Transcription profiling of mouse ES cells expressing fosBd/d alleles which encode exclusively deltaFosB, and vs. phenotypes with fosB-null and wild-type ES cell lines

Among fos family genes modulating the cell fates such as cell proliferation, differentiation and cell death, only the fosB gene produces two forms of mature mRNA for FosB and deltaFosB proteins by alternative splicing of an exonic intron in the exon 4. FosB dramatically enhances the transcription regulation of AP-1 dependent promoters by Jun, while deltaFosB, a truncated form of FosB lacking its C-terminal transactivation domain, suppresses the function of Jun. We and other have shown that FosB and deltaFosB have a distinct function to control the cell fate as well as neuronal functions based on their exogenous expression. To elucidate the authentic function of each protein, it is essential to control each expression separately. We established a mutant mouse embryonic stem (ES) cell line carrying homozygous fosBd/d alleles which encode exclusively deltaFosB, and compared its gene expression profile and phenotypes with fosB-null and wild-type ES cell lines. Both mutant ES cells are devoid of FosB, therefore the common phenotypes between the two mutant ES cells in comparison to wild type depict the effects of FosB deficiency. Opposite phenotypes between the two are considered to be determined by deltaFosB itself. We analyzed the gene expression and cellular function among these two mutant and wild-type ES cells. Experiment Overall Design: Total RNA was purified from ES cells grown in the absence of feeder cells using the Isogen kit (Nippon Gene), according to the manufacturer’s instructions. For DNA microarray experiments, Total RNA (10 microg) were labeled using the Agilent Linear Amplification/Labeling Kit (Agilent Technologies) according to the manufacturer's instructions. One microgram of each Cy3-labeled wild-type and Cy5-labeled mutant cDNA or each Cy3-labeled mutant and Cy5-labeled wild-type cDNA, respectively, were mixed, then hybridized to Agilent Mouse cDNA Microarrays (G4104A, design file number: 000522R000679, Agilent Technologies) with 8500 unique clones from Incyte’s mouse UniGene 1 clone set, according to the manufacturer's hybridization protocol. After the washing step, the microarray slides were analyzed with an Agilent G2565AA microarray scanner system. These experiments were carried out in duplicate using exchanged dye-labeled cDNA probes (i.e., Cy3 and Cy5 dye-swapping experiments).