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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 Mendels laws

at the beginning of the twentieth century. Because these three scien-

tists, as well as Mendel himself, performed their research entirely on

plants, many in the scientific community questioned whetherMendels laws could explain the basis for inheritance in animals, espe-

cially 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 showno 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 first law

even though the traits themselves do not. At the beginning of thetwentieth 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 forgenetic analysis, and foremost among these is the availability of hundreds of single-

gene mutations. These mutations arose during the mouses long history of domesti-

cation 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 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 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 havelarge litters of eight or more pups. They breed readily in captivity. Fathers do not

harm their young. And after centuries of artificial selection, domesticated mice are

docile 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 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 organism-

level human characteristics appear in a limited subset of animals. In fact, manyaspects 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 ofM. musculus. First, because

of the many similarities between mouse and human genomes, researchers can use

Mus musculus:GeneticPortrait of the House Mouse Reference

E

10

A member of the 129 strain of

inbred mice commonly used in

targeted mutagenesis studies.


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E.1 An Overview of Musmusculusin the Laboratory

The Mouse Genome

The most important feature of the mouse genome for con-

temporary geneticists is its close resemblance to the human

genome (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 from

species-specific additions to gene families that already

existed 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 we

saw at the beginning of Chapter 14 of the main textbook,

examinations of mouse and human karyotypes under the

microscope reveal no evidence of chromosome bandingsimilarities between the two species. With the mapping of

thousands of homologous genes in both species, however, a

remarkable 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 one

species, they are said to be syntenic (meaning on the same

thread, or chromosome). When the same set of loci are

also found to be linked in a second species, they are said toexist in a state ofconserved synteny. A comparison of ge-

netic maps of the whole mouse genome with genetic maps

of 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 is

roughly 17.6 Mb. The implication of this finding is that

during the 75 million years that mice and humans havbeen evolving apart from a common ancestor, thegenomes have broken apart and rearranged some 170 time

(17.6 Mb 170 about 3000 Mb the size of the mammalian genome). Conversely, if the proper genome-scal

scissors and glue were available, one could break thmouse genome into about 170 pieces and reassemble thos

pieceslike a puzzlein the form of the human genome

In addition to its powerful evolutionary implication

conserved synteny is a useful tool for practicing geneticistOnce a researcher has mapped a locus in one species, he o

she can look at a homology map and immediately identif

its likely map position in the other species. Of course, fo

genes that have already been cloned, it is possible to us

DNA-DNA hybridization, or computer analysis of a wholegenome sequence, to pick out gene homologs from th

other species. But for loci characterized only by their phe

notypic expression, conserved synteny enables geneticis

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 mic

to excessive consumption of 10% ethanol (the concentra

tion of alcohol found in many wines) provides an exampof the use of conserved synteny for locating human ho

mologs of mouse genes. Mouse geneticists used DNA

markers (as described in Chapter 11 of the main textbookto map theAlcohol-preference-2 (Alcp2) locus to the middle of mouse chromosome 11. Now that the whole mous

genome has been cloned and sequenced, the genes as tran

scription units in Alcpz region have been identified, bu

which one is actually Alcpz is not yet known. Howeve

even though researchers have not yet identified the specifigene, scrutiny of a conserved synteny homology ma

shows that the most likely location for the human homolo

ofAlcp2 is on the short arm of human chromosome 17

close to the centromere (Fig. E.3). With this informatioabout the likely location of an alcohol-preference locu

E.1 An Overview ofMus musculus in the Laboratory 11

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 56 weeks Average, 624728 weeks (1214 years)

Estrus cycle 4 days Average, 28 days

Life span 2 years Average, 78 years


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From this time on, the female progresses through an

estrus cycle that lasts about 4 days in mice and about 28days in humans. During each cycle,primary oocytes (810

in mice, usually only 1 in women) are stimulated to com-

plete the first meiotic division and extrude the first polar

body at the end of this division; the resulting secondary

oocyte begins the second meiotic division but stops atmetaphase and is released from the ovary in a process

known as ovulation. Following ovulation, the secondary

oocyte passes into an oviduct(called afallopian 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 the

ability to reproduce. Many human females, however, live

long enough to pass through menopause, during whichthey stop cycling through estrus, no longer ovulate, and

thus lose the ability to reproduce.

FertilizationJust before and during the estrus phase of the estrus cycle,

female mammals of nonhuman species release species-

specific chemical signals, orpheromones. In a behavioralresponse 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 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 pellucidathe thick

solid shell composed of glycoproteins that surrounds the egg

proper. 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, usuall

only one fuses with the egg. This fusion causes rapid electrochemical changes in the egg membrane that prevent the entr

of additional sperm and activate the newly fertilized egg t

enter the pathway of animal development.

After fusion, the fertilized egg, or zygote, contains twhaploidpronuclei. The two pronuclei never merge; instead

replication occurs within both of the pronuclei. The one

cell embryo carries two replicated pronuclei right up to th

moment of the first mitosis, at which time the membrane

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 th

fertilized egg and thence segregate chromatids into the tw

daughter cells.For the purposes of analysis, scientists divide mous

development into two distinct stages of unequal length

separated by the process of embryonic implantation int

the uterus: a preimplantation stage that lasts 45 days imice, and a postimplantation stage that lasts about 16.

days in mice. During the preimplantation phase, the em

bryo is a free-floating object within the females body. It i

easy to remove this naturally free-floating preimplantatioembryo from the animal, culture it in a petri plate, an

expose it to genetic manipulation before placing it bac

in the reproductive tract of an adult female for developmen

to a newborn animal. After implantation, however, suc

manipulation is no longer possible because the embryo, removed from the adults body, cannot be returned. Th

accessibility of the preimplantation embryo provides th

basis for many of the genetic manipulations researcheuse to study mammalian development.


Ovulation expands

sperm head

Firstcleavage

Secondcleavage

Thirdcleavage

8-cellembryo

Fourthcleavage

outside cells differentiateinto trophectoderm

Blastocystformation

Blastocyst

hatching, andimplantation

Zona pellucida

SpermOne sperm penetrates

Maternal pronucleus

Paternal pronucleus

TrophectodermInner cell mass(ICM)

Blastocoelecavity

Uterine wal

Fertilized egg = zygote = 1-cell embryo

Fertilization2-cellembryo

4-cellembryo

16-cell embryo:

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


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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 matches

the size of the eggs maternal pronucleus. After the paternal

pronucleus has completed its expansion and replicated itschromosomes and the maternal pronucleus has replicated

its chromosomes, the embryo undergoes the first 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 develop-

ing 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 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 be

divided into smaller groups of cells that each have the

potential 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 orDrosophila melanogaster.) In the

laboratory, scientists have obtained completely normal mice

from individual cells that they dissected out of the four-cell-stage mouse embryo and placed back into the female

reproductive 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 ofchimeras, 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 termto designate an embryo or animal composed of cells from

two 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 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 massthat 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 re-

sulting individual is tetraparental, that is, has four parents. It

is also possible to produce hexaparental animals derivedfrom a combination of three embryos. Every organ and tis-

sue 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-

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 of

placental mammals with that of other animals, including

C. elegans andD. melanogaster, shows how different theseprograms can be. In nematodes, embryonic cells are highly

restricted in their developmental potential, or fate, begin-

ning at the two-cell stage; and in fruit flies, polarization of

the egg before fertilization generates distinct cytoplasmicregions dedicated to supporting different developmental

programs within the nuclei that end up in these locations.Consequently, half a nematode embryo or half a fly embryo

can never give rise to a whole animal.

Events Restricting the DevelopmentalPotency of Individual Cells Occur Nearthe End of the Preimplantation Stage

The first differentiation events of mouse embryogenesisoccur in the 16-cell embryo (see Fig. E.5). The cells on the

outside of the embryo turn into a trophectoderm layer that

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

(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-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.


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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, orICM,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 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. Two

more rounds of cell division occur during the blastocyststage, 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-cellzygote and the 64-cell embryo. To accomplish implanta-

tion, the embryo must first hatch from the zona pellucida

so that it can make direct membrane-to-membrane contact

with 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 primitive

egg-laying platypus.

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

Implantation initiates development of the placenta, a mix

of embryonic and maternal tissues that mediates the flow of

nutrients entering the embryo from the maternal blood sup-

ply and the flow of waste products exiting the embryo to

the 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 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 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 after

conception. Newborn animals remain dependent on their

mothers during a suckling period that lasts 1825 days. By

five to eight weeks after birth, mice reach adulthood andare ready to begin the next reproductive cycle.

Two Powerful Transgenic Techniquesfor Analyzing the Mouse Genome

Geneticists have capitalized on certain features of the

mouse 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 MouseGenome by Nuclear Injection

The 1981 development of a method for inserting foreig

DNA into the germ line of mice thrust a primarily observational discipline into the realm of genetic engineerin

with all its implications. Yet the incredibly powerful tran

genic technology is based on a very simple process. Reca

that a transgene is any piece of foreign DNA tharesearchers have inserted into the genome of a comple

organism, such as a mouse or a pea plant, through exper

mental manipulation of early stage embryos or germ cell

any individual carrying a transgene is known as a transgenanimal or plant.

To create a transgenic mouse carrying a foreign DNA

sequence integrated into one of its chromosomes, a re

searcher simply injects foreign DNA into a pronucleus of

fertilized egg and then places the injected one-cell embryback into a female oviduct, where it can continue its deve

opment. Roughly 25% to 50% of the time, for a skilleinvestigator, the injected DNA will integrate at random int

a chromosomal location. Integration can occur while thembryo 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 ha

completed one or two cell divisions; in this case, the mouswill be a mosaic of cells, some with the transgene and som

without it. The relatively high rate of integration appears t

be a consequence of naturally occurring DNA repair en

zymes present in all eukaryotic cells. During evolutionthese 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 othe transgenic procedure.

Up to 50% of the mice born from injected embryohave the foreign DNA stably integrated into their genome

They will thus transmit this DNA to their offspring. Ther

are no limits to the type of DNA that can be incorporated

It can come from any natural sourceanimal, plant, omicrobialor directly from a DNA synthesizer. It is ver

common for investigators to construct DNA molecule

(called DNA constructs) composed of genetic elemen

from different sources. For example, a DNA construcmight have a coding region that is a composite of huma

and Escherichia coli sequences flanked by an upstream

regulatory region that is a composite of mouse and syntheti

sequences.Although embryonic nuclear injection is a powerfu

transgenic tool, it has two significant limitations. First,

can only addnot subtractgenetic material. Second

experimenters cannot target the insertion of foreign DN

to specific genomic locations. Consequently, transgenimice produced by embryonic nuclear injection are usefu

only for the analysis of dominant phenotypes. By 1989

geneticists had developed a way to circumvent thes

limitations.



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Targeted Mutagenesis, a SecondTransgenic Technology, Makes ItPossible 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 an

altered function. The emergence of targeted mutagenesis,which is technically more demanding and more complex

than the nuclear injection technology just described, de-

pended on two advances in cell culture techniques that

occurred during the 1980s.

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

the blastocyst stage into culture such that the embryonic

cells 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 cells

from the ICM. It is possible to grow cultures containing

many millions of ES cells from a single embryo and thenrecover 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 tissuesin the mouse that develops from the embryo. Most impor-

tantly 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 cellsto 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 thetransfection 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 find and re-place its homolog by homologous recombination. The fre-

quency 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 recombina-

tion events do not cause genomic changes. But with the

recombinant DNA technology described in Chapter 9 ofthe main textbook, investigators can modify cloned genes

so that they no longer function; cloned genes modified in

this way are known as knockout constructs. Whenhomologous 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 outspecific genes, researchers developed protocols for identi-

fying and recovering the very rare ES cells in which

homologous recombination occurs.

E.2 How Biologists UseTransgenic Tools to StudyMice and Create a MouseModel for Human Disease

Both add-on and knockout transgenic technologies have

tremendous 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 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 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 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.


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products, characterize genetic regulatory regions, estab-

lish links between mutant phenotypes and particular

transcriptional units as an aid to verifying the identifica-

tion of a cloned gene, and create mouse models of humangenetic diseases.

Using Transgenic Technologyto Determine Gene Function

In Chapter 11 of the main textbook we saw how geneticists

used transgenic technology to demonstrate that the clonedSRY(for sex-determining region on the Y chromosome)

gene could confer maleness on an animal without a

Y chromosome. Incorporation of the SRYgenes coding

region and regulatory sequences into a mouse embryowith two X chromosomes produced an animal with male

genitalia and testes. This result demonstrated that the

product of a single gene on the Y chromosome is all that is

needed to switch the developmental pathway of the fetusfrom female to male (Fig. E. 9).

Biologists have used this same transgenic technology to

examine 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 11

Finding the cell with the knockout allele.Subject culture to drug that kills all cells that do not containselectable 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 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

modified 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).

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

No injection

Pronuclei

Inject SRYDN

FEMALE MALE

19 pairs autosomes,two X chromosomes

19 pairs autosomes,two X chromosomes,and SRYtransgene

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 SRYgene 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 de-

velop as females. But the presence of the SRYtransgene 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 thenatural transgene product in an unnatural manner: at a

higher 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 the

normal function of the wild-type gene.Experiments analyzing the myc gene demonstrate the

power of transgenic technology for uncovering the func-

tion of genes in ectopic expression (that is, expression at

an abnormal place, time, or level). Investigators originallydiscovered the myc gene in the genome of the myelocy-

tomatosis chicken retrovirus; exposure to this virus

caused 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 long

arm of chromosome 8) but not in the genomes of nonver-

tebrate model organisms, such asDrosophila. Moreover,

in human cancer cells obtained from patients withBurkitts 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 ofthese 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 ofmyc gene expression.These findings constitute circumstantial evidence that ab-

normally high levels ofmyc expression might help trans-

form 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 cul-

ture support this hypothesis. In almost every case stud-

ied, however, cultured mammalian cells display

programs of gene expression that do not correspond withthose of the cells they are supposed to model. Thus, the

results of cell culture studies do not necessarily reflect

how 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 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 ex-posure 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 possi-

ble that a phenotype observed in response to the abnor-mal 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 differ-

ently in cultured cells than in cells in vivo. For these rea-sons, it is not possible to rely on cell culture results for

an 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 myc

gene 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 the

precursors to immunoglobulin-producing B cells. In an-

other experiment, they attached tissue-specific promot-ers, 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 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, most

cells 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 myc

gene alone does not cause cells to become cancerous; but

it can operate with other somatic mutational events to


(1) The myclocus found in the mouse genome.

Promoters Exons

5'

5'

3'

3'

(2) Hybrid DNA construct containing the myccoding regionregulated by an inducible promoter

Induciblepromoter

1 kb

(a)

(b)

Figure E.10 Transgenic expression of the mycgene pro-

vides information on the genes role in tumor formation.(a) Construction of a transgene containing the mycgene under

the control of an inducible promoter: (1) structure of the en-

dogenous mycgene and (2) transgene construct with the MTV

(dexamethasone-inducible) promoter attached to a portion of

the mycgene that contains the coding region. (b) Northern

blot showing induction of transgene expression in a range of

adult tissues.


11/23


12/23

embryos at the developmental stage or stages during

which the mouse gene normally undergoes expression. Byexamining the distribution of the colored

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 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 color

than the label, the natural gene product

and the enzyme can be simultaneously ob-served 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 cis-

acting 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 generegulation is the analysis of a gene called T complex protein-

10bt(Tcp10bt). Only differentiating male germ cells in the

process of maturing into spermatozoa express Tcp10bt.Cells in the seminiferous tubules, called sertoli cells, medi-

ate sperm differentiation. When investigators disrupt the

testes and place sperm cells in culture, they cannot simulate

natural 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 six

weeks in mice.

Sperm differentiation is of interest to cell biologists

because the mechanisms that regulate it may differ signifi-cantly from those that control the differentiation of other

cells. 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 round

stem 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; in

contrast, the chromosomes of differentiated sperm cells

have a unique chromatin structure with no histones at-

tached, and they exhibit no gene activity. To understand

these differences, mouse geneticists have tried to character-ize the regulatory regions associated with gene activity in

spermatogenic cells.

To analyze the regulatory region associated with the

Tcp10bt gene, geneticists first made a series of transgene

constructs carrying different lengths of DNA from the

flanking region of the Tcp10bt gene, ligated to the lacZcoding 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 em-bryos, obtaining at least four independent transgenic

5

-galactosidase-galactosidase

-galactosidase

lines of mice for each construct. (It is important to use

multiple transgenic lines in an experiment of this typeto verify that sequences in the transgene construct

itself, rather than sequences that coincidentally flank 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 test-

ing for the presence oflacZtranscripts in Northern blots of

testicular RNA obtained from each transgenic line of mice.As the figure shows, there was no detectable transcription

of the lacZgene in testes from transgenic mice that carried

0.75 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 other

tissue, 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 Tcp10b

t gene. With this informa-

tion, it became possible to design additional experiments to

examine 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

123456

-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 were

made with varying lengths of the flanking sequence adjacent to

the Tcp10bt gene fused to the E. coli gene (which

acts as a reporter). (b) Testicular RNA was obtained from transgenic

mice containing the various lengths of flanking region (shown in

kilobases). With 0.75 kb of flanking region, no transcription of the

reporter gene was observed, but with all larger flanking 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 Identifies Essential Male

Germ Cell Regulatory Sequences, Ewulonu et al., Molecular

Reproduction and Development43: 290297, 1996. Reproduced

by permission of Wiley-Liss, Inc., a subsidiary of John Wiley &Sons, Inc.

5

-galactosidase

5


13/23

transacting proteins that bind to this region. Through these

additional experiments, researchers identified specific testic-ular proteins that activate the Tcp10bt gene.

Using Transgenic Technologyto Link Mutant Phenotypesto Specific Transcription Units

A third significant use of transgenic tools is in establishing

whether a cloned gene corresponds to a locus previously

defined by a mutant phenotype. Consider, for example, the

mouseBrachyury locus, symbolized by a T. This locus isdefined genetically by a dominant, X-ray-induced mutation

that causes heterozygous T/ animals to develop shorttails. Through high-resolution linkage analysis and posi-

tional cloning (described in Chapter 11), researchersshowed that the Tlocus mutation is associated with a 200 kb

deletion on chromosome 17. They also identified a transcrip-

tion unit called pme75 that is located in this same 200 kb

deletion; normally, pme75 is expressed in the embryonictissue that develops into the tail. While highly suggestive,

these data do not prove that the absence of thepme75 gene

causes the short-tail phenotype. It is very likely that the

200 kb deletion also removes other genes, and on the basis

of 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 a

transgene construct containing the complete pme75 codingregion together with its regulatory sequences. You next inject

the construct into wild-type mouse embryos to create a

transgenic line. By breeding this transgenic line to animalswith the T locus mutation, you can obtain offspring that

have the 200 kb deletion over the Tlocus as well as a func-

tionalpme75 gene on a different chromosome (at the locus

where the transgene construct integrated at random). Theseanimals sport a tail of normal length. By demonstrating

that the transgene can correct the mutant phenotype, this

result proves that the Tlocus is the equivalent of thepme75

gene.

Using Targeted Mutagenesis to Createa Mouse Model for Human Disease

Researchers can use targeted mutagenesis, in combination

with 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 of

a mouse model for cystic fibrosis. With the identification

and cloning of the human cystic fibrosis gene (designated

CFTR), the first step toward a mouse model was the

cloning of the mouse homolog. Development of the mous

model also required fabrication of a CFTR knockout con

struct, derivation of an ES cell culture from a mouse blatocyst, transfection of the ES cells with the CFTR knockou

construct, selection of cells in which homologous recom

bination had replaced the wild-type CFTR gene with th

mutant knockout allele, and finally, the production ananalysis of chimeric mice and their offspring. Animal

homozygous for the CFTR knockout allele display a mutan

phenotype that is very similar to that expressed by human

suffering from cystic fibrosis. Thus, in developing drugto alleviate CF symptoms in humans, pharmaceutica

researchers can first test new products in mice to determin

their efficacy.


Transgene constructcontains pme75 gene withits regulatory sequences

Inject into pronucleus of wild-type one-cell mouseembryos

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 Tlocus causes a short tail. A

transgenic animal containing thepme75transgene is mated

with a mutant animal to create animals containing both the

deletion and the transgene. A normal phenotype demonstrate

that the deletion of thepme75gene is responsible for the

short-tail phenotype.


14/23


ES culture

Plasmid clone containing portion ofmouse CFTRlocus with first exon

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

Develop ES cell culture byplacing blastocysts in petridish to undergo many celldivisions without differentiation

Develop DNA constructby adding selectable marker (neo)

andTK

gene toCFTR

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.


15/23


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 EScells 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)


16/23

E.3 The HoxGenes: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 analysis

of the mouse Hox gene family. The Hox genes arehomeotic selector genes, that is, genes that control the de-velopment of body segment characteristics. Members of

the Hoxfamily are distributed among four unlinked gene

clusters that each contain 911 genes (Fig. E.15).

Researchers discovered the mouse Hox gene familythrough cross-hybridization studies using the homeotic

selector genes ofD. melanogaster as probes. In fact,

homeotic selector genes were first identified on the basis

of mutations that produced flies with four wings insteadof two, flies whose mouthparts developed incorrectly aslegs, and flies 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 fly to develop into one type oftissue 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 hasbeen changed into something else. Homeotic genes ap-

pear to control the development of eachDrosophila body

segment (as discussed in the Drosophila portrait, Refer-

ence D on our website). The bizarre phenotypes just

described result when expression of a particular homeoticgene does not occur at the proper time and place. Lack of

appropriate expression flicks the binary switch, trans-

forming the recipient body segment into a different typeof tissue.

Drosophila homeotic genes were first 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 toHomosapiens.

In segmented animals such as flies, homeotic genes are

active 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 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 Hoxloci. This problemwas not unique to understanding the functions of mam-

malian Hoxgenes. Since the discovery of homeotic gene

homologs in the mouse genome, it has become routine for

developmental geneticists to use cross-hybridization proto-cols to look for mouse homologs of every Drosophilagene 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.


Anterior Posterior

Drosophila

Mouse

HoxA, 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

Drosophilahomeotic selector gene3' 5'

Figure E.15 The mouse Hoxgene superfamily contains multiple homologs of each member of the Drosophila homeoticselector gene family. Genes within the four mouse Hoxclusters are lined up according to their homology with each other and specific

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 HoxclustersA, C, and D.


17/23

How Scientists Determine theFunction of a Gene in the Absence ofPreviously Characterized Mutations

Analyses 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 genes ex-

pression. 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 fixed tissue sections obtained from embryos at

different stages of development. When developmental ge-

neticists examined the expression patterns of the Hoxgenes, 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 specific anterior bound-

ary (Fig. E.16). Analysis of the pooled data on Hoxgene

expression 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 th

end of the cluster (for example, A1 and B1) have

broader range of expression that extends further to the anterior region of the embryonic axis. These data suggest tha

differentHoxgenes might be involved in controlling th

development of different sections of the embryonic axi

Expression data alone, however, cannot provide conclusiv

evidence of function.

Ultimately, Only Genetic TestsCan Determine Gene Function

To understand what role a particular Hox gene plays i

development, a scientist must be able to examine em

bryos that do not express that gene at all, or express

outside its normal time or place. Examining the changein development that arise as a result of these geneti

changes makes it possible to decipher the normal role o

the gene and, in the case of the Hoxfamily, test gener

hypotheses concerning the functional interactions of di

ferentHoxgenes. We now present two examples of thapproach.

3

E.3 The HoxGenes: A Comprehensive Example 12

AnteriorPosterior

Caudal Sacral Lumbar Thoracic Cervical Occipital

Extent of expression

12345612341234 56 7 1234567812345678 91011

12

13

atlas

axis

Verte

bra

e

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 representativeHoxgenes 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.


18/23

Validating the Hypothesis That Expressionof the Gene in a HoxCluster Is Epistaticto Expression of the More Genes

The expression data show that someHoxgenes are expressed

across many segments of the embryo, even as those segments

develop 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 is

the most gene in aHoxcluster. In other words, expression

of the most gene is epistatic to expression of the other,

more Hoxgenes. 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 Predictionof a Homeotic Transformation

As one test of this hypothesis, investigators made a trans-gene construct with aHoxA1 regulatory region attached toaHoxD4 coding sequence (Fig. E.17a). According to the

hypothesis,HoxD4 normally controls the development of

the C1 and C2 vertebrae in the cervical region of the spinal

column, while HoxA1 normally controls the development

of the occipital bone at the base of the skull. In a transgenicfetus, however, the presence of the transgene construct

causes expression of the HoxD4 gene in the occipital re-

gion along withHoxA1; and as predicted by the hypothesis,

a homeotic transformation converts the occipital bone intocervical vertebrae (Fig. E.17b and c).

Knockout Studies Confirmed Predictionsof Aberrant Phenotypes

The hypothesis of epistasis also leads to the prediction

that aberrant phenotypes will arise when differentHoxgenesare knocked out by homologous recombination. With each

knockout, one would expect a particular embryonic segment

to become transformed to a more anterior-like structure. The

data obtained from knocking out variousHoxgenes supportthe hypothesis of epistasis. For example, a knockout of

HoxB4 produces a partial homeotic transformation of the

second cervical vertebra from axis to atlas (see Fig. E.16).

Transgenic Studies Lead to anUnderstanding of the DevelopmentalRole of Hoxand Other Homeotic Genes

With the accumulation of transgene and knockout data for

many of the mouseHoxgenes, 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

Hoxgenes and their homologs in other species apparently

do is establish signals identifying the position of each re-

gion along the embryos anterior-posterior axis. The genes

5

5

3

5

5

35


HoxA1 regulatory region HoxD4coding sequence

(a)

(b)

(c)

Figure E.17 Transgenic technology provides support forthe mode of action of the Hoxgene family. (a) A transgenic

construct is produced to missexpress HoxD4 in a more anteriorregion 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 new-

borns, what would have been occipital bone (region E in wild-

type animal) has been transformed into ectopic arches (E1) that

look like cervical vertebrae.


19/23

Solved Problems 12

Connections

Geneticists can now produce transgenic animals by com-

bining the classic tools of mutagenesis with an understand-

ing of molecular biology and embryology. Transgenic

technology has become so sophisticated that, in theory, it is

possible to make any genetic change imaginable to themouse genome and determine its effect on the individual

that emerges.

With the ability to produce mice carrying add-ons and

knockouts ofHoxand other genes that play a role in devel-

opment, mouse geneticists have begun to dissect thprocess by which the mouse embryo develops from th

one-cell zygote, through gastrulation, and into the organ

building stage. Remarkably, the development of all mam

mals is similar, especially in these early stages. Foexample, all the genes important to mouse development ar

conserved in the human genome. Thus, much of what w

learn about the genetic basis for mouse development wi

apply to normal and abnormal human development.

1. The availability of hundreds of single-gene mutationsand a short life cycle contribute to M. musculuss

value 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 the

function 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 create

chimeras.

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 with

specific transcription units.

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

gene function.

6. Studies using transgenic technology have revealedthat theHoxgenes generate signals that identify theposition of each region along a mouse embryos

anterior-posterior axis. Normal development of the

embryo depends on this information.

7. Knowledge of how the Hoxgenes function in micehas elucidated the general role played by homeotic

selector 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 is

made; most, but not all, loss-of-function mutationsare recessive.

a. Which of these types of mutations would yo

study using add-on transgenic technology?b. Which type of mutation would you have to stud

with the implementation of homologous recomb

nation in ES cells? Why?

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 ofparticular 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 theHox-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 bi

lion and 600 million years before the present time.

Thus, the analysis of theHoxgene family is an examplof how detailed genetic studies in one model species can pro

vide a general understanding of gene function across larg

segments of the animal kingdom as well as clues to the con

served mechanisms by which complex developmentaprocesses are carried out.


20/23


21/23

Problems 12

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 more

genetically different embryonic cells

c. inner cell mass 3. undifferentiated cells that serve as a source

of 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 in

two different species

f. chimeras 6. cells destined to become extraembryonic

tissue such as the placenta

g. knockout 7. undifferentiated cells isolated from the

blastocyst that are able to be reconstituted

with 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 geneticistschoose 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 acts

as 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 research

questions, indicate whether you would be more likelyto pursue an answer using yeast or mouse and why you

would choose that organism.

a. Do mutations in different regions of the gene havethe 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 to

open the channel?

d. Are there drugs that can cause an opening of the

channel?

E-4 A mouse gene was localized to a region of chromosome 4. From the synteny map, it appears this gen

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 ap

proach of cloning the gene here.)

b. Briefly outline how you could obtain the huma

clone containing the homolog of this mousgene.

E-5 Why do identical twins occur in mice (and other mammals) but not inDrosophila?

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

E-7 You have discovered a gene, called CTF, that is expressed for several days during development of th

embryonic heart and pituitary in mice. Expressio

continues in the pituitary in the adult and also is seein the germ cells of the adult testis.

a. You bred a male mouse carrying one normal CTallele () and one disrupted CTFallele () withfemale heterozygous for the same alleles and examined 30 of their offspring. (The disrupted allel

contains an insertion within the gene.) Twenty

one of the offspring were/ and 9 were/What would you conclude about the CTFgene ithis case?

b. Suppose instead that you obtained 30 offsprin

and 7 of the mice that had the (/) genotypwere half the normal size. Of these, all 4 male

were sterile, whereas the 3 females were fertileWhat would you conclude about the CTF gen

from these results?c. In the preceding experiments, what technique(

were used to determine if the offspring mice wer(/), (/), or (/)?

E-8 Retinoic acid, which acts on cells via a protein receptor, is thought to be important for limb development i

mammals.

a. Several different strains of mice that have recessivdefects in limb development are available. You hav

tested each of these strains for the presence of th

retinoic acid receptor (RAR protein), RAR mRNA

only portion of the regulatory region that they all

have in common is a 50 bp Sau3A fragment. The

bone-specific regulatory element must be within

this fragment. For muscle expression, there are

three 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 show

that expression of the same gene is subject to di

ferent mechanisms of regulation in two tissutypes that use different regulatory sites in the 5

region flanking the gene.


22/23

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 to

a defect in some gene other thanRAR. Suggest a

simple genetic experiment that would enable youto test this possibility.

c. If you wanted to test the developmental conse-

quences ofRAR gene expression in neurons,

which do not normally make RAR, how would

you 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 an

interesting 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. In

this 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 the

mouse. How could you distinguish between thesetwo 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 chromo-some 2 of mice in a region where a mutation

called limb deformity (ld) had previously been

identified. Mice carrying this mutation are avail-able from a major mouse research laboratory.

How could you tell if the ldmutation was in the

same gene as the transgenic insertion mutation?

d. Analysis of transcripts from the ldgene showedthat many different transcripts (formed by alternate

splicing) were present in both the embryo and

adult. Is this consistent with a role of the ldgene

product 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 first

gene involved in obesity to be cloned. The Ob geneproduct 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 hypothesisthat 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 aWestern 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? 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 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 shownhere?


23/23

e. You have now decided to clone the receptor for

the Ob protein and express the gene in mam-malian 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 nextto 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 thscreen outlined in part e and decided to make

knockout of the gene in mice. What phenotyp

would you predict for mice that lack the Ob re

ceptor? (obese, slim, normal)g. If you inject Ob protein into Ob mutant mice, wh

effect do you predict there will be on the phenotype

Problems 13

Reference E

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