(Investigative Ophthalmology and Visual Science. 2000;41:4080-4084.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
C57BL/6 Mice Lacking Muc1 Show No Ocular Surface Phenotype
Yukitaka Danjo1,
Linda D. Hazlett2 and
Ilene K. Gipson1
1 From the Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and the
2 Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
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Abstract
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PURPOSE. To test the hypothesis that a membrane-spanning mucin, Muc1,
facilitates the spread of tear film and protects against bacterial
adherence.
METHODS. Age-matched, Muc1 null mice and wild-type mice of
C57BL/6 genetic background were used for comparison. Eyes were examined
by slit lamp biomicroscopy with fluorescein solution to assess
epithelial damage and tear film stability. Structure of the ocular
surface epithelia was examined by light microscopy, scanning and
transmission electron microscopy, and wholemount confocal microscopy.
Bacterial adherence assay was performed on in vivo corneas with
Pseudomonas aeruginosa containing a plasmid encoding
green fluorescent protein, followed by wholemount confocal microscopy.
Real-time reverse transcription–polymerase chain reaction was
performed using Muc4-specific primers to quantitate Muc4
mRNA expression in ocular surface tissues.
RESULTS. No differences were found between Muc1 null and control
mice in any parameter tested. Ocular surface epithelia of
Muc1 null mice of the C57BL/6 strain had a normal
appearance of surface microplicae, a well-developed glycocalyx on the
apical cell membrane, and a normal appearance of goblet cell mucin
packets. There was no convincing evidence that bacterial adherence on
the cornea was increased in Muc1 null mice. Muc4 mRNA
expression was not upregulated in Muc1 null mice
compared with control. No ocular surface infections were observed in
Muc1 null mice of the C57BL/6 strain (n =
204), which were housed in the animal facility over a period of 26
months.
CONCLUSIONS. Muc1 null mice of C57BL/6 background appeared normal in
all respects tested. These data differ from the reported phenotype in
the mice of the C57BL/6 x SVJ129 background, which show development of
blepharitis and conjunctivitis.
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Introduction
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Amucus layer is present along the apical surface of the entire
ocular surface epithelium.1
The layer is believed to
provide a barrier to pathogen penetrance, a mechanism for removal and
trapping of particulates, and a means of maintaining hydration at the
ocular surface.
The major constituents of the mucus layer are mucins, which are
exceptionally large glycoproteins that have at least half of their mass
as O-linked carbohydrate. A second defining character of mucins is the
presence in their protein backbone of tandem repeats of amino acids.
These repeated segments are rich in serine and threonine, which are
sites of attachment of the O-linked sugars. These abundant O-linked
carbohydrate side chains provide the very hydrophilic character of
mucins. The length and the amino acid sequence of the tandem repeat
varies between each mucin gene product, and the number of tandem
repeats per mucin molecule can vary in individuals as a result of
genetic polymorphism (for review see Gendler and Spicer2
).
Thirteen distinct human mucin genes have been reported to date; they
can be categorized as either membrane spanning or secreted. Five of the
human mucin genes for which sufficient sequence data are available can
be categorized as membrane spanning (MUCs 1,
3, 4, 12, and
13).3
In general, these mucins have
short cytoplasmic tails, a hydrophobic membrane-spanning domain, and an
extracellular domain, which is primarily the heavily glycosylated
tandem repeat region. It has been estimated that MUC1 and
MUC4 can extend 200 to 500 µm from the cell surface,
depending on the number of tandem repeats present.2
4
These mucins are believed to interact with the large secreted mucins
that are derived from goblet cells or submucosal glands, but the nature
of the interaction is unknown. Extracellular domains of Muc1
and the rat homologue of Muc4 have been shown to be
shed from epithelial surfaces.2
5
Previous studies demonstrate that at least three of the human mucin
genes (MUC1, MUC4, and MUC5AC) are
expressed by the ocular surface epithelium.6
7
MUC1 mucin is a membrane-spanning mucin produced by the
entire ocular surface epithelia. MUC4 mucin, which has also
been recently characterized as a membrane-spanning mucin,8
is expressed by the human conjunctival epithelium. MUC5AC
mucin is a secretory mucin derived on the ocular surface from
conjunctival goblet cells. Although the roles of all the mucins are
assumed to be protection against desiccation and microbial invasion,
the specific role played by each mucin in the tear film remains to be
determined.
We hypothesized that the membrane-spanning mucins, which extend from
the apical cell membranes of epithelial cells, facilitate the spread of
secreted mucins of the goblet cells and the tear film and protect
against adherence of bacteria at the interface between the tear film
and the epithelial surface. We used C57BL/6 mice with targeted
disruption of the Muc1 gene to test this hypothesis but
found no aberrant phenotype. While this report was in preparation,
Kardon et al.9
reported that Muc1 null mice of
a different genetic background displayed a marked propensity for
development of blepharitis and conjunctivitis. These seemingly
contradictory results may be due to differences in facility
maintenance, strain variation in the role of the mucin, or other
environmental or epigenetic differences.
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Materials and Methods
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Animals
Muc1 null mice of C57BL/6 genetic background were
generated by homologous recombination10
and generously
provided by Sandra J. Gendler (Mayo Clinic, Scottsdale, AZ).
Wild-type C57BL/6 mice were obtained from Taconic Farm (Germantown, NY)
and used for control. Mice were housed and fed ad libitum in our
conventional animal facility at the Schepens Eye Research Institute,
which is licensed by the US Department of Agriculture and uses the
Animal Care Assurance Statement of the Public Health Service. The
original breeder mice were confirmed to be Muc1 null by
polymerase chain reaction (PCR) using tail genomic DNA samples (data
not shown). To maintain the Muc1 null strain, the
Muc1 null mice were intercrossed. During this study, August
20, 1997, to date, 204 Muc1 null mice have been weaned. All
procedures conformed to the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
Assessment of Ocular Surface Integrity
Eyes of 9-week-old mice were examined without anesthesia with a
slit lamp biomicroscope. To assess epithelial damage and tear film
stability, fluorescein (Sigma, St. Louis, MO) diluted 2% in
phosphate-buffered saline (PBS; pH 7.4), was instilled onto the cornea,
followed by observation using a blue filter.
Morphologic Assessment
After death, eyes, including lids, of mice aged 9 weeks were fixed
in situ with half-strength Karnovsky’s fixative, dissected, and placed
in the fixative. Tissues for scanning electron microscopy were
processed as described previously.11
For transmission
electron microscopy, they were processed and embedded as described
previously.12
Thick sections were stained with toluidine
blue and viewed by light microscopy.
Bacterial Adherence Assay
Bacterial adherence assays were performed in situ on corneas of
anesthetized mice aged 6 to 7 months, with Pseudomonas
aeruginosa strain 19660 (American Type Culture Collection
[ATCC], Rockville, MD) containing a plasmid, pANT4,13
that encodes green fluorescent protein (GFP). pANT4 was introduced into
P. aeruginosa ATCC 19660 by electroporation as described by
Diver et al.14
Preparation of bacterial inoculum was as
described previously,15
except that bacterial cultures
were grown in peptone-tryptic soy broth medium containing 100 µg/ml
carbenicillin and 50 µg/ml kanamycin. Five or 10 µl of bacteria
solution corresponding to 1 x 107 or 2 x 107 colony-forming units (CFU), respectively,
was placed in situ on corneas of anesthetized mice for 1 hour, with the
help of surface tension. After death, eyes were enucleated carefully to
avoid touching the corneas. Eyes were rinsed three times in either
saline or PBS, fixed in 4% paraformaldehyde, labeled with rhodamine
phalloidin, and processed for wholemount confocal microscopy, as
previously described.16
Dual detection was performed using
fluorescein isothiocyanate (FITC) and tetrarhodamine isothiocyanate
(TRITC) detector channels.
PCR Verification of Muc1 Null Mutation
Tail genomic DNA was isolated using a kit (QIAamp; Qiagen,
Valencia, CA), according to the manufacturer’s instructions. It was
then used for PCR with Muc1-specific primers10
:
sense 5'-ACCTCACACACGGAGCGCCAG-3' (GenBank accession M64928,
nucleotides [nt] 463-483) and antisense 5'-TCCCCCCTGGCACATACTGGG-3'
(nt 724-704) to confirm the homozygous null mutation for the
Muc1 gene. Forty cycles of PCR amplification were performed.
Amplified products were electrophoresed on a 2% agarose gel and
visualized with ethidium bromide.
Real-Time RT-PCR to Determine Relative Muc4
Expression
Techniques for real-time reverse transcription–polymerase
chain reaction (RT-PCR) were essentially the same as previously
reported.17
Total RNA of the ocular surface, including
cornea and conjunctiva, was isolated from 1-year-old Muc1
null mice and wild-type control mice using a commercial reagent
(TRIzol; Life Technologies, Grand Island, NY). The first strand of cDNA
was synthesized from 1 µg of the RNA (DNase treated) with random
primers using a commercial reverse transcriptase (Superscript; Life
Technologies). Real-time PCR was performed using a commercial system
(TaqMan PCR GeneAmp 5700 Sequence Detection System; PE
Biosystems, Foster City, CA). The Muc4-specific primers were
designed from the sequence in GenBank (accession AF218265). The
sense primer was 5'-CTCCAAGAAATGTAGTGGCTTTCAG-3' (nt 2925-2949), the
antisense 5'-CACGGTCTTGGGCTGGAGTA-3' (nt 3066-3047), and the
TaqMan probe sequence was 5'-AACATCCCCAGAAGCGTGTACCCTGG-3'
(nt 2982-3007). The internal standard control gene was amplified
(TaqMan Rodent GAPDH Control Reagents; PE
Biosystems). To verify the validity of using GAPDH as the
internal calibration standard, the efficiencies of the Muc4
and GAPDH amplifications were compared and found to be
equivalent. To verify the identity of the Muc4 PCR product,
it was sequenced at the DNA Sequencing Core Facility of Massachusetts
General Hospital, Boston.
Each PCR reaction contained equivalent amounts of cDNA. Assays were
performed in quadruplicate using a kit (TaqMan PCR; PE
Biosystems) according to the manufacturer’s recommendations.
Quantitation and comparison of amounts of Muc4 mRNA in
Muc1 null mice and wild-type control animals (n =
5 per group) were as previously described.17
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Results
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Gross Appearance of Muc1 Null Mice
The gross views of eyes by slit lamp biomicroscopy appeared normal
in Muc1 null mice of C57BL/6 genetic background. In 10
Muc1 null as well as 4 wild-type mice, punctate fluorescein
staining was detected on all the corneas, indicating that the punctate
stains were not characteristic of Muc1 null mutation (Fig. 1)
. No breakup of the tear film was observed for at least 30 seconds.
During the period of this study, August 20, 1997, to date, 204
Muc1 null mice were weaned, and the age of the mice ranged
to at least a year and a half. No ocular abnormalities—including
conjunctivitis or blepharitis—were found under routine animal care.
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Figure 1. Slit lamp biomicroscopy of the ocular surface of Muc1
null and wild-type mice after fluorescein instillation. Punctate
staining was detected on corneas of both Muc1 null
(A) and wild-type control (B) mice.
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Morphologic Examinations
As demonstrated by scanning electron microscopy, Muc1
null mice possessed normal-appearing surface microplicae, comparable to
those of wild-type control mice, on corneas (Fig. 2A
) as well as on conjunctiva (not shown). Transmission electron
microscopy demonstrated that glycocalyces on the apical epithelial
cells (Fig. 2B)
and mucin packets in the goblet cells (Fig. 2C)
in
Muc1 null mice appeared well developed and were comparable
to those of wild-type control mice. Light microscopy demonstrated that
epithelia of cornea (not shown) and conjunctiva (Fig. 2D)
from
Muc1 null mice were comparable and similar to those of
wild-type control mice, with no inflammation and goblet cell
hypertrophy.
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Figure 2. Morphologic examinations of the ocular surface epithelia derived from
Muc1 null (left) and wild-type control
(right) mice. (A) Scanning electron
microscopy of the apical corneal epithelial cells, demonstrating
comparable surface microplicae. Bar, 5 µm. Transmission electron
microscopy of the apical surface of corneas (B) and the
secretory granules of goblet cells (C), demonstrating
well-developed, comparable glycocalyces and mucin packets,
respectively. Bar, 0.5 µm. (D) Light microscopy of the
conjunctiva demonstrating comparable appearance. Toluidine blue stain.
Bar, 100 µm.
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Bacterial Adherence Assay
In 16 of 20 Muc1-/- eyes (Fig. 3A
) and 8 of 10 wild-type control eyes (data not shown), there was no
adherence of the GFP-expressing P. aeruginosa on in vivo
corneas. On several eyes, a few clusters of adherent bacteria were
observed (Fig. 3B)
, apparently from nonspecific attachment, but no
distinct difference was seen between these clusters on Muc1
null and control mice. Several different rinsing procedures were used,
including vigorous rinse, gentle rinse, and fixation before rinse,
yielding similar results. To make sure that the P.
aeruginosa used in this study was capable of adhering to tissues,
the bacteria were confirmed to adhere to the wound bed of wounded
corneas (data not shown), which was consistent with results described
previously.11
The P. aeruginosa strain (ATCC
19660) has been shown to infect mice of the C57BL/6
strain.15
We could not find convincing evidence that
bacterial adherence was increased on corneas of Muc1 null
mice.
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Figure 3. Bacterial adherence assay on in vivo corneas of Muc1
null mice with P. aeruginosa containing a plasmid
encoding green fluorescent protein. Bacterial adherence was visualized
by wholemount confocal microscopy after labeling with rhodamine
phalloidin. Adherence of the bacteria was not routinely seen
(A). Occasionally, adherent bacteria were observed but no
distinct difference was seen between Muc1 null and control
mice (B). Stacked images of eight optical sections are
shown. Bar, 20 µm.
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Muc4 mRNA in Muc1 Null Mice
To determine whether another membrane-spanning mucin, Muc4, is
upregulated to compensate for loss of Muc1, we compared their mRNA
levels using real-time PCR and PCR primers designed from the carboxyl
terminal region of the mouse MUC4 homologue (GenBank
accession AF218265). These primers amplified a 142-bp product by RT-PCR
of mouse ocular surface RNA. Sequencing of this PCR product showed it
to be identical with the GenBank sequence for Muc4. The
relative amounts of Muc4 expression in Muc1 null
mice compared with that in control animals did not differ (-/- =
0.93, range 0.49–1.75, n = 5; +/+ = 1.0, 0.54–1.86,
n = 5). Rodent GADPH was used as the internal
standard. These data indicate that mRNA expression of another
membrane-spanning mucin, Muc4, is not upregulated in Muc1
null mice.
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Discussion
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This study showed that the ocular surface of Muc1 null
mice of C57BL/6 background appeared normal in all respects tested.
These data are at variance with the report by Kardon et
al.,9
in which a marked propensity for development of
blepharitis and conjunctivitis by Muc1 null mice in C57BL/6
x SVJ129 background, was reported. Several possibilities for the
differences between our study and theirs include the housing conditions
of the animals, mouse strain variation in response to deletion of
Muc1, strain variation of pathogens, or other environmental
or epigenetic differences. Our conventional animal facility, licensed
by the US Department of Agriculture, houses mice that are routinely
tested to assure the absence of pathogenic viruses, bacteria, and
parasites, and is considered murine pathogen free. The facility housing
the animals in the study by Kardon et al.9
is described as
"a conventional animal facility," but it is not clear whether it is
murine pathogen free. Because none of the Muc1 null mice
housed in our facility (>200) displayed development of infection of
the ocular surface, and because the bacterial adherence assay using
P. aeruginosa, a common pathogen seen in eye infections,
gave no definitive evidence that Muc1 null mice were more
susceptible to bacterial adherence, the difference between the two
populations may be due to other factors, including strain variation.
Although there appear to be no reports on susceptibility of the inbred
SVJ129 strain to infection, it is possible that the combination of
C57BL/6 and SVJ129 backgrounds renders Muc1 null mice
susceptible to microbial infections, implying that Muc1
mucin plays a more important role in the latter background in
protecting against infection. Similar phenotypic differences of null
mutation between C57BL/6 and SVJ129 backgrounds are reported, with p53
null mice displaying vitreal opacities, fibrous retrolental tissue, and
retinal folds in the former strain but not in the
latter.18
Because the ocular surface epithelia of rodents express another
membrane-spanning mucin, Muc4, it is possible that this mucin
compensates for loss of Muc1 in some strains of mice. However, the mRNA
level in the ocular surface tissue of the Muc1 null mice was
not upregulated when compared by real-time PCR with that of control
mice. These data are consistent with that in a previous paper reporting
no difference in expression levels of Muc4 using slot-blot analysis of
mRNA of mammary gland, salivary gland, lung, stomach, and colon of
Muc1 null mice.10
Perhaps the Muc4 protein is
sufficiently protective without enhanced expression levels.
Direct comparison of the two different strains of mice under the same
experimental, laboratory, and care and handling conditions would be
useful to sort out the variance in response to the null mutation. The
comparison was impractical, because it is impossible to "duplicate
the mice because they were mixed outbred mice" (Sandra J. Gendler, personal communication, May 1999). The outbred mice
were retrieved and maintained for approximately a year in the animal
facility of the laboratory that originally developed both
Muc1 null strains, they reportedly did not observe the same
eye problems, but comment that "our facility is very clean"
(Gendler, personal communication, May 1999). Although the data obtained
regarding absence of an ocular surface phenotype in the C57BL/6 strain
of mice represent a report of negative findings, they indicate that in
some circumstances, MUC1 is not necessary for protection of
the ocular surface. Whether these data are relevant to the human ocular
surface is not known. In conjunctiva and cornea of rats and mice, there
is a high level of expression of the membrane-spanning mucin
Muc4 (Bartman et al., unpublished
data)19
20
In humans, the level of expression of
MUC4 in central cornea is much less.5
7
Perhaps
the concentration of Muc4 on rodent eyes is so high that loss of Muc1
has a negligible effect. It is possible that, in human ocular surface,
MUC1 may have a protective role.
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Acknowledgements
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The authors thank Norm Michaud for technical assistance in scanning
electron microscopy, Rob Moccia and Sandra Spur–Michaud for work on
the Muc4 cDNA sequence, and Jeffery Hobden for providing
the GFP-labeled bacteria.
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Footnotes
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Supported by Grants R37-EY03306 (IKG) and R01-EY02986 (LDH) from the National Eye Institute.
Submitted for publication December 13, 1999; revised May 30 and August 16, 2000; accepted August 22, 2000.
Commercial relationships policy: N.
Corresponding author: Ilene K. Gipson, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. gipson{at}vision.eri.harvard.edu
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References
|
|---|
-
Nichols, BA, Chiappino, ML, Dawson, CR (1985) Demonstration of the mucous layer of the tear film by electron microscopy Invest Ophthalmol Vis Sci 26,464-473[Abstract/Free Full Text]
-
Gendler, SJ, Spicer, AP (1995) Epithelial mucin genes Annu Rev Physiol 57,607-634[Medline][Order article via Infotrieve]
-
Williams S, Wreschner D, McGuckin M. Structure and expression of a
novel transmembrane epithelial mucin gene. Mucins in Health and
Disease. Proceedings of the Sixth International Workshop on
Carcinoma-Associated Mucins. London, UK: Imperial Cancer Research Fund;
2000:29.
-
Nollet, S, Moniaux, N, Maury, J, et al (1998) Human mucin gene MUC4: organization of its 5'-region and polymorphism of its central tandem repeat array Biochem J 332,739-748
-
Pflugfelder, SC, Liu, Z, Monroy, D, et al (2000) Detection of sialomucin complex (MUC4) in human ocular surface epithelium and tear fluid Invest Ophthalmol Vis Sci 41,1316-1326[Abstract/Free Full Text]
-
Inatomi, T, Spurr–Michaud, S, Tisdale, AS, Gipson, IK (1995) Human corneal and conjunctival epithelia express MUC1 mucin Invest Ophthalmol Vis Sci 36,1818-1827[Abstract/Free Full Text]
-
Inatomi, T, Spurr–Michaud, S, Tisdale, AS, Zhan, Q, Feldman, ST, Gipson, IK (1996) Expression of secretory mucin genes by human conjunctival epithelia Invest Ophthalmol Vis Sci 37,1684-1692[Abstract/Free Full Text]
-
Moniaux, N, Nollet, S, Porchet, N, Degand, P, Laine, A, Aubert, J-P. (1999) Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin Biochem J 338,325-333
-
Kardon, R, Price, RE, Julian, J, et al (1999) Bacterial conjunctivitis in Muc1 null mice Invest Ophthalmol Vis Sci 40,1328-1335[Abstract/Free Full Text]
-
Spicer, AP, Rowse, GJ, Lidner, TK, Gendler, SJ (1995) Delayed mammary tumor progression in Muc-1 null mice J Biol Chem 270,30093-30101[Abstract/Free Full Text]
-
Spurr–Michaud, SJ, Barza, M, Gipson, IK (1988) An organ culture system for study of adherence of Pseudomonas aeruginosa to normal and wounded corneas Invest Ophthalmol Vis Sci 29,379-386[Abstract/Free Full Text]
-
Gipson, IK, Kiorpes, TC, Brennan, SJ (1984) Epithelial sheet movement: effects of tunicamycin on migration and glycoprotein synthesis Dev Biol 101,212-220[Medline][Order article via Infotrieve]
-
Lee, AK, Falkow, S. (1998) Constitutive and inducible green fluorescent protein expression in Bartonella henselae Infect Immun 66,3964-3967[Abstract/Free Full Text]
-
Diver, JM, Bryan, LE, Sokol, PA (1990) Transformation of Pseudomonas aeruginosa by electroporation Anal Biochem 189,75-79[Medline][Order article via Infotrieve]
-
Kwon, B, Hazlett, LD (1997) Association of CD4+ T cell dependent keratitis with genetic susceptibility to Pseudomonas aeruginosa ocular infection J Immunol 159,6283-6290[Abstract]
-
Danjo, Y, Gipson, IK (1998) Actin. "purse string" filaments are anchored by E-cadherin-mediated adherens junctions at the leading edge of the epithelial wound, providing coordinated cell movement J Cell Sci 111,3323-3331[Abstract]
-
Fink, L, Seeger, W, Ermert, L, et al (1998) Real-time quantitative RT-PCR after laser-assisted cell picking Nat Med 4,1329-1333[Medline][Order article via Infotrieve]
-
Ikeda, S, Hawes, NL, Chang, B, Avery, CS, Smith, RS, Nishina, PM (1999) Severe ocular abnormalities in C57BL/6 but not in 129 Sv p53-deficient mice Invest Ophthalmol Vis Sci 40,1874-1878[Abstract/Free Full Text]
-
Tei, M, Moccia, R, Gipson, IK (1999) Developmental expression of mucin genes ASGP (rMuc4) and rMuc5AC by the rat ocular surface epithelium Invest Ophthalmol Vis Sci 40,1944-1951[Abstract/Free Full Text]
-
Tei, M, Spurr–Michaud, SJ, Tisdale, AS, Gipson, IK (2000) Vitamin A deficiency alters the expression of mucin genes by the rat ocular surface epithelium Invest Ophthalmol Vis Sci 41,82-88[Abstract/Free Full Text]
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Abstract
Introduction
