Establishment of Human DCLK1 Isoform 2 Isogenic Cells Using HCT116 Cells
Valeria Campbell Brown, Lianna Li
Biology Department, Tougaloo College, Tougaloo, MS
DoubleCortin-like Kinase (1) (DCLK1) is a cancer stem cell (CSC) marker that is over-expressed in CSCs and epithelial-mesenchymal transition (EMT) cells of many cancers. Due to its expression in the therapy-resistant, tumorigenic subpopulation in cancer tissue, DCLK1 plays critical roles in indefinite cell proliferation, tumorigenesis, tumor metastasis, and recurrence of cancer. Further evidence has shown that the de-regulation or inhibition of DCLK1 directly causes a decrease in cancer succession and reduces the possibility of relapse. Since DCLK1 is so crucial to overall cancer progression, it’s important to better understand how its over-expression specifically impacts the stages and development of human colorectal cancer (hCRC). However, DCLK1 has five isoforms and association of each isoform with hCRC is unclear. For the current project, we aim to establish the DCLK1 isoform 2 (DCLK1-S) isogenic cell clone to further investigate how DCLK1-S affects tumorigenesis of hCRC. In order to achieve our goal, we transformed the Myc-DDK-tagged human DCLK1-S cDNA into the TOP10 competent cells and plated onto the Kanamycin-selection LB agar plate. Four colonies were selected for DNA plasmid extraction and the extracted DNA were sent for DNA sequencing. The DNA with correct sequencing were transfected into the HCT116 cells using the Lipo3000. Forty eight hours after transfection, cells were selected using neomycin for two weeks. Afterwards, neomycin-resistant cells were trypsinized into single cell suspension and plated into 96-well plate with 1 cell per well in the neomycin-selection medium. After isogenic cells were transferred into T-25 flask, whole cell lysate was prepared to confirm DCLK1-S expression using Western Blot. Our results demonstrated that of the 30 neomycin-resistant isogenic cell clones, DCLK1-S expression was significantly increased in 3 of them. These three clones will be used to further evaluate effect of DCLK1-S on the tumorigenesis of hCRC.