Phenotypic variability and incomplete penetrance of spontaneous fractures in an inbred strain of transgenic mice expressing a mutated collagen gene (COL1A1)

(1)

Phenotypic variability and incomplete

penetrance of spontaneous fractures in an

inbred strain of transgenic mice expressing a

mutated collagen gene (COL1A1).

R Pereira, … , J S Khillan, D J Prockop

J Clin Invest.

1994;

93(4)

:1765-1769.

https://doi.org/10.1172/JCI117161

.

Phenotype variability and incomplete penetrance are frequently observed in human

monogenic diseases such as osteogenesis imperfecta. Here an inbred strain of transgenic

mice expressing an internally deleted gene for the pro alpha 1(I) chain of type I procollagen

(COL1A1) was bred to wild type mice of the same strain so that the inheritance of a fracture

phenotype could be examined in a homogeneous genetic background. To minimize the

effects of environmental factors, the phenotype was evaluated in embryos that were

removed from impregnated females 1 d before term. Examination of stained skeletons from

51 transgenic embryos from 11 separate litters demonstrated that approximately 22% had a

severe phenotype with extensive fractures of both long bones and ribs, approximately 51%

had a mild phenotype with fractures of ribs only, and approximately 27% had no fractures.

The ratio of steady-state levels of the mRNA from the transgene to the level of mRNA from

the endogenous gene was the same in all transgenic embryos. The results demonstrated

that the phenotypic variability and incomplete penetrance were not explained by variations

in genetic background or levels in gene expression. Instead, they suggested that phenotypic

variation is an inherent feature of expression of a mutated collagen gene.

Research Article

Find the latest version:

http://jci.me/117161/pdf

(2)

Phenotypic

Variability

and

Incomplete

Penetrance

of

Spontaneous Fractures

in

an

Inbred

Strain

of

Transgenic Mice Expressing

a

Mutated

Collagen

Gene

(COLlAl)

Ruth Pereira, Kenneth Halford, Boris P. Sokolov, Jaspal S. Khillan, and Darwin J. Prockop

DepartmentofBiochemistry and Molecular Biology, Jefferson Institute ofMolecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Abstract

Phenotype variability and incomplete penetrance are fre-quently observed in humanmonogenic diseases such as

osteo-genesis imperfecta. Here an inbred strainoftransgenicmice

expressinganinternallydeleted geneforthe proal(I)chainof

typeIprocollagen

(COLIAl)

wasbred towild type mice of the

same strain so that the inheritance ofa fracture phenotype could beexamined inahomogeneous genetic background. To minimize theeffects ofenvironmental factors, the phenotype wasevaluated in embryos that were removedfrom impregnated females1dbefore term. Examinationofstained skeletons from 51 transgenic embryosfrom 11 separate litters demonstrated that - 22% hada severephenotype withextensivefracturesof both long bones andribs, - 51% hada mild phenotype with fractures of ribs only, and - 27% had nofractures.Theratio of steady-state levels of the mRNAfromthe transgene to the level

ofmRNAfromtheendogenousgenewasthesamein all

trans-genic embryos.The results demonstrated that the phenotypic

variability and incomplete penetrance were not explained by variations ingenetic background or levels in gene expression. Instead, they suggested thatphenotypic variationisaninherent featureofexpression ofamutatedcollagen gene.(J. Clin.

In-vest. 1994.93:1765-1769.) Key words: osteopenia * gene

ex-pression* predisposition to fractures

Introduction

Phenotypic variability and incomplete penetrance are fre-quently observedinmonogenic diseasesin humans (see

refer-ences 1 and2). Thephenomenaareusuallyexplained by

dif-ferencesingenetic backgroundorenvironmental factors. How-ever, thesamephenomenaarefrequentlyseenwith mutations insimpler organisms with homogeneous geneticbackgrounds and in whom environmental factors can be rigorously

con-trolled. Forexample,twodifferent dominant negativealleles of

let-60locus in Caenorhabitis

elegans

produced variable

pene-Address correspondence to Dr.Darwin J. Prockop, Department of

Bio-chemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University,

Philadelphia,PA 19107.

Receivedfor publication 16August 1993 andin revised form 10 December 1993.

trance of avulvalessphenotype so that about halfof the

prog-eny who inherited one of the mutated alleles were eggless, whereas abouthalf were not (3). As another example, similar marked variations inphenotypewere seen inthe same

organ-ism with several mutations in the unc-5, unc-6, and unc-40 genesthatguide circumferential migrationsofpioneeraxons

and mesodermal cells (4).

Ofspecial interestto ushas been thephenotypic variation andincomplete penetrance seen in somefamilies with

osteo-genesisimperfecta.Forexample, de Vries anddeWet(5)

re-portedon afamilyin which three affected members of thefirst

and third generationswere of short stature and hadmultiple fractures and skeletal deformities. The family member from the secondgenerationwho transmitted thedisease, however, wasof normal stature and had no evidence of either fractures

or skeletal deformities.Similarly, Sippolaetal.(6)described a

largethree-generation familyinwhich nine affected members had a variable phenotype in thatsomehadonlyslightlyshort

stature and bluesclerae, whereas others had multiplefractures

and permanent dislocations of large joints.

Toexplore the causes ofincompletepenetranceand pheno-typic variationseenwith mutatedcollagen genes,weexamined

herean inbred strain oftransgenicmice (7, 8)expressingan

internally deleted gene for the proal (I) chain of typeI procol-lagen(COL1 A 1). The construct was designedsoas to cause

synthesis ofshortened proaI(I) chains that associated with

normal proa1(I) chains from the endogenous mouse gene. Theassociation ofthechainscauseddegradationofboth the normal and the mutated chainsthrough a phenomenon

re-ferredto asprocollagensuicideoras adominant negativeeffect (9). Aspreviouslyreported(7,8), lines of miceexpressing high

levelsoftheinternallydeleted genedeveloped a lethal pheno-type with multiple fractures and other features similarto pro-bands with lethal variants ofosteogenesis imperfecta.Linesof

miceexpressinglowerlevelsof thesamegenewereeither phen-otypicallynormalorhadamilder phenotype. The results here

demonstratethataninbred line oftransgenicmiceexpressinga

moderate levelof the mutated geneproducesavariable

pheno-type of fractureseveninembryosexamined inutero.

Methods

Preparation andbreeding oftransgenic mice. Transgenicmice were prepared inaninbredstrain of mice (FVB/N)asdescribedpreviously

(7). To identify transgenic mice, genomicDNA wasextractedfrom tissuesbydigestionwithproteinase K,extraction with phenol:chloro-form:isoamyl alcohol, and ethanol precipitation. Because the copy number in thelinewas>15, the presenceorabsenceof the transgene

wasreadily detectedby slot blottingof theDNA with aprobe that consisted of the nick-translated 32P-radiolabeled transgene(7).

J.Clin. Invest.

0021-9738/94/04/1765/05 $2.00

(3)

Skeletalstaining. To define the phenotypes of thetransgenic mice,

viscera and skin were removed and the mouse carcasses were fixed in 95% ethanol for 3 d. The samples were thendehydratedin 100% eth-anol for 2 d. Theywereimmersed in 1% KOH for 1 dtodissolve the soft tissues and the skeletonswerestained with 1% KOHcontaining 0.001%Alizarin red S for 1 d (10). The samples were incubated with 25, 50, and 80%glycerolfor 1 d each and stored in 100%glycerol.The presence of fractures in the skeletons was assessed by light microscopy. Assaysof levels ofexpression. To assay the steady-state levelsof

mRNA,total cellular RNAwasisolated from tissues by extraction with acidicguanidiniumthiocyanate-phenol-chloroform( 1 1). Theamount

ofmRNA from the transgene relativetothe mouse mRNAfrom

endog-enous COL 1 Al genewasmeasured byaquantitative reverse

transcrip-tase(RT)-PCRmethod ( 12 ). 1-5 ugof total RNAwasreverse

tran-scribed in 20Mlof reaction mixture using primer BS33 (5'-ATCAAG-TTTGA'3') (200pmol)for the proa l (I) chain and apreamplification system for first-strand cDNAsynthesis (SuperScript<>,GIBCO BRL,

Gaithersburg, MD). Thesamplewastreated with RNase H and the cDNAwasamplifieddirectly usingaPCR reagent kit(GeneAmpo,

Perkin-Elmer Cetus,Cochranville,PA), and primers BS31 (5'-TTG-GCCCTGTCTGCTT-3') and BS32

(5'-TGAATGCAAAGGAAA-AAAAT-3') using4pmolof each in a

100-Al

reaction mixture (12). One of theprimersused in the PCRwas5' end labeled with[32PdATP using T4 polynucleotide kinase terminus labeling system (GIBCO

BRL). PCRconditionswere 80 s at940C, I min at470C,and 20 s at

720Cfor 15cycles.After thePCR,

5Al

ofreaction mixturewasdigested

with 12UofBstNIfor 15hat 60'C. 10

Al

ofthe product was denatured andsubjectedtoelectrophoresisin 15% PAGEcontaining6 M urea. Thegelwasfixed anddried.Theappropriatebands on the gel were then assayed byexposingthegel toaphotostimulable storagephosphor

imaging plate (400SPhosphorlmager; Molecular Dynamics, Sunny-vale, CA). As reported previously ( 12), the assay was linear when carriedoutwith standard samples in which the ratio of mRNA from the transgene and the endogenous gene varied from 0.1 to1.0.

Assayofmineralcontentof embryos.After thestainedskeletons were evaluatedmicroscopically,the same samples were taken for assay

ofmineralcontent.Thesampleswerewrappedin aluminum foil and reduced to mineral byheatingat600Cin an insulated oven (Type 1400 furnace;Barnstead-Thermolyne; Dubuque, IA) for 18 h, after which they were weighed again. Because the small amounts of glycerol in the samplesignitedduring the first few minutes at 600C, the oven

wasplaced in a fume hood. Preliminary experiments indicated there wasnofurther decrease in weight if samples were incubated at600C for>18h.

1 ~~~~2

IV

II 0a

123 4 5 6~~~1

III

IV

$it>

,

Results

Evaluation of phenotype

in transgenic mice. The

transgenic

micewerepreparedwith aninternally deleted construct of the humangenefor theproa 1

(I)

chain oftype Iprocollagen (9).

The geneconstruct contained 2.3kbofthepromoter and2 kb

of the 3

'-flanking

sequence.Intron 5

of

the genewas

joined

to

intron 46sothat therewas anin-frame

deletion

ofexons6-46.

The line selected for study here (line V in reference 7)

ex-pressed moderate levels of the transgene.

Ultrastructural

data

onthe linewererecently

reported

(13).

Toobtain embryos for

analysis

here,

transgenic

malesfrom

generations

III,

IV,and V(Fig.

1)

werebredtowildtype

fe-males from the same inbred strain. Embryos were removed from

impregnated

females 18dafter the vaginal plugwasseen

and,

therefore,

1dbefore

expected

normal delivery. The

skele-tons were

stained

and

examined microscopically.

Some ofthe

mice hadasevere phenotype with

extensive

fractures of both long bones and ribs (Fig.2

left).

Other micehad amild

pheno-typewith fractures of ribsbut not anylongbones

(Fig.

2

right).

Still other mice hadno evidence of fractures.Asindicated in

Table

I,

51

transgenic

embryos from 11 separate litterswere

examined.

About22% hadtheseverephenotype, - _{51% had}

the mild

phenotype,

and - 27% hadanormal

phenotype.

Level of

expression

ofthe

transgene.To

determine

whether the

variability of

phenotypewasrelatedtolevel of

expression

in thetransgene, mRNA from tissues of the embryoswas

assayed

bya

quantitative

RT-PCR method. The method used identical

primers

for both the human transgene and the endogenous

mouse gene, but DNA fragments of different size were

ob-tained after

cleavage with the

restriction endonuclease

BstNI

(12).Asindicated in Fig. 3, the fragments fromthePCR

prod-uctsfrom the transgene and the endogenousgene werereadily

distinguished

by

polyacrylamide

gel

electrophoresis.

Theratio of thetwo fragmentswas used as a measure of the level of

expression

ofthe transgene

relative

to

expression

ofthe

endoge-nousgene.Asindicated in Table II, there were no

significant

differencesin the ratios of themRNAs amongmice that had

theseverephenotype, the mild phenotype, andanormal

pheno-type.

Figure1.Scheme for matings of the

transgenicmice. Forexamination

ofphenotypic

variation

in 18-d-old

transgenicembryos, cesarean

sec-tionswereperformedonthemating

(4)

jf,

rt

.... (S ....

W-S

%.1

"P

k

*^S

*A 5

low

.\

and_ffike

F

Figure 2. Stained skeletons from18-d-oldtransgenic embryos. (Left) Embryowith fracturesof both ribs andlongbones.(Right)Embryowith

fractures of ribsonly.

Previous experiments (8) demonstrated thatthe mineral

content offemurs from 6-wk-old transgenic mice of the line

was reduced to 68% ofthe values for control littermate (P

<0.01).Here,thesameskeletons usedtoevaluate the

pheno-type were subsequently assayed for mineral content. There

were nosignificant differencesbetweenembryos withno

frac-tures,those withthe mildphenotype, and those with thesevere

phenotype(Table II). The standard deviations forthemean

values, however,wererelatively large.

Effect of uterine position. One possible explanation forthe

variability in phenotype was theuterine position ofthe

em-bryos. Therefore, the severity of the phenotypewasexamined as a function ofwhether the embryos were at the tip of the

uterinehorn,inthe middle, or nearthe base. Asindicated in Table III, there was only a slight effect of uterine position.

Embryos with the mild and severe phenotypes were about

equally distributedinthe uterine horns. Embryos witha

nor-mal phenotypewere located primarilyat the base and lower

regionsof theuterus.

Discussion

Phenotypicvariation andincompletepenetrancein

transmis-sion ofmonogenicdiseases presentperplexing problemsin

deal-ingwith the molecularetiologyof human diseases.When

en-countered withinfamilies, theyalsopresentdifficultproblems

ingenetic counseling.Inosteogenesis imperfecta,marked phe-notypic variation has been seen among affected members of

some families (5, 6). The phenotype variability reportedin

families withosteogenesis imperfectaisgenerallylessthanseen

here with transgenic mice, but data are available only on a limited number ofvery large families, and most parents of

children withsevereformsofthe disease elect not to have

addi-tional children. Also, markedphenotypic variation has been *o

a.

-S.4

a*

?I/

%M.O-'--48qw

.0-*"""w

-

t,!Im-,.V.

.0-W

/I

I

N

(5)

TableI.Phenotypes

of

18-day-oldTransgenic Embryos

Phenotypes of transgenic embryos Severe

(limb and Mild(rib

Matings* Litters rib fractures) fractures) Nofractures

tgIII-2 xWt 1 2 1

tgIII-4XWt 1 1 1 1

tgIV-2XWt 2 4 5 4

tgIV-5XWt 1 1 1

tgIV-7XWt 1 2 2 2

tgIV-OxXWt 2 2 6 2

tgV-2xWt 1 1 3 1

tgV-3XWt 1 2 2

tgV-6XWt 1 1 4

Totals 11 11 26 14

*_{Transgenic mice}

(tgIII-2,

_etc.)_identified_as_{indicated in Fig.} _{1. Wt,}

wild type mice from the same inbred strain (FVB/N).

seen incomparingtheeffects of similarmutations in the same gene(14, 15). Forexample, somesingle-base mutations that replacecodonsfor glycine withcodonsforbulkier amino acids produce lethal phenotypes, whereas othersimilar glycine

sub-stitutions produce phenotypessomild theyaredifficultto dis-tinguish from postmenopausal osteoporosis (16). Although > 150suchglycine substitutionshave been analyzed, no sim-plerelationshiphas been apparent between theseverityof the phenotype and the nature ofthe amino acid substituted for glycineorthelocationofthesubstitutionamong the 338

obli-gate glycine residues in each of the proa 1(I) and proa2(I)

chainsoftype Iprocollagen ( 14, 15 ).

Breeding lines oftransgenic mice expressing a mutated col-lagen geneprovideanopportunitytodeterminewhether or not

phenotypic variation produced by expression of a mutated gene can be explained by variations in genetic background.

Previously, the line oftransgenic mice examined here was shown toproduceavariablephenotypeinthat - 6% of new-born transgenic pups had a lethal phenotype with extensive

Transgenic

C

N

a

M

s

som

_M

"m O

"o

-M-Transgenic

C

N M

S

M

. . * * s

_

_{__"}

_{_.*0-}

--Figure 3. Assay ofexpression of thetransgeneand theendogenous

geneforproa I(I) chains oftype Iprocollagen. Theassay wascarried

outbyaquantitativeRT-PCRassaydescribed in thetext.Results fromtwoindependent experimentsarepresented. C, controlmouse; N,transgenicembryo with normal phenotype; S, transgenic embryo witha severephenotype;M,transgenic embryo withamild

pheno-type;H,fragmentfrom the humantransgene;M,fragment from the endogenousmousegene.

TableII. BoneMineralContentandSteady-StateLevelsof

mRNAfromMutatedExogenous COLJAIGene andEndogenous COLIAJ Gene in18-d-old Embryos

Ratio of mRNAs (exogenous/endogenous)* Transgenic

phenotype Tail Skin Mineral content* mg

Normal 0.61±0.20 0.44±0.25 6.69±1.55

(n= 12) (n= 11) (n= 13) Mild 0.58±0.19 0.36±0.17 6.99±1.60

(n = 13) (n = 12) (n =23)

Severe 0.55±0.12 0.51±0.28 6.01±0.70 (n=7) (n =6) (n =7)

*Values aremeans±standarddeviation(n=_{number of}_samples).

fractures, - _{33% had a moderate phenotype with fractures,}

and theremaining mice had no apparent fractures (8). The

phenotypic variationwasobserved even though thetransgenic miceweregenerated inaninbred strainand thelinewas propa-gated in the same inbred strain in which the genetic back-ground can beassumedtobe homogeneous by the usual

crite-rion that thestrainwaspreviously maintained for>20

genera-tions of brother-to-sistermatings ( 17).Inparallel experiments

( 18), we observedsimilar variability in expression ofa pheno-typeof cleft palate andskeletal changes similartothose seen withchondrodysplasiasin an inbred transgenic lineexpressing

amutatedgenefortype IIprocollagen (COL2A1).Therefore,

thevariationsinphenotypeareunlikelytobeexplained bythe transgenesdisrupting loci that may modulate skeletal

pheno-types.

Onepossible explanationfor thevariabilityinthe fracture

phenotype previously seen in the transgenic mice expressing themutated COL1Al gene (8)was differences in incidental

trauma tothe miceduringbirth orshortlythereafter. Another

possible explanation wasdifferences in levels ofexpression of the transgene. Therefore,here wecarriedoutfurtherbreeding ofthetransgenic mice inthe sameinbred strainandexamined

embryos by caesarian section. The results demonstrated that the same

phenotypic

variation was present in 18-d-old

em-bryos.Aboutone-fifth oftheembryoshad a severephenotype withextensivefractures oflongbones andribs.Abouthalfhad

TableIII.Effect ofUterine Position onPhenotype

Uterineposition*

Base Abovethe base Transgenic

phenotype 1 2 3 4 5 6

Normal 5 4 3 1

Mild 4 4 8 3 7

Severe 2 1 3 1 2 2

*_{Embryos removed by the}_cesarean_section_were_{scored for}

pheno-type(Table I)anduterineposition.Thepositionsof embryos in the

uterus werenumbered from the lowestpositionin theuterus tothe

(6)

a mildphenotype with fractures only in ribs, and the remaining mice had no fractures. Thephenotypicvariation was not ex-plained byvariationsin the level ofexpression ofthetransgene inthemice.Asassayed by aquantitativeRT-PCR method, the level ofexpressionwasthe same in embryos withthe severe

phenotype, embryos with the mild phenotype, and embryos

withanormalphenotype. Also, there was only aslight effect of

uterine position on theseverity ofthephenotype inthat em-bryos with a normal phenotypewere found primarily in the base and the lowerportion of theuterine horn, butembryos

with themildand severephenotypeswere aboutequally distrib-uted.

Thevariations in phenotypeobserved here may have been

determined by subtle differences in micro-environment that were not related by thepositionofthe embryos in the uterus.

Alternatively, they may have been determined by stochastic eventsduring embryonic development thatwere notrelatedto the micro-environment. For example, stochastic events may haveproducedvariations in expression ofboththeendogenous gene andmini-genein someembryos withoutalteringthe min-eral contentof bone. Regardless ofthemolecular mechanisms, however,the resultsindicated that variabilityinphenotypewas aninherent feature of expression ofthe mutatedcollagengene eveninahomogeneousgenetic background.Theobservations, therefore, may be an

important

consideration in counseling families with

osteogenesis imperfecta, particularly

families in

whichthefirstaffected memberhas amild phenotype.

Acknowledgments

Supportedin partbygrants fromN.I.H.(AR-38 188),the Marchof

Dimes/Birth Defect Foundation, and the Lucille P. Markey Charitable Trust.

References

1.Lalouel, J. M., and R. White. 1990. Analysis ofGenetic Linkage. In Princi-plesand PracticeofMedical Genetics, Vol. 1. A. E. H. Emery, D. L. Rimoin, J. A. Sofaer, I. Black, and V.A.McKusick,editors.Churchill-Livingston Inc., New York.149-164.

2.Ott, J. 1991. Penetrance.InAnalysisof Human GeneticLinkage.Revised Edition. The JohnsHopkins UniversityPress, Baltimore. 146-164.

3. Han, M., R.V.Aroian,andP. W.Sternberg.1990. Thelet-60locus controls theswitch between vulval and nonvulval cell fates inCaenorhabditiselegans. Genetics. 126:899-913.

4.Hedgecock,E.M., J.G.Culotti, and D. H. Hall. 1990. The unc-5, unc-6,

and unc-40genesguidecircumferential migrations ofpioneeraxonsand

meso-dermal cellsontheepidermis in C. elegans. Neuron. 4:61-85.

5. de Vries, W. N., and W. J. deWet.1987.Cysteine ina Ichains of human

typeIcollagen producesaclinical heterogeneous form ofosteogenesisimperfecta.

InUCLASymposiumonMolecularBiologyand CellularBiology:NewSeries,

Vol 46.A.Sen,and T. Thornhill, editors. Liss, New York. 56-64.

6.Sippola,M.,S.Kaffe,and D. J.Prockop.1984. Aheterozygousdefect for

structurallyalteredproa2chains oftypeI procollagen inamildvariant of

osteo-genesis imperfecta. The alteredstructuredecreases the thermal stability of procol-lagen and makes it resistant to procollagen N-proteinase. J. Biol. Chem. 259:14094-14100.

7.Khillan, J. S.,A.S. Olsen, S. Kontusaari, B. Sokolov, and D.J.Prockop. 1991.Transgenicmice thatexpressamini-geneversionof the humangenefor

typeIprocollagen (COLIAI ) developaphenotype resemblingalethal form of osteogenesisimperfecta. J. Biol. Chem. 266:23373-23379.

8.Pereira, R., J. S. Khillan, H. J. Helminen, E. L. Hume, and D. J.Prockop.

1993.Transgenicmiceexpressingapartiallydeletedgenefortype Iprocollagen

(COLIAI ). A breeding line withaphenotype ofspontaneousfractures and de-creased bonecollagenandmineral. J. Clin. Invest. 91:709-716.

9.Olsen, A. S., A. E. Geddis,andD. J. Prockop. 1991.High levelsof

expres-sion ofaminigene version of the humanproa I(I) collagengenein stably

trans-fectedmousefibroblasts. Effects of deleting putative regulatorysequencesin the firstintron. J. Biol. Chem. 266:1117-1121.

10.Peters, P. W. J. 1977. Double staining of fetal skeletons for cartilage and bone.In Methods in Prenatal Toxicology. D. Neubert, H.-J. Merker, and T. E. Kwasigroch, editors. Georg Thieme Verlag, Stuttgart. 153-154.

1 1.Chomzynski, P., and N. Sacchi. 1987. Single-step method ofRNA isola-tionby acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Bio-chem. 162:156-159.

12.Sokolov, B. P., P.K.Mays,J.S.Khillan, and D.J.Prockop. 1993. Tissue-anddevelopment-specific expression in transgenic mice in thetypeIprocollagen

(COLAIA ) mini-geneconstructwith 2.3kb of thepromoterregion and 2 kb of the3-flanking region. Specificity is independent of putative regulatorysequences

of thefirst intron. Biochemistry. 32:9242-9249.

13.Cassella, J. P., R. Pereira, J. S. Khillan,D. J.Prockop, N. Garrington, and S. Y.Ali. 1994. An ultrastructural, microanalytical and spectroscopic study ofa

transgenicmousemodelfor osteogenesis imperfecta. J. Bone. Inpress.

14.Kuivaniemi, H., G. Tromp, andD.J.Prockop. 1991.Mutations in

colla-gen colla-genes: causesofrareandsome commondiseases in humans. FASEB (Fed. Am.Soc. Exp. Biol.) J. 5:2052-2060.

15.Byers, P. H., G.A.Wallis, and M. C. Willing. 1991. Osteogenesis imper-fecta: translation of mutation into phenotype. J. Med. Genet. 28:433-442.

16.Spotila, L. D., C.D.Constantinos,L.Sereda, A. Ganguly, B. L. Riggs, and D.J.Prockop. 1991. Mutations inthegenefor typeIprocollagen (COLIA2)in a

woman with post-menopausal osteoporosis: evidence for a phenotypic and

geno-typic overlap with mildosteogenesis imperfecta. Proc. Nati. Acad. Sci. USA. 88:5423-5427.

17.Hogan, B.,F.Constantini, and E. Lacy. 1986. Manipulating the Mouse Embryo:ALaboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,NY. 7pp.

18.Helminen, H. J.,K.Kiraly,A.Pelttari, M. I. Tammi,P.Vandenberg, R. Pereira,R.Dhulipala,J. S.Khillan, L. Ala-Kokko, E.L.Hume,B.P. Sokolov, andD. J. Prockop. 1993. An inbred line of transgenic mice expressingan inter-nallydeletedgenefortypeIIprocollagen (COL2A 1 ). Young mice haveavariable phenotype ofachondrodysplasiaand older micehave osteoarthritic changes in