Transcription factors c-jun c-fos Egr-1 Kid 1
Cytokines JE KC
PTHrP Endothelin 1 Endothelin 3
Schematic representation of differential display. In a complex organ like the kidney, ischemic renal injury triggers altered expression of various cell factors and vascular components. Depending on the severity of the insult, expression of these genes can vary in individual cells, leading to their death, survival, or proliferation. A better understanding of the various factors and the signal transduction pathways transduced by them that contribute to cell death can lead to development of therapeutic strategies to interfere with the process of cell death. Similarly, identification of factors that are involved in initiating cell migration, dedifferentiation, and proliferation may lead to therapy aimed at accelerating the regeneration program. To identify the various factors involved in cell injury and regeneration, powerful methods for identification and cloning of differentially expressed genes are critical. One such method that has been used extensively by several laboratories is the differential display polymerase chain reaction (DD-PCR).
In this schematic, mRNA is derived from kidneys of sham-operated (controls) and ischemia-injured rats, some pretreated with insulin-like growth factor (IGF-I). The mRNAs are reverse transcribed using an anchored deoxy thymidine-oligonucleotide (oligo-dT) primer (Example: dT-MX, where M represent G, A, or C, and X represents one of the four nucleotides). An anchored primer limits the reverse transcription to a subset of mRNAs. The first strand cDNA is then PCR amplified using an arbitrary 10 nucleotide-oligomer primer and the anchored primer. The PCR reaction is performed in the presence of radioactive or fluorescence-labeled nucleotides, so that the amplified fragments can be displayed on a sequencing gel. Bands of interest can be excised from the gel and used for further characterization. ARF—acute renal failure.
Schematic representation of a differential display gel in which mRNA from kidneys is reverse-transcribed and polymerase chain reaction (PCR) amplified (see Figure 17-25). The PCR amplification is conducted in the presence of radioactive nucleotides. The cDNA fragments corresponding to the 3' end of the mRNA species are displayed by running them on a sequencing gel, followed by autoradiography. The arrows show bands corresponding to mRNA transcripts that are expressed differentially 1) in response to insulin-like growth factor (IGF-I) treatment and induction of ischemic injury; 2) in an IGF-I-dependent manner; 3) in response to induction of ischemic injury; and 4) to genes that are down-regulated after induction of ischemic injury. ARF—acute renal failure.
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