Large-scale Sequencing Of Human Dendritic Cell CDNA Library And Characterization Of A Novel Cytokine CX1

Large scale sequencing of cDNA library has been proven to be a powerful tool for evaluation of the gene expression profile and identification of novel genes. For better understanding the biological functions and their related mechanism of dendritic cells (DCs), we established a system for large scale sequencing of human DC cDNA library.Human DCs were generated by culture of peripheral monocytes for 7 days with GM-CSF and IL-4. The monocyte-derived DCs with the purity of more than 90% were demonstrated to express high levels of HLA-ABC, HLA-DR, B7, CD40, CD54 and markers of CD1a and CD83, and potent stimulators to allogenic T cells. mRNA was extracted and purified from DCs and analyzed for the integrity of DCs specifically expressed molecules including DC-CK1 and DEC205. The cDNA plasmid library was constructed using SuperscriptTM cDNA synthesis and plasmid cloning system, and transformed into DH10B cells. More than 93% of the clones from the constructed library contained inserts at the average size of 1.6 kb. Random clones were picked from DC cDNA library and subjected to large-scale sequencing for cDNA 5'termini using BigDye Terminator Cycle Sequencing kit. The raw data were sent to Sun450 server and analyzed with Wu-blast and GCG programs. An in–house database with 14,218 DC ESTs was successfully generated. BLASTN search demonstrated that 10,356 EST were from known genes.Among the known gene ESTs, 6.83% (708) represents 233 non-redundant immune-related genes including MHC, cluster of differentiation, immune receptors, adherence molecules, cytokines, complements and signaling molecules. ESTs representative of MHC accounted for 48.3% of total immune-related ESTs, which is in accord with the high expression of MHC I, MHC II and the unique antigen presenting capacity of DCs. ESTs representative of CDla,CD1c,CD68,TNF-α,AIF-1,DAP12 and GM-CSF receptorβchain were found abundantly in DC EST database. In addition, some immunologically relevant genes were detected, including CD86, ICAM-1, CD11b, IL-16, MIP-1α, MCP, CCR6, CXCR2 and CXCR4, etc. To our knowledge, some known genes are initiately observed to be expressed by DCs, which may indicate their potential roles in DC functions and the new functions mediated by DCs.