Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro alpha 1(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a beta-galactosidase reporter gene. A construction containing a 3,000 bp promoter and a 3,020 bp intron 1 fragment directed high levels of beta-galactosidase expression specifically to chondrocytes. Expression of the transgene coincided with the temporal expression of the endogenous gene at all stages of embryonic development. Successive deletions of intron 1 delineated a 182 bp fragment which targeted beta-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. Transgenic mice harboring a 309 bp Col2a1 promoter lacking intron 1 tester sequences showed no beta-galactosidase expression in chondrocytes. Reduction of the 182 bp fragment to a 73 bp subfragment surrounding a decamer sequence previously reported to be involved in chondrocyte specificity, resulted in loss of transgene expression in chondrocytes. When the Col2a1 promoter was replaced with a minimal beta-globin promoter, the 182 bp intron 1 sequence was still able to target expression of the transgene to chondrocytes. We conclude that a 182 bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression on a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression.

REFERENCES

Cheah
K. S.
,
Lan
E. T.
,
Au
P. K.
,
Tam
P. P.
(
1991
).
Expression of the mouse1(II) collagen gene is not restricted to cartilage during development.
Development
111
,
945
953
Friedrich
G.
,
Soriano
P.
(
1991
).
Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.
Genes Dev
5
,
1513
1523
Garofalo
S.
,
Vuorio
E.
,
Metsäranta
M.
,
Rosati
R.
,
Toman
D.
,
Vaughan
J.
,
Lozano
G.
,
Mayne
R.
,
Ellard
J.
,
Horton
W.
,
de Crombrugghe
B.
(
1991
).
Reduced amount of cartilage collagen fibrils and growth plate anomalies in transgenic mice harboring a glycine-to-cysteine mutation in the mouse type II procollagen1-chain gene.
Proc. Nat. Acad. Sci. USA
88
,
9648
9652
Horton
W.
,
Miyashita
T.
,
Kohno
K.
,
Hassell
J. R.
,
Yamada
Y.
(
1987
).
Identification of a phenotype-specific enhancer in the first intron of the rat collagen II gene.
Proc. Nat. Acad. Sci.USA
84
,
8864
8868
Lawn
R. M.
,
Efstratiadis
A.
,
O'Connell
C.
,
Maniatis
T.
(
1980
).
The nucleotide sequence of the human-globin gene.
Cell
21
,
647
651
Lee
B.
,
Vissing
H.
,
Ramirez
F.
,
Rogers
D.
,
Rimoin
D.
(
1989
).
Identification of the molecular defects in a family with spondyloepiphyseal dysplasia.
Science
244
,
978
980
Lefebvre
V.
,
Garofalo
S.
,
Zhou
G.
,
Matsäranta
M.
,
Vuorio
E.
,
de Crombrugghe
B.
(
1994
).
Characterization of primary cultures of chondrocytes from type II collagen/-galactosidase transgenic mice.
Matrix Biol
14
,
329
335
Mercer
E. H.
,
Hoyle
G. W.
,
Kapur
R.
,
Brinster
R. L.
,
Palmiter
R. D.
(
1991
).
The dopamine-hydroxylase gene promoter directs expression of E. coli LacZ to sympathetic and other neurons in adult transgenic mice.
Neuron
7
,
703
716
Metsäranta
M.
,
Toman
D.
,
de Crombrugghe
B.
,
Vuorio
E.
(
1991
).
Mouse type II collagen gene. Complete nucleotide sequence, exon structure and alternative splicing.
J. Biol. Chem
266
,
16862
16869
Pourquie
O.
,
Caltey
M.
,
Teillet
M.
,
Ordahl
C.
,
Le Douarin
N. M.
(
1993
).
Control of dorsoventral patterning of somitic derivatives by notochord and floor plate.
Proc. Nat. Acad. Sci. USA
90
,
5242
5246
Sandell
L. S.
,
Morris
N.
,
Robbins
J. R.
,
Goldring
M. B.
(
1991
).
Alternatively spliced type II procollagen mRNA define distinct populations of cells during vertebral development: differential expresssion of the amino-propeptide.
J. CellBiol
114
,
1307
1319
Savagner
P.
,
Miyashita
T.
,
Yamada
Y.
(
1990
).
Two silencers regulate the tissue-specific expression of the collagen II gene.
J. Biol. Chem
265
,
6669
6674
Southern
E.
(
1975
).
Detection of specific sequences among DNA fragments separated by gel electrophoresis.
J. Mol. Biol
98
,
503
517
Vikkula
M.
,
Metsäranta
M.
,
Syvänen
A.C.
,
Ala-Kokko
L.
,
Vuorio
E.
,
Peltonen
L.
(
1992
).
Structural analysis of the regulatory elements of the type II procollagen gene.
Biochem. J
285
,
287
294
Wang
L.
,
Balakir
R.
,
Horton
W. E.
Jr.
(
1991
).
Identification of a cis-acting sequence in the collagen II enhancer required for chondrocyte expression and the binding of a chondrocyte nuclear factor.
J. Biol. Chem
266
,
19878
19881
Yee
S.
,
Rigby
P. W. J.
(
1993
).
The regulation of myogenin gene expression during the embryonic development of the mouse.
Genes Dev
7
,
1277
1289
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