Second Excursion Validation

The validation of a target or a biomarker before proceeding to further development usually requires the unambiguous demonstration that the candidate really plays the proposed role. One way to achieve this is by the genetic manipulation of biological systems, which turns on or off the expression of candidate proteins, related pathways or associated proteins by site-directed mutagenesis, knock-out or transfection experiments. Recently, one of the most attractive approaches is that ofRNA silencing (siRNA), which avoids the obfuscating effects of pleiotropic and redundant signaling pathways which are affected in more global ways by mutational approaches [31-33]. In particular, with regard to highly popular transgenic animal models, siRNA technologies provide a much more targeted and focused approach to validation by aiming at specific m-RNAs at specific stages of development, differentiation, or under well-defined functional conditions (Fig. 8.3).

Embryonic stem cells fulfill these requirements; they can be differentiated under very well controlled conditions to various organotypic cell types, which then represent different embodiments of a single genome, thus providing a perfect substrate for initial validation. Quite a number of recent studies have applied siRNA protocols to hESC and mESC [34-36]. A short description of siRNA procedures is provided in Figure 8.3. Together with the cutting-edge analytical technologies mentioned above, the corresponding use of hESC and mESC could provide a most powerful ensemble for integrated discovery, validation and screening, developing and applying protein biomarker signatures in novel in-vitro methods for toxicology and efficacy testing of chemicals and pharmaceuticals. ESC-based in-vitro systems can, moreover, be applied directly for the generation of corresponding in-vivo models [34-36].

Fig. 8.2 Pattern control of ischemic effects in a neural murine embryonic stem cell (ESC) culture model by quantitative and differential analysis of protein expression: high-resolution 2D gels [151] were used to compare the protein expression pattern of neurons exposed to chemical ischemia (CI) with control neurons (CO). The two single gels in (A) and (B) were obtained without prior fractionation; the labeling pattern is explained in the upper part of the figure. Labeling controls (C) and (D) are not shown. In the lower part of the figure a more detailed view of surrogate two-color representation of radioactively cross-

Fig. 8.2 Pattern control of ischemic effects in a neural murine embryonic stem cell (ESC) culture model by quantitative and differential analysis of protein expression: high-resolution 2D gels [151] were used to compare the protein expression pattern of neurons exposed to chemical ischemia (CI) with control neurons (CO). The two single gels in (A) and (B) were obtained without prior fractionation; the labeling pattern is explained in the upper part of the figure. Labeling controls (C) and (D) are not shown. In the lower part of the figure a more detailed view of surrogate two-color representation of radioactively cross-

labeled duplicate gel frames comparing phosphoproteomes of control neurons versus neurons exposed to chemical ischemia is shown. The left panel shows control in blue versus ischemia in orange; the right panel shows control in orange versus ischemia in blue. The two phosphoproteins appearing after the induction of chemical ischemia shown here, have the same quantities and positions in both series of the labeling procedure (125I and 131I), and thus represent novel post-translational surrogate markers, in an ESC model related to human neurodegeneration [28].

Fig. 8.3 An overview of the silencing mechanisms by small interfering RNA (siRNA). The key event is the generation of double-stranded RNA (dsRNA); this can occur endo-genously in the cell, by genomic transcription of antisense RNA to microRNA (miRNA) or long dsRNA or by direct experimental introduction into target cells. The dsRNA is cleaved by intrinsic enzymes to siRNAs at cleavage sites indicated by orange triangles. These

Fig. 8.3 An overview of the silencing mechanisms by small interfering RNA (siRNA). The key event is the generation of double-stranded RNA (dsRNA); this can occur endo-genously in the cell, by genomic transcription of antisense RNA to microRNA (miRNA) or long dsRNA or by direct experimental introduction into target cells. The dsRNA is cleaved by intrinsic enzymes to siRNAs at cleavage sites indicated by orange triangles. These siRNA pieces form different types of RNA-induced silencing complexes (RISC, with single-stranded siRNA), which subsequently cause mRNA degradation, chromatin methy-lation, or translational inhibition [152, 153]; siRNA approaches are versatile because they can be applied in vitro, and also used for corresponding systemic or local in-vivo mammalian gene silencing.

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