| Introduction
DNA microarrays are a key technology in modern molecular biotechnology.
Due to their high degree of parallelism, they allow the simultaneous analysis
of complex genetic changes, thereby enabling a deeper insight into processes
such as differentiation, growth and cell development.
In our project we are developing DNA microarrays for the identification
of fetal and somatic liver stem cells. The microarrays will be used for
the examination of the differentiation status of these cells, which is
influenced by certain factors of the extra-cellular microenvironment in
vitro.
The results are of great significance for an understanding of embryonic
and fetal organ development. They provide the basics for both the understanding
of developmental biology and for the development of toxicologically and
clinically applicable organ replacement systems, which are of great importance
for tissue engineering technologies.
This technology field focuses on the development of biological substituents
that maintain, regenerate and improve tissue and organ functions. Excluding
ethical and technical problems, modified tissue or tissue cultures will
be optimized in terms of material and time for tissue and organ transplantation.
In this respect, the genetic background of cell differentiation has developed
into a focal point of research interest in the last few years.
Goal
In our project, fetal hepatocytes of the rat should undergo the most
extensive proliferation and differentiation possible. This will occur
in connection with the optimization of culture conditions such as matrix,
media and supplements. DNA microarrays specially developed for the identification
of fetal hepatocytes will be used. The CreativeChip™ 3D Slides (Eppendorf®)
will be tested for its suitability as a base substrate for these DNA microarrays.
Material and methods
Cell culture
Fetal hepatocytes were cultivated under various conditions. The culture
conditions result from the use of various growth factors (individually
or combined) and an optimal matrix or "feeder" cells.
Microarray design and production
All genes relevant for the differentiation and growth of the liver have
been identified from various databases. Oligonucleotides (50mers) were
chosen from areas near the 3' ends of the mRNAs, which represent these
genes sequence-specifically. The specificity of these oligonucleotides
was tested with PCR*, Southern and Northern blots (not shown). The spotting
of the oligonucleotides onto CreativeChip 3D Slides (Eppendorf) was performed
using a GMS 417 array system (Affymetrix) according to manufacturer instructions.
Lyophilized oligonucleotides were dissolved in water and adjusted to
a concentration of 100 µM. Prior to printing, they were mixed 1:1
with spotting buffer (200 mM phosphate buffer (pH 9.0) / 10% Na2SO4 /
2 M betaine).
RNA isolation
Total RNA was isolated from the cells cultivated under various conditions
with the RNeasy® Kit (Qiagen®). In order to obtain the cleanest
and most DNA-free RNA possible, DNase digestion, phenol/chloroform/isoamyl
alcohol purification and ethanol precipitation were performed.
Reverse transcription with direct fluorescence labeling
A reverse transcription with direct labeling, meaning the incorporation
of Cy3-dCTP or Cy5-dCTP during cDNA synthesis, was subsequently performed.
100 µg total RNA, SuperScript™ II Reverse Transcriptase (Invitrogen™)
and gene-specific primer were used for this reaction. In order to remove
the RNA at the end of the reaction, an alkaline RNA digest was performed
at 65°C.
Purification of the labeled cDNA
To avoid hybridization artifacts, the generated cDNA strands had to be
separated from free fluorescence labeled nucleotides. By using YM30 columns
(Millipore), it was possible to simultaneously remove the free nucleotides
and attain a concentration of the cDNA.
Hybridization
For the hybridization reaction, the labeled cDNA was mixed with hybridization
buffer (250 mM phosphate buffer / 1 x SSC / 1 mM EDTA / 4.8% SDS) and
denatured for 3 min at 95°C. The hybridization mix was then added
to the hybridization area of the microarray and covered with a cover slip,
avoiding air bubbles. The hybridization reaction was performed for 16
hours at 42°C.
Detection of the hybridization signals
The detection and quantification of the hybridization events were done
using a GMS 418 laser scanner (Affymetrix).
 |
Fig. 1: Hybridization of a Rat Liver
Chip printed on Eppendorf CreativeChip 3D Slide
Cy5 labeled cDNA: synthesized from 100 µg total RNA from hepatocytes
cultivated on collagen 1 w/o HGF (hepatocyte growth factor)
Cy3 labeled cDNA: synthesized from 100 µg total RNA from hepatocytes
cultivated on collagen 1 w/ HGF |
 |
Fig. 2: Hybridization of a Rat Liver
Chip printed on Eppendorf CreativeChip 3D Slide
Cy3 labeled cDNA: synthesized from 100 µg total RNA from hepatocytes
cultivated on collagen 1 w/o HGF
Cy5 labeled cDNA: synthesized from 100 µg total RNA from hepatocytes
cultivated on collagen 1 w/ HGF |
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