Functional Characterization of Two Novel Human Prostate Cancer Metastasis Related Genes
Annual rept. 16 Jan 2004-15 Jan 2005
TULANE UNIV HEALTH SCIENCE CENTER NEW ORLEANS LA
Pagination or Media Count:
We propose to identify the functional characterization of two novel cancer-specific, metastasis-related genes whose constitutive expression may be pivotal for prostate cancer progression. Work accomplished was performed based on the proposed statement of work. We have characterized the full-length cDNAs of the Seq1 and Seq2 genes using at least three 5 and 3 rapid amplifications of cDNA ends RACE commercial kits Invitrogen Carlsbad, CA, BD Bioscience Clontech Inc, and Seegene, Rockville, MD. To optimize the PCR conditions for each kit, we had designed several sets of gene-specific primers GSP23-28 nt long with 50-70 GC and Tm of 55 to 75 degrees C for each gene. We have also designed several sets of nested GSPs to verify our cloned genes. Because of unique secondary structures, high GC content, short SSH sequences, and low levels of expression of these genes in prostate cancer cell lines, we had great deal of difficulty in accomplishing this task in a timely fashion. As such, we devised different strategies for the first-strand synthesis using a modified oligodT primers 5-CDS primer or 3-CDS primer, and Smart oligo II primer under various conditions. The full-length cDNA sequences were subcloned into mammalian expression vectors Invitrogen and ready to be used for generation of recombinant proteins and antibody production.
- Medicine and Medical Research