行政院國家科學委員會專題研究計畫成果報告:基因槍在眼表層細胞中之研究
Date Issued
1999
Date
1999
Author(s)
胡芳蓉
DOI
882314B002372
Abstract
The regulatory cis-DNA elements of the
cornea-specific keratin 12 gene has been
studied for a long time. Conventional ways of
identifying this cell-specific promoter using
cultured rabbit corneal epithelial cells derived
from cornea explants is studied by many other
authors and had following conclusions. First,
for unknown reasons , these primary corneal
epithelial cells could not be effectively
transfected using liposomes. Second, the
subcultured epithelial cells from cornea
explants could be transfected but they dedifferentiated
and ceased to express keratin 12.
Third, SV40 large T-antigen transformed
rabbit corneal epithelial cell line is also used to
test K12 promoter-b-gal DNA constructs,
because they could be efficiently transfected by
liposomes or electroporation. This established
2
corneal epithelial cell line maintains many
epithelial cell characteristics, but they no
longer express keratin 12. However, this cell
line is still of value to examlne K12 promoter-
b-gal DNA constructs for their functionality,
because it is easy to maintain and still
possesses many epithelial cell characteristics,
e.g. stratification, expression of keratins, etc.
From the studies of Dr. Kao, a 0.2 kb element
in the 5'-flanking region of the keratin 12 gene
is identified that could direct the expression of
b-galactosidase by using this cell line. As the
5'-end flanking sequences increased in the
promoter-reporter gene constructs, the levels of
b-galactosidase expression decreased in the
transfected rabbit corneal epithelial cells.
These observations imply that 5’ end sequence
of the Krt1.12 gene may contain sufficient and
essential regulatory cis-DNA elements for
corneal epithelial cell-specific expression of
the reporter gene(s), e.g. CAT
(chloroamphenicol acetyltransferase) and b-
galactosidase. Thus, a selective group of the
reporter DNA constructs is tested with the
transgenic mouse technique. Unfortunately,
none of the transgenic mouse lines expressed
reasonable levels of the CAT activity in cornea
or other tissues.
In order to circumvent these problems,
we have tried the newly developed in vivo
particle-mediated gene transfer techniques
(Helios™ Gene Gun System") for a few trials
to examlne the promoter activities of reporter
gene constructs. The results of these "Gene
Gun" experiments indicate that our reporter
gene constructs (1.0 KZ, 2.5 KZ) contain
regulatory cis-DNA elements for cornea
epithelial cell expression of b-galactosidase.
However, more experiments are needed to
identify and characterize the DNA elements
which are essential and sufficient for cornea
epithelial cell-specific expression of reporter
gene(s). Thus, the purpose of this study is to
apply the "Gene Gun" system in the study of
ocular surface epithelium. Then we can
identify the exact structure of cis-regulatory
DNA element of Krt1.12 gene. The
identification of such DNA elements is a
prerequisite for creating transgenic mouse
models which may over-express cytokines, or
carry domlnant negative mutations of receptors
for future studies of corneal physiology, and
development of a gene therapy strategy for
treating corneal disease.
In the first year study, we found that
the activity of k12 promoter increased to a
constant level from 1.0 to 2.5 kb by in vivo
Gene Gun delivery transfection. This result
encourageed us to investigate the more detailed
structure with k12 promoter. At first we
focused on the Pax-6 pair boxes located within
2.5 KZ. There are four Pax-6 pair boxes
located upstream from the transcriptional
initiation site to 2.5 kb. We construct several
deletion constructs, including 2.0 KZ / 0.2 KZ,
2.0 KZ /0.4 KZ, and 2.0 KZ /0.6 KZ deletion
of Pax-6 elements, and we found that the
promoter activities decreased when the number
of Pax elements decreased. It suggests that the
Pax pair boxes in the promoter of k12 did play
important roles in the regulation of k12
expression.
Subjects
Research Project
Report Style
National Science Council
Publisher
臺北市：國立臺灣大學醫學院眼科
Type
journal article
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