Testing of SHIRPA, a mouse phenotypic assessment protocol, on
Jill A. Rafael,
Kay E. Davies
Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
Mammalian Genetics Unit, Medical Research Council Harwell, Didcot, Oxfordshire OX11 ORD, UK
Received: 5 August 1999 / Accepted: 14 April 2000
Abstract. The SHIRPA protocol was proposed as a rapid, com-
prehensive screening method for qualitatively abnormal pheno-
types in the mouse (Rogers et al., Mamm Genome 8, 711, 1997).
This screening technique is currently being used to identify mu-
tants induced by N-ethylnitrosourea (ENU) mutagenesis (Brown
and Nolan, Hum Mol Genet 7, 1627, 1998). SHIRPA can be used
to identify mutants with neuromuscular abnormalities, but the sen-
sitivity of the protocol is unknown. We tested two dystrophin-
deficient mutants Dmd
, both of which are in-
distinguishable from wild-type by a simple visual assessment, at
different ages, using the primary screen of the SHIRPA protocol.
The most dramatic observation was that both Dmd
mice showed extreme fatigue after testing, while mice
from the same C57BL strains appeared unaffected. Each strain of
dystrophin-deficient mice showed a different profile in locomotor
activity and deficiencies in the wire maneuver, righting reflex, and
negative geotaxis tests. Furthermore, the wire maneuver test indi-
cated an earlier onset of muscular impairment in Dmd
mice. These data suggest that the SHIRPA primary
screen is effective not only in identifying subtle neuromuscular
mutants, but also in distinguishing qualitative differences between
mutants with neuromuscular abnormalities.
mouse was initially identified as a spontaneous mu-
tant in a colony of C57BL/10 mice by increased serum pyruvate
kinase levels (Bulfield et al. 1984). These mice possess a mutation
in the 5Ј coding region of the dystrophin gene that affects the
expression of only the full-length dystrophin protein which is nor-
mally expressed in skeletal muscle and at low levels in brain
(Hoffman et al. 1987; Chelly et al. 1990). Dmd
/Y skeletal muscle shows large amounts of degenerating
fibers accompanied by lymphocytic infiltration, newly regenerated
fibers, and finally the replacement of muscle mass with connective
tissue (Bulfield et al. 1984). These changes result in a vast reduc-
tion of the normalized force produced from dystrophic muscles
compared with aged-matched wild-type controls (Cox et al., 1993;
Petrof et al. 1993). These physiological deficiencies increase with
age, yet these mice are still able to walk normally around their
cages under laboratory conditions up until two years of age (Lynch
et al. 1997). Dmd
mice show deficiencies in voluntary wheel
running exercise (Carter et al. 1995) and do not run as far or as fast
as age-matched C57BL/10 controls (Hayes and Williams 1996).
An elevated level of pyruvate kinase in serum and histopatholog-
ical muscle lesions initially identified the Dmd
et al. 1984); however, these assays are time-consuming and require
sophisticated measuring equipment.
mouse was identified by elevated levels of
muscle proteins in serum in a screen for X-linked muscular dys-
trophy mutations after an ENU mutagenesis procedure (Chapman
et al. 1989). This mouse strain has a mutation in the 3Ј coding
region of the dystrophin gene that disrupts expression of all iso-
forms of the protein (Cox et al., 1993), including Dp71 (71 kilo-
dalton Dystrophin protein) that is expressed at high levels in brain
and other non-muscle tissues (Rapaport et al. 1992). Dmd
mice have been suggested to have difficulty rearing their young,
possibly owing to mental impairment (Cox et al., 1993). Recent
behavioral studies suggest that the Dmd
mice do not show
any differences from controls in long-term potentiation or in radial
maze experiments (Vaillend et al. 1998).
The SHIRPA primary screen is designed to provide a behav-
ioral and functional profile in less than 10 min per mouse and does
not require any sophisticated measuring equipment. However, it
has not been shown whether this screening would also become a
useful tool in identifying qualitative differences between mutants.
We tested a total of 76 animals, including homozygous and hemi-
mice and mice from the inbred
strain C57BL/6 to determine whether we could identify any dif-
ferences, using the SHIRPA primary screen, between these well-
characterized mutants and their background strain. We were able
to observe striking differences in the performance of mutant ani-
mals versus age-matched controls. Dmd
showed deficiencies in wire maneuver, righting reflex, catalepsy,
and locomotor activity tests and were severely fatigued after test-
ing. These data suggest that SHIRPA screening will be a useful
tool in identifying and characterizing mice with novel mutations
that affect skeletal muscle. Thus, SHIRPA provides a simple test to
identify the Dmd
mouse, whereas previously muscle histology
was the easiest assay. With the production of targeted knockout
mutations of several other members of the dystrophin-associated
protein complex, this assay might also serve as a simple test to
determine qualitative differences between mutants.
Materials and methods
mutation originally arose on a C57BL/10 background
and has been maintained as an inbred strain (Bulfield et al. 1984). The
mutation was recovered after ENU mutagenesis of a C3H line
congenic for the Hprt
marker alleles (Chapman et al. 1989).
After the identification of the Dmd
mutation, this strain was back-
crossed an unknown number of times (according to The Jackson Labora-
tory) onto a C57BL/6 background and then maintained as an inbred line.
, and C57BL/6 mice were maintained by sib mating,
* The first two authors contributed equally to this manuscript.
** Present address: Dept. of Molecular and Cellular Biochemistry, The
Ohio State University, College of Medicine, 410 Hamilton Hall, 1645 Neil
Avenue, Columbus, OH 43210, USA.
Correspondence to: K.E. Davies E-mail: firstname.lastname@example.org
Mammalian Genome 11, 725–728 (2000).
© Springer-Verlag New York Inc. 2000