BMC Cancer 2004, 4:78 http://www.biomedcentral.com/1471-2407/4/78 cFiDifgicNusAree-qPu5CeRncaenparlyimsiserosf gene expression was done using spe- cDNA-PCR analysis of gene expression was done using specific sequence primers. We used SMART cDNAs from 7–10 different tumor samples (patients B84, B85, B87, B89, B90, B91, B92, B93, B95, B96) as template, from single metastasized lymph node indicated as LMB82 (patient B82) and from normal breast, normal testis, lym- phocytes from healthy donors. cDNAs were normalized by amplification of β-actin gene. There are agarose gels with ubiquitously-expressed genes in Figure 5A, underexpressed genes in Figure 5B, overexpressed ones in Figure 5C. tested in comparison with normal breast, although it was also transcribed in testis and normal lymphocytes (Figure 5C). T7-1 (KIAA1288) was found to be overexpressed in 50% of the primary breast carcinomas and in the metasta- sis specimen tested. In order to obtain an evaluation of the accuracy of the method used for the analysis of gene expression, we performed PCR amplification of neu/ HER2, a known tumor marker overexpressed in breast cancer. We observed that neu/HER2 is overexpressed in 2 primary tumors among the 7 tested (≈29%) in accordance with the literature on breast carcinoma [19,20]. Discussion In the present study we report the construction of MCF-7 and MDA-MB-468 cell lines, breast carcinoma and testis cDNA phage-displayed libraries expressed as fusions to bacteriophage lambda gpD. The new phage vectors bear a flexible GS linker between the cloned protein domain and protein D, so as to facilitate lambda head assembly. More- over, a new efficient protocol to synthesize cDNA was applied. We primed cDNA synthesis on mRNA template with random oligonucleotides containing a constant 5' end. After complete synthesis of double-stranded cDNA, a second round of random priming was applied to generate oriented fragments of cDNA suitable for library construc- tion. This protocol, in comparison with previous version, increases the presence of authentic protein domains in the library twofold, because of correct cDNA orientation. Moreover, some of the clones isolated from our previous libraries were results of chimerical fusion of two or more different genes, generated through double random priming on ds cDNA template. The new protocol has reduced this problem significantly. We also confirmed the advantage of N-terminal fusion for domain library construction in phage display vectors for screening with sera, because a significant amount of false positive cross-reactive clones, containing stop codons downstream of the fusion site giving rise to short mimo- tope sequences, were selected from the C-terminal fusion library (T6). Only 4 clones with specific tumor-related reactivity were isolated from the T6 library. However, C- terminal fusion might allow efficient display and selec- tion for some antigenic C-terminal protein domains. In fact, the C-terminal fragment of fucosyltransferase (clone T6-7) was isolated from the T6 library. The panel of selected TAAs in Table 2 [see Additional file 1] contains several functionally defined gene products, previously unknown as tumor antigens. AKAP450 and Sos1 proteins, corresponding to clones T5-8 and T5-13, are intracellular components of the signal transduction pathway. Sos1 is a well-known guanine nucleotide exchange factor for Ras oncogene [21]. Transgenic mice expressing a dominant form of Sos in basal keratinocytes Page 7 of 10 (page number not for citation purposes)