Splicing Regulations of CD44v3in Breast Cancer Metastasis
The objective of this project is to investigate the molecular mechanisms of the alternative splicing patterns of CD44v3 and how they are regulated in the metastasis of breast cancer cells. The primary goal was to construct a minigene containing the CD44v3 plasmid. The protocol involved multiple steps of cloning, including polymerase chain reaction experiments, gel purification, enzyme digestion, ligation, transformation, and mini-prep procedures. The cloning process was followed by the transfection of the newly constructed minigene into breast cancer cells. The breast cancer cells used were non-metastatic MCF-7 cells and metastatic MDA-MB-231 cells. Data from previous reverse transcription-polymerase chain reaction (RT-PCR) experiments indicated that the MDA cells contain a higher concentration of CD44v3, suggesting that a CD44v3 minigene may be able to serve as a model system of the gene for further study of splicing regulation. Two minigenes were constructed. The first minigene contained the exontrap vector and the variable exon 3. The second minigene experienced more difficulty during cloning than the v3 minigene. The construction of the E5-v3-E16 minigene required a new strategy of inserting exons 5 and 16 individually into the v3 plasmid in order to obtain the complete minigene. Once they were successfully cloned, both the v3 and the E5-v3-E16 minigenes were transfected into breast cancer cells. After the transfection and RT-PCR experiments yield results, the project will continue and begin to investigate the molecular mechanisms of the splicing regulation of CD44v3. Link to PowerPoint? presentation
Baines, Hayley, "Splicing Regulations of CD44v3in Breast Cancer Metastasis " (2003). URC Student Scholarship.
Ren-Jang Lin, M.D. (City of Hope)
Howard Hughes Medical Institute Fellowshipat City of Hope Medical Center