ARTICLE

Modulating Skeletal Muscle Mass by Postnatal,Muscle-Specific Inactivation of the Myostatin GeneLuc Grobet,1 Dimitri Pirottin,1 Frederic Farnir,1 Dominique Poncelet,1 Luis Jose Royo,1Benoıt Brouwers,1 Elisabeth Christians,2 Daniel Desmecht,3 Freddy Coignoul,3 Ronald Kahn4,and Michel Georges1*1Department of Genetics, Faculty of Veterinary Medicine, University of Liege (B43), Liege, Belgium2Department of Histology-Embryology, Faculty of Veterinary Medicine, University of Liege (B43), Liege, Belgium3Department of Pathology, Faculty of Veterinary Medicine, University of Liege (B43), Liege, Belgium4Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

Received 13 November 2002; Accepted 5 January 2003

Summary: By using a conditional gene targeting ap-proach exploiting the cre-lox system, we show thatpostnatal inactivation of the myostatin gene in striatedmuscle is sufficient to cause a generalized muscularhypertrophy of the same magnitude as that observed forconstitutive myostatin knockout mice. This formallydemonstrates that striated muscle is the production siteof functional myostatin and that this member of theTGF� family of growth and differentiation factors regu-lates muscle mass not only during early embryogenesisbut throughout development. It indicates that myostatinantagonist could be used to treat muscle wasting and topromote muscle growth in man and animals. genesis35:227–238, 2003. © 2003 Wiley-Liss, Inc.

Key words: myostatin; conditional knockout; cre-lox; mus-cular hypertrophy

INTRODUCTION

Myostatin (MSTN) is a recently discovered member ofthe TGF� superfamily of growth and differentiation fac-tors (McPherron et al., 1997). Constitutive loss of MSTNfunction results in a dramatic increase in skeletal musclemass as a result of combined muscular hyperplasia andhypertrophy. MSTN knockout mice (McPherron et al.,1997), as well as mice (Szabo et al., 1998) and cattle(Grobet et al., 1997,; Kambadur et al., 1997; McPherronand Lee, 1997, Grubet et al., 1998) homozygous fornaturally occurring MSTN loss-of-function mutations areindeed sharing a phenotype commonly referred to as“double-muscling.” More recently, transgenic mice thatconstitutively overexpress dominant negative MSTN al-leles under the dependence of strong skeletal muscle-specific promoters were shown to be “double-muscled”as well (Lee and McPherron, 2001; Yang et al., 2001; Zhuet al., 2001).

It remained unknown, however, whether the inhibi-tion of MSTN at later stages of development retains the

potential to promote muscle growth. An answer to thisquestion is essential to justify the development and ad-ministration of MSTN antagonist for the treatment ofmuscle wasting or as a means to enhance meat produc-tion in farm animals.

We herein describe the generation of conditionalMSTN knockout mice using gene-targeting techniques,and demonstrate that the postnatal inactivation of theMSTN gene in striated muscle tissue causes a muscularhypertrophy phenotype. This result is a promise fornovel therapeutic opportunities and applications in ani-mal breeding.

RESULTS

Generation of Transgenic Mice Allowing forConditional Inactivation of the Myostatin Gene

In order to generate a MSTN allele that would allowfor its conditional inactivation, we elected to flank thethird exon of the MSTN gene — known to encode thebioactive carboxyterminal domain of the protein — witha pair of loxP sites.

To prevent disruption of putative cis-acting elements,the loxP sites were inserted in regions of low similaritywith the bovine orthologous sequence. These were iden-tified by aligning the murine and bovine MSTN genesequences using the algorithm of Needleman and Wun-sch (1970) implemented with the BESTFIT program

* Correspondence to: Michel Georges, Department of Genetics, Facultyof Veterinary Medicine, University of Liege (B43), 20 Boulevard de Colon-ster, B-4000 Liege. Belgium.

E-mail: michel.georges@ulg.ac.beContract grant sponsor: the Belgian Ministere de l’Agriculture et des

Classes Moyennes, Contract grant number: S-5990, Contract grant sponsors:SYNXPharma Inc., Toronto, and BASF.

DOI: 10.1002/gene.10188

© 2003 Wiley-Liss, Inc. genesis 35:227–238 (2003)

(GCG Wisconsin Package™). Two major insertionspresent in the bovine but not in the murine sequenceand detected by dotplot analysis (Fig. 1) were eliminatedbefore performing the BESTFIT alignment. A similarityprofile was generated by sliding a 200-bp windowthrough the aligned sequences and computing the per-centage similarity between the bovine and murine se-quence for each window (Fig. 1).

Using standard cloning procedures, we constructed atargeting vector comprising 1) a loxP site in a poorlyconserved region of intron 2; 2) a “floxed” neomycineresistance cassette inserted in a poorly conserved region3� of the main polyadenylation site; 3) 8.2 and 3.3 Kb of5� and 3� homology arms, respectively; and 4) two thy-midine kinase selection cassettes at either end of theconstruct (Fig. 1).

After electroporation of RI embryonic stem cells, pos-itive-negative selection with neomycin and gancyclovir,and analysis of candidate clones by PCR and Southernblotting, we identified two clones in which proper genereplacement had taken place (data not shown). Follow-ing transient expression of cre-recombinase, we thenderived five clones having lost the neomycine resistancecassette but maintained a floxed MSTN exon III (Fig. 1).Cells from one of these clones were aggregated withdepellucidated CD1 morulae which were transferred tofoster mothers to yield three chimeric males. Whencrossed to CD1 females, one of these transmitted the RIgenome to all its 13 offspring as judged from their coat

color. As expected, approximately half of these (threemales and four females) also inherited the floxed myo-statin allele (genotype: MSTN�/flox). These were inter-crossed to produce a homozygous MSTNflox/flox line.

To produce mice harboring a constitutive deletion ofthe third MSTN exon, we mated MSTNflox/flox males withFBV females and transiently expressed cre in the result-ing zygotes by microinjection of the pCAGGS-Cre plas-mid (Araki et al., 1995).

We obtained 17 offspring, of which four had receivedthe nonrecombined MSTNflox allele while eight had in-herited a recombined MSTN� allele (Fig. 1). Two of thesewere intercrossed to generate MSTN�/� descendants.

Generation of a MCKcre�/- MSTN�/flox Intercross

MSTNflox/flox females were mated to transgenicmales expressing cre-recombinase under the depen-dence of the murine muscle creatine kinase (MCK)promoter (Bruning et al., 1998). Resulting MCKcre�/-

MSTN�/flox F1 mice were intercrossed to generate anF2 population of 134 individuals. Their MCKcre andMSTN genotypes were determined by PCR, revealingthe segregation ratios expected for a dihybrid cross(Table 1; P � 0.65).

All F2 animals were weighed at 2, 3, 4, and 5 monthsof age; 81 randomly selected animals were then sacri-ficed and dissected. Tissue samples were collected fromeight F2 animals (two MCKcre�/? MSTN�/�, three MCK-

FIG. 1. Composition of the targeting construct. The murine and bovine MSTN genes are aligned. The three exons (I, II, and III) arerepresented as cylinders with the 5� and 3� UTR sequences shown in light and the coding sequences in dark. The dotted arrows correspondto SINE sequences. “ID1” and “ID2” correspond to insertion/deletion events present in the bovine sequence but not in its murine ortholog.A similarity profile, corresponding to the sequence similarity of a 200 bp window slid across the aligned murine and bovine sequences isplotted. The approximate positions in the targeting construct of the two thymidine kinase cassettes (TK), the neomycin resistance cassette(NEO), the three loxP sites (�), the 5� and 3� homology arms, and the vector sequences (pNEB193) are shown. The vertical arrows pointtowards the similarity dips in which the floxed neomycine resistance cassette and the isolated 5� loxP site were inserted. The location ofthe two primer pairs used in the assay to monitor cre-mediated excision of the third exon are shown as white arrows. The deletions obtainedby transient expression of cre in RI embryonic stem cells and yielding the MSTN� and MSTNflox alleles are shown.

228 GROBET ET AL.

cre�/? MSTN�/flox, three MCKcre�/? MSTNflox/flox, allmales) for morphometric and histological analyses.

The Lox P Sites Flanking Exon III Do Not Affectthe Functionality of the MSTNflox Allele

We first verified whether the insertion of the loxP siteson either side of exon III might interfere with the func-tionality of the MSTN gene. MSTN mRNA levels werecompared between MCKcre-/- MSTN�/�, and MCKcre-/-

MSTNflox/flox in a range of tissues of 2-month-old mice(Fig. 2). In both genotypes, MSTN specific RT-PCR prod-uct was detected in skeletal muscle (pectoralis and gas-trocnemius plantaris), but not in heart, liver, spleen, andabdominal fat. The intensity ratios between the bandscorresponding to MSTN and the �-actin control werecomparable in both genotypes. These results indicatethat the loxP sites did not affect the tissue-specific ex-pression pattern of MSTN nor its expression level inskeletal muscle.

A possible effect of loxP sites was further assessed bycomparing live weight, carcass weight, and weight ofindividual organs from MCKcre-/- MSTN�/�, MCKcre-/-

MSTN�/flox, and MCKcre-/- MSTNflox/flox F2 mice. Fornone of the analyzed traits did we find any evidence foran effect of MSTN genotype within the MCKcre-/- geno-type (Fig. 3, Table 2).

Altogether, these results indicate that the functionalityof the MSTNflox allele is essentially equivalent to that ofits wildtype counterpart.

Evidence for a Slightly Deleterious Effect of theMCKcre Transgene

We then examined whether the MCKcre transgenemight on its own have an effect on the examined pheno-types. This was achieved by comparing the phenotypes ofMCKcre�/? MSTN�/� versus MCKcre-/- MSTN./. mice. Al-though not statistically significant sensu stricto (P � 0.07),it is noteworthy that there was some evidence for a slightlydeleterious effect of the MCKcre�/? genotype on liveweight, carcass weight, as well as the weight of severalindividual organs (Fig. 3, Table 2) If real, this could eitherbe due to the expression of cre in skeletal muscle, or be an

effect of the transgene insertion, or of a QTL allele linked tothe transgene insertion site.

The MCKcre Transgene Induces a Postnatal,Muscle-Specific Inactivation of the MSTN Gene

To study the spatio-temporal pattern of cre-mediatedexcision of the third exon of the MSTNflox allele wedeveloped a PCR assay that allowed for the coamplifica-tion of 1) a “control” 335 bp fragment located �3 Kbupstream of exon I; and 2) a 195 bp fragment from aMSTNflox allele having undergone a cre-mediated dele-tion of exon III. The distance separating the latter primerpair is too large (�4 Kb) to allow amplification of thecorresponding PCR products from the intact MSTNflox

allele or the wildtype MSTN� allele (Fig. 1).The assay was calibrated on genomic DNA extracted

from MSTN�/�, MSTN�/�, MSTN�/� mice, as well as onsamples comprising varying proportions of MSTN�/� andMSTN�/� DNA (1/1 to 1/63). This indicated that anexcision rate �3% could reliably be detected (Fig. 4A).

We first applied this assay to genomic DNA extractedfrom a range of tissues of 5-month-old MCKcre�/? MSTN-flox/flox animals. As expected, the �-specific 195 bp frag-ment was detected in skeletal muscle and cardiac muscle,but not in any of the other tissues that were analyzed(kidney, lung, liver, spleen, and testis) (Fig. 4B). The inten-sity ratios of the 353 bp and 195 bp fragments indicatedthat at least 50% of the MSTNflox molecules have undergonecre-mediated deletion in skeletal muscle and slightly less inthe heart. As myotubes only contribute approximately halfof the nuclei from skeletal muscle (Schmalbruch and Hell-hammer, 1977), we speculated that the excision rate inmyotubes might well be essentially complete.

We then applied our test to genomic DNA extractedfrom skeletal muscle of MCKcre�/? MSTNflox/flox 18 dayspostcoitum (pc) fetuses, as well as 1-, 2-, and 15-day-oldyoung. The �-specific 195 bp fragment was absent in the18-day pc fetuses, as well as in the 1- and 2-day-oldnewborns. This indicates that the vast majority of theMSTNflox alleles would still be intact, and hence func-tional at these developmental stages. The 195 bp frag-ment, however, was present in the 15-day-old young, atlevels similar to those found in 5-month-old animals (Fig.4B). These results are in agreement with previous re-ports by other groups using the same MCKcre transgene(Martin Holzenberger, pers. commun.).

We then examined the effect of the cre-mediated ex-cision on MSTN expression using RT-PCR. No MSTN-specific RT-PCR products could be amplified from skel-etal muscle of 2-month-old MCKcre�/? MSTNflox/flox

mice, contrary to what was observed in MCKcre-/- MST-Nflox/flox and MCKcre-/- MSTN�/�, but identical to thesituation in constitutive MSTN knockouts (MSTN�/�)(Fig. 2). This strongly suggests that the excision rate isindeed essentially complete in myotube nuclei.

Table 1Segregation of the MSTN and MCKcre Genotypes

in the MCKcre�/-MSTN�/flox Intercross

F–M (Exp.)MSTN�/�

13–20 (16.75)MSTN�/flox

38–30 (33.5)MSTNflox/flox

25–17 (16.75)

MCKcre�/?

56–48 (50.25)10–14 (13.4) 28–24 (26.8) 18–10 (13.4)

MCKcre/

20–19 (16.75)3–6 (4.5) 10–6 (8.9) 7–7 (4.5)

Observed number of females and males with a given genotype.Numbers in parentheses correspond to the expected numbers as-suming that the MCKcre and MSTN loci are autosomal and un-linked.

229CONDITIONAL MSTN KNOCKOUT

Postnatal, Muscle-Specific Inactivation of theMSTN Gene Causes a “Double-Muscling”Phenotype

Monitoring growth in the intercross populationclearly revealed a major effect of the MSTN./. genotypewithin the MCKcre�/? subpopulation (Table 2, Fig. 3). At2 months of age, MCKcre�/? MSTNflox/flox animals ofboth sexes weighed on average 5.6 g more (�23%; P 0.0001) than their MCKcre�/? MSTN�/� counterparts. At5 months of age, this difference had increased to 7.9 g(�26%; P 0.0001). A modest effect was detectable in

MCKcre�/? MSTNflox/� individuals as well, weighing2.2 g more (�9%; P 0.05) at 2 months and 3 g more(�10%; P 0.001) at 5 months.

Visual examination of the carcasses of MCKcre�/flox

MSTNflox/flox animals revealed a marked, generalizedmuscular hypertrophy (Fig. 5). We quantified this im-pression by showing that the carcasses of MCKcre�/?

MSTNflox/flox and MCKcre�/? MSTNflox/� animalsweighed on average 6.3 g (�37%; P 0.0001) and 1.9 g(�11%; P 0.001) more than that of their MCKcre�/?

MSTN�/� counterparts (Table 2, Fig. 3). The increase in

FIG. 2. Detection of MSTN transcripts by RT-PCR in a range of tissues from 2-month-old mice of four different MCKcre./. MSTN./.

genotypes. The 397 bp fragment is specific for MSTN transcripts containing the IInd and IIIrd MSTN exons. The 698 bp product serves asan internal control for the assay and is specific for �-actin transcripts containing the IVth and VIth �-actin exons. “MW” and “NC” stand formolecular weight marker and negative control, respectively.

230 GROBET ET AL.

carcass weight therefore accounted for 80% and 64% ofthe increase in weight of MCKcre�/? MSTNflox/flox andMCKcre�/? MSTNflox/� animals, respectively.

We showed that this increase in carcass weight wasprimarily due to an increase in muscle mass by 1) com-paring the weight of the pectoralis muscle, and 2) com-paring the cross-sectional area of lower leg muscles.When compared to MCKcre�/? MSTN�/� animals, theweight of the pectoralis was shown to be increased by77% (P 0.0001) in MCKcre�/? MSTNflox/flox and 14%(P 0.05) in MCKcre�/? MSTNflox/� animals (Table 2).The total cross-sectional area of the lower leg was shownto be increased by 74% (P 0.05) in MCKcre�/? MSTN-flox/flox animals (Fig. 5, Table 2). Superficial muscle layersseemed to be more profoundly affected than the deepermuscle layers. Indeed, the cross-sectional area of theexternal biceps femoris group and internal adductorgroup of muscles (cf. Fig. 7) were increased, respec-tively, two- (�209%) and five-fold (�566%) in MCK-cre�/? MSTNflox/flox animals, while the areas occupied bythe deeper laying anterior tibialis cranialis group andposterior gastrocnemius plantaris group were increasedby only 33% and 65%, respectively (Table 2).

It is noteworthy that the weight of several internalorgans (particularly liver, lung, and to a lesser extentheart) was also increased in MCKcre�/? MSTNflox/flox

when compared to MCKcre�/? MSTN�/� animals (Table2).

Taken together, these results clearly demonstrate thatpostnatal inactivation of MSTN in skeletal muscle still hasa major effect on muscle growth in the mouse.

The Conditional “Double-Muscling” Phenotype IsPrimarily Due to Muscle Fiber Hypertrophy

To determine whether the observed muscular hyper-trophy resulted from an increase in the number (hyper-plasia) and/or an increase in the size of individual myo-tubes, we measured the cross-sectional area of individualmuscle fibers in the tibialis cranialis and gastrocnemiusplantaris group of muscles in MCKcre�/? MSTN�/�,MCKcre�/? MSTNflox/�, and MCKcre�/? MSTNflox/flox in-dividuals. Figure 6 shows the frequency distribution ofindividual fiber area in the MCKcre�/? MSTN�/� andMCKcre�/? MSTNflox/flox genotypes. In MCKcre�/? MST-Nflox/flox individuals, average myofiber area is increasedby 52% (P 0.0001) in the tibialis cranialis musclegroup and by 49% (P 0.0001) in the gastrocnemius

FIG. 3. Live weight at 5 months(g), carcass weight (g), and weightof the pectoralis muscles (g) in theMCKcre�/- MSTN�/flox intercross.Animals are sorted by sex, MCKcregenotype (circles: MCKcre-/-, dia-monds: MCKcre�/?), and MSTNgenotype. The shaded symbolscorrespond to individual mea-surements, the white symbols tothe average of the correspondinggenotype class.

231CONDITIONAL MSTN KNOCKOUT

Tab

le2.

Gen

otyp

icV

alue

sin

the

MC

Kcr

e�/

MS

TN�

/flo

xIn

terc

ross

MC

Kcr

e/

MC

Kcr

e�/?

MS

TN�

/�M

STN

�/fl

ox

MS

TNflo

x/fl

ox

MS

TN�

/�M

STN

�/fl

ox

MS

TNflo

x/fl

ox

Live

wei

ght

2m

onth

s(g

)27

.47

�1.

20(8

)25

.71

�0.

85(1

6)25

.95

�0.

94(1

3)24

.82

�0.

71(2

3)27

.01

�0.

47(5

1)[�

12%

]***

**30

.44

�0.

69(2

4)[�

33%

]***

*Li

vew

eigh

t3

mon

ths

(g)

30.2

2�

1.20

(8)

28.6

9�

0.88

(15)

28.0

8�

1.02

(11)

27.3

3�

0.69

(24)

30.2

8�

0.49

(48)

[�11

%]*

****

**34

.51

�0.

66(2

7)[�

26%

]***

*Li

vew

eigh

t4

mon

ths

(g)

31.3

9�

1.18

(8)

30.3

4�

0.86

(15)

30.3

4�

1.00

(11)

29.3

0�

0.68

(24)

31.8

7�

0.48

(48)

[�9%

]**

****

36.9

0�

0.64

(27)

[�26

%]*

***

Live

wei

ght

5m

onth

s(g

)32

.35

�1.

23(8

)31

.42

�0.

90(1

5)30

.76

�1.

10(1

0)30

.06

�0.

71(2

4)33

.13

�0.

51(4

7)[�

10%

]***

****

37.9

5�

0.67

(27)

[�26

%]*

***

Car

cass

wei

ght

(g)

18.9

4�

0.78

(4)

17.8

2�

0.56

(8)

17.9

7�

0.55

(8)

17.3

1�

0.42

(14)

19.2

6�

0.27

(33)

[�11

%]*

****

**23

.66

�0.

40(1

6)[�

37%

]***

*W

eigh

tof

pec

tora

lis(m

g)27

5�

24(4

)27

4�

17(8

)27

7�

18(7

)26

4�

13(1

4)30

1�

8(3

3)[�

14%

]***

**46

7�

13(1

5)[�

77%

]***

*W

eigh

tof

shou

lder

(mg)

860

�41

(4)

823

�30

(8)

809

�31

(7)

776

�22

(14)

896

�14

(33)

[�15

%]*

***

****

1,15

4�

22(1

5)[�

49%

]***

*W

eigh

tof

leg

(mg)

478

�28

(4)

458

�20

(8)

465

�21

(7)

434

�15

(14)

517

�10

(33)

[�19

%]*

***

****

658

�15

(15)

[�52

%]*

***

Wei

ght

ofhe

art

(mg)

150

�9

(4)

146

�7

(8)

141

�7

(7)

133

�5

(14)

143

�3

(33)

146

�5

(15)

[�10

%]

Wei

ght

ofsp

leen

(mg)

95�

19(4

)82

�14

(8)

84�

14(7

)81

�10

(14)

99�

7(3

3)83

�10

(15)

Wei

ght

oflu

ngs

(mg)

178

�10

(4)

172

�7

(8)

169

�8

(7)

166

�6

(14)

177

�7

(33)

186

�5

(15)

[�12

%]*

Wei

ght

ofliv

er(m

g)1,

285

�85

(4)

1,14

8�

61(8

)1,

171

�64

(7)

1,11

5�

46(1

4)1,

215

�30

(33)

1,27

3�

45(1

5)[�

14%

]*W

eigh

tof

kid

neys

(mg)

420

�26

(4)

397

�19

(8)

410

�20

(7)

402

�14

(14)

407

�9

(33)

402

�14

(15)

Tota

lare

aof

the

leg

(mm

2)

ND

ND

ND

33.9

0�

5.50

(2)

42.2

8�

4.49

(3)

*59.

15�

4.49

(3)[�

74%

]*tib

.cr

an.

area

(mm

2)

ND

ND

ND

8.34

�1.

13(2

)9.

38�

0.92

(3)[�

12%

]11

.08

�0.

92(3

)[�33

%]

gast

r.p

lant

.ar

ea(m

m2)

ND

ND

ND

14.7

7�

1.36

(2)

18.1

6�

1.11

(3)[�

23%

]**

24.9

3�

1.11

(3)[�

69%

]**

add

uct.

area

(mm

2)

ND

ND

ND

1.26

�1.

90(2

)4.

30�

1.56

(3)

8.39

�1.

56(3

)[�56

6%]*

bic

.fe

m.

area

(mm

2)

ND

ND

ND

1.09

�0.

92(2

)1.

33�

0.92

(3)

3.37

�0.

75(3

)[�20

9%]

Fib

erar

eain

tib.

cran

.(�

m2)

ND

ND

ND

1,07

8�

42(2

)1,

299

�42

(3)[�

20%

]***

****

1,64

1�

42(3

)[�52

%]*

***

Fib

erar

eain

gast

r.p

lant

.(�

m2)

ND

ND

ND

1,37

7�

45(2

)1,

492

�45

(3)[�

8%]

****

2055

�45

(3)[�

49%

]***

*

Leas

tsq

uare

mea

nsof

the

six

geno

typ

ecl

asse

s�

stan

dar

der

rors

ofth

ees

timat

es.P

heno

typ

esar

eas

defi

ned

inM

ater

ials

and

Met

hod

s.N

umb

ers

inp

aren

thes

esco

rres

pon

dto

the

resp

ectiv

esa

mp

lesi

ze.T

heb

ackg

roun

dco

loro

fthe

cells

mar

ksgr

oup

sof

geno

typ

esth

atd

ono

tdiff

ersi

gnifi

cant

lyam

ong

each

othe

r,b

utd

iffer

from

the

geno

typ

esw

itha

diff

eren

tb

ackg

roun

dco

lor.

The

stat

istic

alsi

gnifi

canc

eof

(i)th

eM

STN

�/fl

ox

-M

STN

�/�

cont

rast

isgi

ven

atth

erig

htof

the

geno

typ

eva

lue

inth

eM

CK

cre�

/?M

STN

�/fl

ox

colu

mn,

(ii)

ofth

eM

STN

flo

x/fl

ox

MS

TN�

/flo

xco

ntra

stat

the

left

ofth

ege

noty

pe

valu

ein

the

MC

Kcr

e�/?

MS

TNflo

x/fl

ox

colu

mn,

(iii)

ofth

eM

STN

flo

x/fl

ox

-M

STN

�/�

cont

rast

atth

erig

htof

the

geno

typ

eva

lue

inth

eM

CK

Cre

�/?

MS

TNflo

x/fl

ox

colu

mn.

****

P

0.00

01,

***P

0.

001,

**P

0.

01,

*P

0.05

.Fo

rsi

gnifi

cant

cont

rast

s,nu

mb

ers

inb

rack

ets

exp

ress

the

incr

ease

inp

heno

typ

icm

ean

with

resp

ect

toth

eM

CK

cre�

/?M

STN

�/�

clas

sas

ap

erce

ntag

eof

the

MC

Kcr

e�/?

MS

TN�

/�m

ean.

ND

:no

td

one.

232 GROBET ET AL.

plantaris muscle group when compared to the MCK-cre�/? MSTN�/� genotype (Table 2). It is noteworthythat in MCKcre�/? MSTNflox/flox individuals, individualfiber area seems to be distributed bimodally (Fig. 6).

Comparing the effect on the area of the muscle groupwith that on the area of individual muscle fibers withinthe corresponding muscle group indicates that the hy-pertrophy of individual myotubes accounts entirely forthe increase in muscle mass of the tibialis cranialisgroup, while accounting for 71% of the increase in mus-cle mass of the gastrocnemius plantaris group, the re-mainder reflecting myofiber hyperplasia.

DISCUSSION

It was previously reported that constitutive loss of MSTNfunction causes a generalized skeletal muscle hypertro-phy in mice and cattle (e.g., McPherron et al., 1997;Grobet et al., 1997). It was not known, however,whether this results from a lack of MSTN activity duringearly myogenesis only or whether MSTN secretion isrequired throughout development (including after birth)to regulate muscle mass. Using a conditional gene tar-geting approach based on the cre-loxP system, we dem-onstrate in this work that postnatal loss of MSTN func-

FIG. 4. Monitoring Cre-mediated excision of the MSTN third exon. A: Calibration of the test. The 353 bp amplification product serves asinternal control and corresponds to a segment of the MSTN promoter. The 195 bp amplicon is specific for the MSTNflox allele havingundergone cre-mediated deletion of exon III (Fig. 1). The lane marked “MW” corresponds to a molecular weight marker. The three lanes onthe left of “MW” correspond to animals of MSTN�/�, MSTN�/�, and MSTN�/� respectively. The lanes on the right correspond to varyingproportions of MSTN�/� and MSTN�/� DNA (1/1 to 1/63). B: Utilization of the test. The developmental stage/age of the analyzed animalsis indicated above the gel images, while the analyzed tissue and genotype of the individuals are indicated below.

233CONDITIONAL MSTN KNOCKOUT

tion in striated muscle results in a generalizedmuscular hypertrophy that is of the same magnitudeas that observed for constitutive MSTN knockout mice(McPherron et al., 1997). These results complementrecent studies indicating that 1) postnatal systemicadministration of myostatin causes cachexia in themouse (Zimmers et al., 2002); and 2) antibody-medi-

ated blockade of endogenous myostatin results in anincrease in muscle mass and reduction of muscle de-generation in mdx mice (Bogdanovitch et al., 2002).Altogether, these studies indicate that MSTN does notonly act during early embryogenesis, but that it oper-ates as a functional “chalone” at later stages of devel-opment as well.

FIG. 5. Comparison between a MCKcre�/? MSTN�/� and MCKcre�/? MSTNflox/flox individual, of (A) the thoracic portion of the carcass, (B)the caudal portion of the carcass, (C) hematoxylin-eosin-stained cross-sections of the lower leg (cf. Fig. 7).

234 GROBET ET AL.

We demonstrate that the induced muscular hypertro-phy is primarily due to an increase in size of individualmuscle fibers and to a lesser extent to an increase in theirnumber (hyperplasia). This is in essence similar to whatwas observed for constitutive MSTN knockout mice(McPherron et al., 1997), but is strikingly different fromthe “double-muscling” condition in cattle, which is vir-tually exclusively due to myofiber hyperplasia (e.g., Han-set et al., 1982; Wegner et al., 2000). We also show thatall muscle groups are not equally affected by the loss ofMSTN function, superficial muscle layers exhibiting amore severe hypertrophy than the deeper layers. Thesame observation was previously made in “double-mus-cled” cattle (Hanset et al., 1982).

It could be argued that the induced myostatin inacti-vation, although postnatal, occurred at a stage wherefiber number is not yet set. It does not prove, therefore,that inactivation at an older age would yield the sameeffect. However, the fact that the observed muscularhypertrophy primarily reflects an increase in the diame-ter of muscle fibers rather than in their number suggeststhat the effect is fiber-number independent and couldtherefore operate at any stage of development. To for-mally prove this, we are performing a similar experiment

using transgenic mice that express cre in skeletal muscleunder the dependence of a tetracycline-inducible pro-moter (Utomo et al., 1999).

It is noteworthy that we observed an increase in theweight of lung and liver and to a lesser extent heart (P 0.08) of MCKcre�/? MSTNflox/flox animals (Table 2). It isunclear at present whether this is due to a direct effecton these organs of reduced concentrations of circulatingMSTN, or whether it reflects the indirect physiologicaladaptation of the organism to the increased muscle mass.Sharma et al. (1999) suggested that cardiac muscle mayproduce low levels of MSTN that may act in a paracrineway. We were not able in this work to detect MSTNmRNA in cardiac muscle of 2-month old mice (Fig. 2).Note that all internal organs of “double-muscled” cattlewere shown to be reduced in size when compared tothose of wildtype controls (Ansay and Hanset, 1979).

McPherron and Lee (2002) and Lin et al. (2002) re-cently showed a suppression of fat accumulation inMSTN knockout mice. Similar observations were previ-ously made in “double-muscled” cattle (e.g., Ansay andHanset, 1979; Hanset et al., 1982). As suggested byMcPherron and Lee (2002), the MSTNflox/flox mice gen-erated as part of this work could be used to test whetherthe inactivation of the MSTN gene in adipose tissue isessential for the suppression of fat accumulation. Con-trary to the findings of McPherron et al. (1997), how-ever, we were not able to detect MSTN transcripts inabdominal fat tissue in this work (Fig. 2).

Our results strongly suggest that myostatin antagonistscould be used for the treatment of muscle wasting asso-ciated with many chronic disease states as well as topromote muscle growth in farm animals.

MATERIALS AND METHODS

Generation of Transgenic MSTNflox/flox Mice byGene Targeting

Construction of the targeting vector. The target-ing vector was constructed using standard cloning pro-cedures (Sambrook and Russel, 2001) and is schemati-cally represented in Fig. 1. The building blocks used forits construction were:

1) A lambda clone (�-MMMSTN-1) containing theentire murine MSTN gene plus 4 and 6.5 Kb of upstreamand downstream sequences. This clone was isolatedfrom a murine genomic library (ML1043J, ClonTech,Palo Alto, CA) constructed from C57Bl6/6N genomicDNA. The 15,382 bp insert of �-MMMSTN-1 has beencompletely sequenced and has been submitted to Gen-Bank under accession number AY204900.

2) A cassette containing a bacterial neomycine phos-photransferase gene driven by a thymidine kinase pro-moter, isolated from the pMC1neoPolyA vector (Strat-agene, La Jolla, CA).

3) A cassette containing the herpes simplex thymi-dine kinase gene under the dependence of the CMV

FIG. 6. Frequency distribution of myofibers with a given cross-sectional area in the tibialis cranialis and gastrocnemius plantarismuscle groups for the MCKcre�/? MSTN�/� (white) and MCKcre�/?

MSTNflox/flox (black) genotypes.

235CONDITIONAL MSTN KNOCKOUT

promoter isolated from the pcDNA3 vector (Invitrogen,Carlsbad, CA).

4) Oligonucleotide adapters containing loxP se-quences.

5) A slightly modified pNEB193 (New England Bio-labs, Beverly, MA) cloning vector.

The targeting construct was completely sequenced priorto use to verify the integrity of the MSTN sequences. Theregions corresponding to the 5� and 3� homology armswere shown by sequencing to be perfectly identical inthe targeting vector (C57Bl6/6N origin) and the RI em-bryonic stem (ES) cell line (SV129 origin) (Nagy et al.,1993).

Gene targeting by homologous recombination inES cells. Gene targeting was performed in RI cells(Nagy et al., 1993) using standard procedures (Torresand Kuhn, 1997). Briefly, the targeting vector was lin-earized with PacI and 25 �g of the resulting productwere used to electroporate 107 RI ES cells. Positive-negative selection was performed using G418 (300 �g/ml) and gancyclovir (2 �M). Resistant clones werepicked in triplicate in 96-well plates (one copy for freez-ing, two for DNA extraction). Screening for the ex-pected targeting event was performed by PCR and con-firmed by Southern blotting. Positive clones wereexpanded and 107 cells transfected with 5 �g of super-coiled pMC-cre plasmid (Gu et al., 1993) in order todelete the neomycin resistance cassette and obtain a“MSTNflox” allele (Fig. 1). The resulting clones wereplated at low density and subsequently picked in tripli-cate in 96-well plates (one copy for freezing, one copy tomonitor the G418 sensitivity, and one for DNA extrac-tion). G418 sensitive clones were analyzed by PCR toidentify those having undergone the deletion event char-acterizing the MSTNflox alleles.

Generation of transgenic mice. 2.5-day-old CD1morulae were harvested and aggregated with targeted EScells as described previously (Torres and Kuhn, 1997).Uterine transfer was performed the next day in C57BL CBA pseudopregnant mothers using standard proce-dures (Hogan et al., 1994). Resulting male chimerasexhibiting a patched coat color were mated to CD1females. Offspring inheriting the MSTNflox allele wereidentified by PCR using DNA extracted from tail tips ofcolored individuals and subsequently intercrossed toproduce MSTNflox/flox mice.

Generation of Transgenic MSTN�/� Mice

A MSTNflox/flox male was mated to an FVB female.Resulting zygotes were harvested using standard proce-dures and microinjected with 1 ng/�l of supercoiledpCAGGS-Cre plasmid in which the expression of cre isdriven by the chicken beta actin promoter (Araki et al.,1995). The resulting offspring were screened by PCR forthe cre-mediated deletion of the third exon (MSTN�

allele) (Fig. 1). Heterozygous MSTN�/� individuals wereintercrossed to generate a MSTN�/� line.

Analyses of the MCKcre�/- MSTN�/flox Intercross

Monitoring cre-mediated excision of the thirdexon of the MSTNflox allele. To monitor the in vivocre-mediated excision of exon III, we developed a mul-tiplex PCR assay amplifying a 353 bp control fragmentlocated �3 Kb upstream of the MSTN transcription startsite (UP-primer: 5�-AGTGGAAGAAATTCTCTCTTCACTC-3�; DN-primer: 5�-GTAGCTCACCTCACCCTGCATGTTC-3�), as well as a 195 bp fragment specific for the deletedMSTNflox allele (UP-primer: 5�-CCATATAGTGCTCAGA-AAGAGCTAC-3�; DN-primer: 5�-TGGGCTAATTATGAAT-TATTCACTC-3�). The approximate positions of the cor-responding primer pairs are shown in Figure 1.

Monitoring MSTN expression by RT-PCR analy-sis. MSTN expression was monitored by RT-PCR ampli-fication of a 397 bp fragment using primers located,respectively, in exons II (5�-AGACTCCTACAACAGTGTT-TGT-3�) and III (5�-TCCCATCCAAAGGCTTCAAAATC-3�). As positive control, a �-actin RT-PCR product of 698bp was simultaneously amplified with primers locatedrespectively in exons IV (5�-ACCTTCAACACCCCAGC-CATGTACG-3�) and VI (5�-CTGATCCACATCTGCTG-GAAGGTGG-3�) (Wu et al., 2000). Total RNA was ex-tracted from heart, liver, spleen, peritoneal fat,gastrocnemius plantaris muscle, and pectoralis muscleusing Trizol� (Invitrogen). First strand cDNA synthesiswas carried out in a reaction volume of 20 �l startingfrom 2 �g total RNA per sample using an oligo(dT)16 asprimer and PowerScript™ reverse transcriptase (BD Bio-sciences/ClonTech, Palo Alto, CA).

Weight measurements. Live weight was recordedat 2, 3, 4, and 5 months of age. Animals were sacrificedat 5 months and dissected. We determined the weight ofthe carcass (skinned body minus all internal organs andassociated fat and connective tissue), “shoulder weight”(skinned left forelimb cut at wrist-level), “leg weight”(skinned left leg cut at knee-level), weight of the dis-sected pectoralis muscles, and weight of individual or-gans including heart, lungs, kidneys, liver, and spleen.

Morphometric analyses. Legs of 5-month-old ani-mals were stretched on a solid support and fixed in 4%buffered formaldehyde following standard procedures.After fixation, a 5 mm-wide transversal slice centered onthe widest part of the lower leg was treated in EDTA-saturated phosphate buffered saline for 1 week. Afterparaffin embedding, transversal sections taken from thecenter of the slices were stained with hematoxylin-eosin.Sections were photographed with Nomarski optics and aLeica digital camera. Image processing was realized us-ing the analySIS� 3.0 image analysis software (Soft Imag-ing System, Munster, Germany). “Total leg” area, area ofthe “tibialis cranialis” muscle group (tibialis cranialis,extensor digitorum longus and lateralis, peroneus lon-gus), area of the “gastrocnemius plantaris” muscle group(gastrocnemius caput laterale and mediale, soleus, flexordigitorum longus), area of the “biceps femoris” and areaof the “adductor” muscle group (adductor, gracilis, semi-

236 GROBET ET AL.

membranosus, and semitendinosus) defined as illus-trated in Figure 7 were measured for each animal.

We photographed one microscopic field at 20 mag-nification for each of the four muscles in the “tibialiscranialis” group and for three muscles in the “gastrocne-mius plantaris” group (gastrocnemius caput laterale andmediale, flexor digitorum longus) (Fig. 7). A grid defin-ing 20 identical squares was superimposed on each im-age and the area occupied by the myofiber spanning thecenter of each of the 20 squares was measured. Thisyielded 20 4 � 80 measurements per animal for thetibialis cranialis group and 20 3 � 60 measurementsper animal for the gastrocnemius plantaris group.

Statistical Analysis

Phenotypic data were analyzed using a linear modelincluding an overall mean, a fixed effect correspondingto sex (male, female), a fixed effect corresponding togenotype (MCKcre-/- MSTN�/�, MCKcre-/- MSTN�/flox,MCKcre-/- MSTNflox/flox, MCKcre�/? MSTN�/�, MCK-cre�/? MSTN�/flox, and MCKcre�/? MSTNflox/flox) and anerror term. Statistical analyses were performed using theGLM procedure of the SAS package (SAS Institute, Cary,NC).

ACKNOWLEDGMENTS

The experimental design was approved by the ethics com-mittee of the Faculty of Veterinary Medicine, University of

Liege, and conducted under licence LA1610475 from theBelgian Ministere des Classes Moyennes et de l’Agriculture.We thank A. Nagy for kindly providing the R1 ES cells andP. Vassali for providing the pcAGGS-cre plasmid. We thankG. Vassart, M. Parmentier, C. Ledent, and C. Dessy forhelpful discussions, and to K. Sarlet for technical assistance.

LITERATURE CITED

Ansay M, Hanset R. 1979. Anatomical, physiological and biochemicaldifferences between conventional and double-muscled cattle inthe Belgian Blue and White breed. Livestock Prod Science 6:5–13.

Araki K, Araki M, Miyazaki J, Vassali P. 1995. Site-specific recombina-tion of a transgene in fertilized eggs by transient expression of crerecombinase. Proc Natl Acad Sci USA 92:160–164.

Bogdanovitch S, Krag TO, Barton ER, Morris LD, Whittemore LA,Ahima RS, Khurana TS. 2002. Functional improvement of dystro-phic muscle by myostatin blockade. Nature 420:418–421.

Bruning JC, Michael MD, Winnay JN, Hayashi T, Horsch D, Accili D,Goodyear LJ, Kahn CR. 1998. A muscle-specific insulin receptorknockout exhibits features of the metabolic syndrome of NIDDMwithout altering glucose tolerance. Mol Cell 2:559–569.

Grobet L, Royo Martin LJ, Poncelet D, Pirottin D, Brouwers B, Riquet J,Schoeberlein A, Dunner S, Menissier F, Massabanda J, Fries R,Hanset R, Georges M. 1997. A deletion in the myostatin genecauses double-muscling in cattle. Nat Genet 17:71–74.

Grobet L, Poncelet D, Royo Martin LJ, Brouwers B, Pirottin D, MichauxC, Menissier F, Zanotti M, Dunner S, Georges M. 1998. Moleculardefinition of an allelic series of mutations disrupting the myostatinfunction and causing double-muscling in cattle. Mamm Genome9:210–213.

Gu H, Zou YR, Rajewsky K. 1993. Independent control of immuno-globulin switch recombination at individual switch regions evi-denced through Cre-loxP-mediated gene targeting. Cell 73:1155–1164.

Hanset R, Michaux C, Dessy-Doize C, Burtonboy G. 1982. Studies onthe 7th rib cut in double-muscled and conventional cattle. In: KingJWB, Menissier F, editors. Muscle hypertrophy of genetic originand its use to improve beef production. The Hague: MartinusNijhoff. p 341–349

Hogan B, Beddington R, Costantini F, Lacy E. 1994. Manipulating themouse embryo, a laboratory manual, 2nd ed. Cold Spring Harbor,NY: Cold Spring Harbor Laboratory Press.

Kambadur R, Sharma M, Smith TPL, Bass JJ. 1997. Mutations in myo-statin (GDF8) in double-muscled Belgian Blue Cattle. Genome Res7:910–916.

Lee S-J, McPherron AC. 2001. Regulation of myostatin activity andmuscle growth. Proc Natl Acad Sci USA. 98:9306–9311.

Lin J, Arnold HB, Della-Fera MA, Azain MJ, Hartzell DL, Baile CA. 2002.Myostatin knockout in mice increases myogenesis and decreasesadipogenesis. Biochem Biophys Res Commun 291:701–706.

McPherron AC, Lee J. 1997. Double muscling in cattle due to mutationsin the myostatin gene. Proc Natl Acad Sci USA 94:12457–12461.

McPherron AC, Lee S-J. 2002. Suppression of body fat accumulation inmyostatin-deficient mice. J Clin Invest 109:595–601.

McPherron AC, Lawler AM, Lee SJ. 1997. Regulation of skeletal musclemass in mice by a new TGF� superfamily member. Nature 387:83–90.

Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. 1993.Derivation of completely cell culture-derived mice from earlypassage embryonic stem cells. Proc Natl Acad Sci USA 90:8424–8428.

Needleman SB, Wunsch CD. 1970. A general method applicable to thesearch for similarities in the amino acid sequence of two proteins.J Mol Biol 48:443–453.

Sambrook J, Russel DW. 2001. Molecular cloning, a laboratory manual, 3rded. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Schmalbruch H, Hellhammer U. 1977. The number of nuclei in adult

FIG. 7. Schematic representation of a cross-section of the widestpart of the lower leg. The muscle groups considered in this study arethe “tibialis cranialis” group (T.C.), the “gastrocnemius plantaris”group (G.P), the “adductor” and the “biceps femoris” (B.F.). Approx-imate locations of the areas in which individual myofiber area wasrecorded are indicated: a) tibialis cranialis, b) extensor digitorumlongus, c) extensor digitorum lateralis, d) peroneus longus, e) gas-trocnemius caput lateralis, f) flexor digitorum longus, g) gastrocne-mius caput mediale.

237CONDITIONAL MSTN KNOCKOUT

rat muscles with special reference to satellite cells. Anat Rec189:169–175.

Sharma M, Kambadur R, Matthews KG, Somers WG, Devlin GP, Cona-glen JV, Fowke PJ, Bass JJ. 1999. Myostatin, a transforming growthfactor beta superfamily member, is expressed in heart muscle andis upregulated in cardiomyocytes after infarct. J Cell Physiol 180:1–9.

Szabo G, Dallmann G, Muller G, Patthy L, Soller M, Varga L. 1998. Adeletion in the myostatin gene causes the compact (Cmpt) hyper-muscular mutation in mice. Mamm Genome 9:671–672.

Torres RM, Kuhn R. 1997. Laboratory protocols for conditional genetargeting. New York: Oxford University Press.

Utomo AR, Nikitin AY, Lee WH. 1999. Temporal, spatial, and celltype-specific control of Cre-mediated DNA recombination in trans-genic mice. Nat Biotechnol 17:1091–1096.

Wegner J, Albrecht E, Fielder I, Teuscher F, Papstein H-J, Ender K.

2000. Growth- and breed-related changes of muscle fiber charac-teristics in cattle. J Anim Sci 78:1485–1496.

Wu S-Q, Hopfner RL, McNeill JR, Wilson TW, Gopalakrishnan V. 2000.Altered paracrine effect of endothelin in blood vessels of thehyperinsulinemic, insulin resistant obese Zucker rat. CardiovascRes 45:994–1000.

Yang J, Ratovitski T, Brady JP, Solomon MB, Wells KD, Wall RJ. 2001.Expression of myostatin pro domain results in muscular transgenicmice. Mol Reprod Dev 60:351–361.

Zhu X, Hadhazy M, Wehling M, Tidball JG, McNally EM. 2000. Domi-nant negative myostatin produces hypertrophy without hyperpla-sia in muscle. FEBS Lett 474:71–75.

Zimmers TA, Davies MV, Koniaris LG, Haynes P, Esquela AF, Tomkin-son KN, McPherron AC, Wolfman NM, Lee S-J. 2002. Induction ofcachexia in mice by systemically administered myostatin. Science296:1486–1488.

238 GROBET ET AL.