BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
233, 227–230 (1997)
ARTICLE NO.
RC976432
Ontogeny of Glutamic Acid Decarboxylase Gene
Expression in the Mouse Pancreas
Jean-Marie Pleau,
1
Mark Throsby, Anne Esling, and Mireille Dardenne
CNRS URA 1461, Universite´ Paris V, Hoˆpital Necker, 161 rue de Se`vres, 75015 Paris, France
Received February 26, 1997
weeks of age, and GAD 67 specific T cells appear one
Using two RT–PCR quantitative assays, we mea-
week later (12-13), suggesting a role for GAD 67 during
sured the pancreatic expression of the two isoforms
the development of the autoimmune process leading to
of glutamic acid decarboxylase in the foetus, new-
diabetes.
born and 14-, 21- and 35-day-old male and female NOD
In previous studies, we developed two competitive
and C57BL/6 mice. In the C57BL/6 mouse, GAD 67 pan-
RT-PCR assays to measure the expression of the two
creatic expression is stable; in NOD mice, GAD 67 ex-
GAD isoforms in pancreases from 5-, 10- and 15-week-
pression is similar to that found in control mice, ex-
old male and female NOD, BALB/C and C57BL/6 mice
cept at 5 weeks of age, when pancreatic GAD 67 ex-
(14-15). Similarly, we measured the expression of the
pression is about 2.5 times higher than in C57BL/6
two GAD isoforms in brains from 5-week-old mice. In
mice. The pancreatic expression of GAD 65 is under
the mouse brain, the GAD 67 isoform was expressed
the detection limit of the assay until 5 weeks of age.
at levels 1000 times higher than GAD 65; however,
The overexpression of GAD 67 characterized in pan-
the expression of the two GAD isoforms was similar
creas from 5-week-old NOD mice could be the result
whatever the age, sex or strain studied (15). Con-
of
b
cell hyperactivity, previously reported in this
versely, in comparison with nonautoimmune mouse
mouse strain.
᭧ 1997 Academic Press
strains, we observed a sexual dimorphism for GAD 65
expression in NOD mice (14) and an overexpression of
GAD 67 in the pancreas (15).
In the present study, we analyzed the pancreatic ex-Insulin dependent diabetes mellitus (IDDM) is a ge-
netically influenced autoimmune disease in which in- pression of GAD 67 in male and female foetuses at 18
days of gestation, in newborns and in 14-, 21- and 35-sulin producing cells in pancreatic islets are progres-
sively destroyed (1). Several
b
cell autoantigens have day-old NOD and C57BL/6 mice. We also measured
GAD 65 expression in the same samples.been described, such as insulin (2), carboxypeptidase-
H (3), heat shock protein (4), peripherin (5), tyrosine
phosphatase (6) and glutamic acid decarboxylase
MATERIALS AND METHODS
(GAD) (7). GAD is an enzyme responsible for the pro-
duction of the neurotransmitter
g
-amino butyric acid
(8). It exists in two forms which differ by their molecu-
Animals
lar weight, GAD 65 and GAD 67 (9). The two GAD
NOD and C57BL/6 mice used in this study were obtained from
isoforms are encoded by two non-allelic genes (10).
our locally inbred colonies maintained under specific pathogen-free
Nonobese diabetic (NOD) mice spontaneously develop
conditions.
diabetes and serve as a model for human type I diabetes
(11). Previous studies have demonstrated that an im-
Preparation of RNA
mune response to GAD develops in NOD females, con-
current with the onset of insulitis. Autoreactive T cells
Total RNA was isolated from each mouse pancreas and from pools
specific to GAD 65 have been characterized at three
of four pancreases from foetuses and from newborn mice by the gua-
nidium isothiocyanate/cesium chloride method (16). RNA concentra-
tion andintegrity were estimated by spectrophotometry and by dena-
turant gel electrophoresis, respectively. Prior to expression analysis,
1
To whom correspondence should be adressed. Fax: 33 (1) 44 49
06 76. E-mail: dardenne@infobiogen.fr. RNA preparations were submitted to RT-PCR using specific primers
for mouse
b
actin (table I) and only sample preparations with similarAbbreviations: GAD: glutamic acid decarboxylase; stRNA: stan-
dard RNA; RT: reverse transcription; PCR: polymerase chain reac- levels of
b
actin amplicon were used for the competitive RT-PCR
assays.tion; NOD: nonobese diabetic.
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