Construction of a quantitative PCR system to determine expression of tumor associated antigen

Cancer immunotherapy is a strategy for cancer treatment by induction of anti tumor responses. The identification of candidate tumor associated antigens (TAA) suggested their potential use as immunogens for vaccination studies. Quantification of a TAA expression by cancerous cells is an important factor in determination of induced immune response efficiency against tumors and thus enables us to devise optimal immunotherapy protocol to cure cancer. The quantitative polymerase chain reaction (PCR) enables us to compare the TAA expression in highly metastatic tumor clones with that in less metastatic ones and in normal cells. It allows us to gain more insight into genome rearrangements that occur in malignant transformation as well as broaden our knowledge about tumor cell gene expression regulation. One of the peptides isolated previously in our lab from a murine lung carcinoma is the mutated Connexin 37 (cx37), a gap-junction protein. Research is underway to determine the expression level of the TAA in various Lewis lung carcinoma cells. This evaluation is achieved by means of quantitative PCR. A quantitative PCR experiment includes preparation of a control template, which is added in known amounts together with the target template in a series of amplification reactions. The control template uses the same primers as the target sequence, yet their PCR products differ in size so as to be distinguishable. Two methods were used to produce this control template. The first one included specific deletion of a sequence of approximately 100 bp that lay between the two primers, insertion of the new template into a plasmid vector, transformation of competent bacteria, detection of transformed bacterial colonies and isolation of the plasmid DNA in a large quantity. The non-mutated, deleted Connexin 37 cDNA was also isolated from bacteria and used for another experiment aimed at producing deleted, mutated Connexin 37 cDNA by means of primer mutagenesis. (Fig. 5, Ref 8.)