Essential Cell Biology 5th edition
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Essential Cell Biology 5th edition

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356 CHAPTER 10 Analyzing the Structure and Function of Genesreplaced by the altered one. In this way, the function of the mutant proteincan be analyzed in the absence of the normal protein. A common wayof doing this in mice makes use of cultured mouse embryonic stem (ES)cells (discussed in Chapter 20). These cells are first subjected to targetedgene replacement before being transplanted into a developing embryo toproduce a mutant mouse, as illustrated in Figure 10–28.Using a similar strategy, the activities of both copies of a gene can beeliminated entirely, creating a “gene knockout.” To do this, one caneither introduce an inactive, mutant version of the gene into cultured EScells or delete the gene altogether. The ability to use ES cells to producesuch “knockout mice” revolutionized the study of gene function, and the(A)ES cells growingin culture(B)pregnant mousealtered versionof target geneconstructed bygeneticengineeringINTRODUCE A DNAFRAGMENT CONTAININGALTERED GENE INTOMANY CELLSLET EACH ES CELLPROLIFERATE TOFORM A COLONYINJECT ALTEREDES CELLSINTO EARLYEMBRYOISOLATE EARLYEMBRYOFigure 10–28 Targeted gene replacementin mice utilizes embryonic stem (ES)cells. (A) First, an altered version of thegene is introduced into cultured ES cells.In a few rare ES cells, the altered gene willreplace the corresponding normal genethrough homologous recombination (asdescribed in Chapter 6, pp. 220−222 andFigure 6−31). Although the procedure isoften laborious, these rare cells can beidentified and cultured to produce manydescendants, each of which carries analtered gene in place of one of its twonormal corresponding genes. (B) Next, thealtered ES cells are injected into a very earlymouse embryo; the cells are incorporatedinto the growing embryo, which thendevelops into a mouse that contains somesomatic cells (colored orange) that carry thealtered gene. Some of these mice may alsohave germ-line cells that contain the alteredgene; when bred with a normal mouse,some of the progeny of these mice willcontain the altered gene in all of their cells.Such a mouse is called a “knock-in” mouse.If two such mice are bred, one can obtainprogeny that contain two copies of thealtered gene—one on each chromosome—in all of their cells.TAKE CELLS FROM THERARE COLONY IN WHICHTHE DNA FRAGMENTHAS REPLACED ONECOPY OF THENORMAL GENEES cells with one copy of targetgene replaced by altered genesome of theseoffspring havegerm-line cellscontaining altered genethe offspring willinclude males andfemales with one copyof target genealtered in all cellsEARLY EMBRYO FORMEDPARTLY FROM ALTEREDES CELLSINTRODUCE EARLYEMBRYO INTOPSEUDOPREGNANTMOUSEBIRTHMATE WITHNORMAL MOUSEMATINGTRANSGENIC MOUSEIN WHICH BOTH COPIES OFTARGET GENE ARE ALTERED

Exploring Gene Function357(A)(B)Figure 10–29 Transgenic mice with a mutant DNA helicase show prematureaging. The helicase, encoded by the Xpd gene, is involved in both transcriptionand DNA repair. Compared with a wild-type mouse (A), a transgenic mouse thatexpresses a defective version of Xpd (B) exhibits many of the symptoms of prematureaging, including osteoporosis, emaciation, early graying, infertility, and reduced lifespan.The mutation in Xpd used here impairs the activity of the helicase and mimicsECB5 e10.36/10.29a human mutation that causes trichothiodystrophy, a disorder characterized by brittlehair, skeletal abnormalities, and a greatly reduced life expectancy. These resultssupport the hypothesis that an accumulation of DNA damage contributes to theaging process in both humans and mice. (From J. de Boer et al., Science 296:1276–1279, 2002. With permission from AAAS.)technique is now being used to systematically determine the function ofevery mouse gene (Figure 10–29).A variation of this technique can be used to produce conditional knockoutmice, in which a known gene can be disrupted more selectively—only ina particular cell type or at a certain time in development. The strategyinvolves the introduction of an enzyme, called a recombinase, that canbe directed to selectively excise—and thus disable—a gene of interest(Figure 10−30). Such conditional knockouts are useful for studying geneswith a critical function during development, because mice missing thesecrucial genes often die before birth.IN NON-TARGET TISSUES (e.g., MUSCLE), THE GENE OF INTEREST IS EXPRESSED NORMALLYCre recombinase geneLoxP sitegene of interestLoxP siteliver-specific promoterINACTIVEIN TARGET TISSUE (e.g., LIVER), THE GENE OF INTEREST IS DELETEDliver-specific promoterACTIVEGENE OFFGENE ONgene of interest removedfrom chromosome andlost as cells divideCre recombinase madeonly in liver cellsGENE ONaltered chromosomeGENE OFFprotein ofinterestCre RECOMBINASEBINDS TO LoxP SITESCre CATALYZESRECOMBINATIONBETWEEN LoxP SITESFigure 10–30 In conditional knockouts,a gene can be selectively disabled in aparticular target tissue. The approachrequires the insertion of two engineeredsegments of DNA into an animal’s germ-linecells. The first contains the gene encoding arecombinase (in this case, Cre recombinase)that is under the control of a tissue-specificpromoter. This promoter ensures thatrecombinase will be produced only in thetarget tissue. The second DNA moleculecontains the gene of interest flanked bynucleotide sequences (in this case, LoxPrecombination sites) that are recognized bythe recombinase. The mouse is engineeredso that this version of the gene of interest isthe only copy the animal has.In non-target tissues, no recombinase willbe produced and the gene of interest willbe expressed normally. In the target tissue,however, the tissue-specific promoter willbe activated, allowing the recombinase tobe produced. The enzyme will then bind tothe LoxP sites and catalyze a recombinationreaction that will excise the gene ofinterest—thus disabling it specifically in thetarget tissue.

Exploring Gene Function

357

(A)

(B)

Figure 10–29 Transgenic mice with a mutant DNA helicase show premature

aging. The helicase, encoded by the Xpd gene, is involved in both transcription

and DNA repair. Compared with a wild-type mouse (A), a transgenic mouse that

expresses a defective version of Xpd (B) exhibits many of the symptoms of premature

aging, including osteoporosis, emaciation, early graying, infertility, and reduced lifespan.

The mutation in Xpd used here impairs the activity of the helicase and mimics

ECB5 e10.36/10.29

a human mutation that causes trichothiodystrophy, a disorder characterized by brittle

hair, skeletal abnormalities, and a greatly reduced life expectancy. These results

support the hypothesis that an accumulation of DNA damage contributes to the

aging process in both humans and mice. (From J. de Boer et al., Science 296:1276–

1279, 2002. With permission from AAAS.)

technique is now being used to systematically determine the function of

every mouse gene (Figure 10–29).

A variation of this technique can be used to produce conditional knockout

mice, in which a known gene can be disrupted more selectively—only in

a particular cell type or at a certain time in development. The strategy

involves the introduction of an enzyme, called a recombinase, that can

be directed to selectively excise—and thus disable—a gene of interest

(Figure 10−30). Such conditional knockouts are useful for studying genes

with a critical function during development, because mice missing these

crucial genes often die before birth.

IN NON-TARGET TISSUES (e.g., MUSCLE), THE GENE OF INTEREST IS EXPRESSED NORMALLY

Cre recombinase gene

LoxP site

gene of interest

LoxP site

liver-specific promoter

INACTIVE

IN TARGET TISSUE (e.g., LIVER), THE GENE OF INTEREST IS DELETED

liver-specific promoter

ACTIVE

GENE OFF

GENE ON

gene of interest removed

from chromosome and

lost as cells divide

Cre recombinase made

only in liver cells

GENE ON

altered chromosome

GENE OFF

protein of

interest

Cre RECOMBINASE

BINDS TO LoxP SITES

Cre CATALYZES

RECOMBINATION

BETWEEN LoxP SITES

Figure 10–30 In conditional knockouts,

a gene can be selectively disabled in a

particular target tissue. The approach

requires the insertion of two engineered

segments of DNA into an animal’s germ-line

cells. The first contains the gene encoding a

recombinase (in this case, Cre recombinase)

that is under the control of a tissue-specific

promoter. This promoter ensures that

recombinase will be produced only in the

target tissue. The second DNA molecule

contains the gene of interest flanked by

nucleotide sequences (in this case, LoxP

recombination sites) that are recognized by

the recombinase. The mouse is engineered

so that this version of the gene of interest is

the only copy the animal has.

In non-target tissues, no recombinase will

be produced and the gene of interest will

be expressed normally. In the target tissue,

however, the tissue-specific promoter will

be activated, allowing the recombinase to

be produced. The enzyme will then bind to

the LoxP sites and catalyze a recombination

reaction that will excise the gene of

interest—thus disabling it specifically in the

target tissue.

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