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.
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
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 M. Prior to printing, they were mixed 1:1 with spotting buffer (200 mM phosphate buffer (pH 9.0) / 10% Na2SO4 / 2 M betaine).
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 g 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 65C.
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.
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 95C. 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 42C.
Detection of the hybridization signals
The detection and quantification of the hybridization events were done
using a GMS 418 laser scanner (Affymetrix).
To do this, fetal hepatocytes were cultivated under 2 different culture conditions (collagen 1 without HGF and collagen 1 with HGF, respectively). The expression of liver-specific markers, such as albumin, Cytochrome P 450, GST, etc., in the various cultivated hepatocytes was subsequently tested with the Rat Liver Chip.
The culture condition 2 (collagen 1 with HGF) led to an up-regulation of liver-specific genes in both experiments.Fig. 3: Two-dimensional scatter plot analysis (Cy3 against Cy5 fluorescence signal, logarithmic) of a typical experiment according to Figure 1.
The developed Rat Liver Chip includes 240 liver-specific genes, which primarily encode phase I and phase II enzymes and matrices. Oxidation, reduction, methylation and desulphurizing reactions, as well as hydrolytic cleavages occur in phase I of the liver metabolism. The biological function of these reactions is the transformation of toxic substances or external materials and thereby the reduction of their biological activity, as well as the increase of their polarity. The Cytochrome P 450 system is mainly involved in this process. In phase II, various substrates are coupled to very polarized, negatively charged molecules. The resulting conjugates are considerably more water soluble in comparison to unconjugated compounds, and thereby are easier to eliminate. The Glutathione S-Transferase, for example, belongs to the family of phase II enzymes. Fetal hepatocytes can be stimulated to increased proliferation by the creation of optimal culture conditions. The initial cell number can be increased up to 30 times, depending upon the combination of growth factors. With the microarrays specially developed for this process, it was demonstrated that the liver-specific functions (phase I and phase II enzymes) are expressed for up to 10 days in culture under optimal conditions.
The CreativeChip 3D Slides are characterized by a constantly low intrinsic fluorescence so the dynamic range of measurable signals is very high. Binding the probes (in this case: 50mer oligonucleotides) terminally, modified with a linker molecule (5'aminoalkane molecule), to the slide surface results in the advantage that the entire length of spotted oligonucleotides is available for the hybridization process. In combination with an exact probe positioning this results in optimal signal and sensitivity yields and low background. This is of particular importance because most of the genes have been ascertained to be regulated only to a minor extent.References