Biology Reference
In-Depth Information
15
Enhancer Analysis
of the
2(XI) Collagen Genes
in Transfected Chondrocytes and Transgenic Mice
α
1(II) and
α
Noriyuki Tsumaki, Ying Liu, Yoshihiko Yamada,
and Paul Krebsbach
1. Introduction
Extracellular matrix (ECM) plays a critical role in normal development,
tissue repair, and is altered in several disease states. During these processes,
expression of ECM genes is regulated temporally and spatially. Identification
of regulatory elements that direct tissue- and stage-specific expression is
important for understanding of the role of ECM genes in cellular differentia-
tion and function. A significant number of ECM genes have been cloned and
their regulatory elements have been identified. Reporter gene systems are
widely used to study gene regulation and function
(1-3)
. In this chapter, we
describe methods for the identification of promoters and enhancers of cartilage
collagen genes for the
2(XI)
chains using transgenic mice and tran-
sient transfection in cell cultures
(4-9)
.
Transient transfection of plasmid DNA into cultured cells is a well estab-
lished method for studying gene regulation. Traditionally, the CAT (chloram-
phenicol acetyltransferase) gene has been used as a reporter gene to analyze
activity of promoter and enhancer elements in transfection assays
(1
,
2)
. More
recently, the
lacZ
(
α
1(II)
and
α
-galactosidase) and luciferase genes have often been used
because of their high sensitivity and nonradioactive methods of analyzing these
gene products. In addition, several reagents and equipment (e.g., Helios Gen
Gun and Biolistic PDS-1000/He System from Bio-Rad, Richmond, CA) for
DNA transfection have been developed to improve transfection efficiency
(10
,
11)
. Although the transfection in cell cultures is rapid and convenient, it is
difficult to evaluate comprehensive cell-type specificity of promoter and
β
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