filters tofind, through hybridization, a genomic clonecontaining the gene.

c. To study developmental expression of this gene,the fusion construct would be transferred into themouse genome, using transgenic techniques. Atdifferent stages in development the tissues of themouse can be tested for the presence of themRNA or protein using the lacZ DNA or anti-body that recognizes the proteinrespectively.

d. To examine the transcripts in these two cell types,you would prepare mRNA from isolated muscleand bone cells and use the cDNA clone to probeseparated RNAs that have been transferred to a fil-ter (Northern hybridization analysis). Tran-scripts may be different sizes because of differenttranscription start sites or different processing(splicing) events.

e. The presence or absence of bone or muscle tran-scripts in mice that carry different transgene con-structs provides a way to identify DNAfragments containing tissue-specific regulatorysequences. There are three constructs (4, 5, and6) that express the transgene in bone tissue. The

�-galactosidase

15¿

DNA in subclone Expressionin bone

Expressionin muscle

1.2.3.4.5.6.7.

–––+++–

––––+++

350300 400 50 100 Coding region

BamHIBamHI

S = Sau 3AS S S S

bp

S

har06584_refE_109-132 11/06/2006 06:11 PM Page 128

Problems 129

Problems

E-1 Choose the matching phrase in the right column foreach of the terms in the left column.

a. stem cells 1. cells destined to become the fetus

b. conserved synteny 2. animals derived from two or moregenetically different embryonic cells

c. inner cell mass 3. undifferentiated cells that serve as a sourceof two or more types of differentiated cells

d. trophectoderm 4. experimental elimination of gene function

e. embryonic stem cells 5. the same genes are genetically linked intwo different species

f. chimeras 6. cells destined to become extraembryonictissue such as the placenta

g. knockout 7. undifferentiated cells isolated from theblastocyst that are able to be reconstitutedwith normal cells in an embryo and differ-entiate normally into every tissue type

E-2 The mouse genome (3000 Mb) is 30 times larger thanthe 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 withcystic fibrosis, encodes a membrane protein that actsas a channel for the passage of Cl�. Although one par-ticular mutation (a deletion called D508) is predomi-nant in Caucasians, more than 200 different mutationsin the gene have been identified that cause the disease.Many Cl� channel genes, including CFTR homologs,have been identified in other organisms such as C. ele-gans and yeast. For each of the following researchquestions, indicate whether you would be more likelyto pursue an answer using yeast or mouse and why youwould 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 af-

fect channel function in all organs normally in-volved in cystic fibrosis?

c. What portion of the protein receives a signal toopen the channel?

d. Are there drugs that can cause an opening of thechannel?

E-4 A mouse gene was localized to a region of chromo-some 4. From the synteny map, it appears this genewould localize to chromosome 8 in humans.a. How could you determine if the gene is in this re-

gion of human chromosome 8? (Do not use the ap-proach of cloning the gene here.)

b. Briefly outline how you could obtain the humanclone containing the homolog of this mousegene.

E-5 Why do identical twins occur in mice (and other mam-mals) but not in Drosophila?

E-6 How are different coat color alleles used in the proto-col for making a gene knockout in mice?

E-7 You have discovered a gene, called CTF, that is ex-pressed for several days during development of theembryonic heart and pituitary in mice. Expressioncontinues in the pituitary in the adult and also is seenin the germ cells of the adult testis.a. You bred a male mouse carrying one normal CTF

allele (�) and one disrupted CTF allele (�) with afemale heterozygous for the same alleles and ex-amined 30 of their offspring. (The disrupted allelecontains an insertion within the gene.) Twenty-one of the offspring were � / � and 9 were � / �.What would you conclude about the CTF gene inthis case?

b. Suppose instead that you obtained 30 offspringand 7 of the mice that had the (� / �) genotypewere half the normal size. Of these, all 4 maleswere sterile, whereas the 3 females were fertile.What would you conclude about the CTF genefrom 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 inmammals.a. Several different strains of mice that have recessive

defects in limb development are available. You havetested each of these strains for the presence of theretinoic acid receptor (RAR protein), RAR mRNA,

only portion of the regulatory region that they allhave in common is a 50 bp Sau3A fragment. Thebone-specific regulatory element must be withinthis fragment. For muscle expression, there arethree clones (5, 6, and 7) that express the trans-gene in muscle tissue and these share the adja-

cent 350 bp Sau3A fragment. This analysis showsthat expression of the same gene is subject to dif-ferent mechanisms of regulation in two tissuetypes that use different regulatory sites in the 5�region flanking the gene.

har06584_refE_109-132 11/06/2006 06:11 PM Page 129

and the RAR gene using Western (protein), North-ern (RNA), and Southern (DNA) blots, respectively.Based on the following data, give a reasonable hy-pothesis regarding the defect in each mutant.

Strain RAR protein RAR mRNA RAR gene

A Normal* Normal NormalB Absent Normal NormalC Absent Short NormalD Absent Short Altered†

E Absent Absent Altered†

F Absent Absent Absent*Normal means that the protein, RNA, and DNA bands werenormal in size and abundance; normal does not refer to func-tion of the protein.†One of the bands hybridizing with the RAR cDNA clone mi-grated to a different position in strains D and E.

b. The limb deformity in strain A could be due toa defect in some gene other than RAR. Suggest asimple genetic experiment that would enable youto test this possibility.

c. If you wanted to test the developmental conse-quences of RAR gene expression in neurons,which do not normally make RAR, how wouldyou do this?

E-9 When DNA is injected into the fertilized mouse egg,the DNA can insert at random in any of the chromo-somes. Subsequent matings produce animals homozy-gous for the transgene insertion. Sometimes aninteresting mutant phenotype is generated by the inser-tion event. In one case, after injection of DNA contain-ing the mouse mammary tumor virus (MMTV)promoter fused to the c-myc gene, investigators identi-fied a recessive mutation that causes limb deformity. Inthis mouse, the distal bones were reduced and fused to-gether; the mutation also caused kidney malfunction.a. The mutant phenotype could be due to insertion of

the transgene in a particular region of the chromo-some or a chance point mutation that arose in themouse. How could you distinguish between thesetwo possibilities?

b. The mutation in this example was in fact causedby insertion of the transgene. How could you usethis transgene insertion as a tag for cloning?

c. The insertion mutation was mapped to chromo-some 2 of mice in a region where a mutationcalled limb deformity (ld) had previously beenidentified. Mice carrying this mutation are avail-able from a major mouse research laboratory.How could you tell if the ld mutation was in thesame gene as the transgenic insertion mutation?

d. Analysis of transcripts from the ld gene showedthat many different transcripts (formed by alternatesplicing) were present in both the embryo andadult. Is this consistent with a role of the ld geneproduct in limb development in the embryo?

E-10 Several different mouse mutants have been identi-fied that have an obese phenotype. The Ob gene, de-fective in one class of these mutants, was the firstgene involved in obesity to be cloned. The Ob geneproduct is made in fat cells and is transported to thebrain, where it informs the brain that the animal issatiated (full).a. You made an antibody to the Ob protein and

used this to test predictions of the hypothesisthat Ob is a satiety factor. You isolated proteinfrom mice that had been eating normally, frommice that had been starved, and from mice thathad been force-fed a high-calorie diet. You did aWestern analysis using your antibody as a probeagainst proteins from these animals. Results areshown here. Are these results consistent with thehypothesis for the role of the Ob protein? Whyor why not?


Normal Starved Force-fed

Wild type A B C D

b. How could you determine if the Ob gene is tran-scriptionally regulated?

c. If the amount of mRNA was the same for all threetypes of mice, what type of regulation is involved?

d. You isolated RNA from several different Obmutants and analyzed the RNA using Northernanalysis. What conclusions could you reachabout each mutation based on the results shownhere?

har06584_refE_109-132 11/06/2006 06:11 PM Page 130

e. You have now decided to clone the receptor forthe Ob protein and express the gene in mam-malian cells. Which is the correct alternative foreach 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 identified the Ob receptor clone using thescreen outlined in part e and decided to make aknockout of the gene in mice. What phenotypewould you predict for mice that lack the Ob re-ceptor? (obese, slim, normal)

g. If you inject Ob protein into Ob mutant mice, whateffect do you predict there will be on the phenotype?

Problems 131

har06584_refE_109-132 11/06/2006 06:11 PM Page 131

Documents

Mus musculus:Genetic Portrait of the House Mouse Referencelibrary01.forbiddenmousecity.com/L028.pdf · 110 Reference EMus musculus: Genetic Portrait of the House Mouse homology analysisto