关键词: Cas9, 荧光素酶, 胰腺癌, 活体成像, 基因编辑

Abstract: Objective To establish human and mouse pancreatic cancer cell strains stably expressing Cas9 protein, green fluorescent protein, red fluorescent proteins, luciferase-tdTomato, and to validate the activity of luciferase and gene editing of Cas9 function for pancreatic cancer research using luciferase and CRISPR/Cas9 system. Methods In human pancreatic cancer cells (AsPC-1, CFPAC-1, HPAC, BxPC-3, HS 766T, MIA PaCa-2, PANC-1, and SW 1990), and mouse pancreatic cancer cell (Pan02), the cells were infected with Cas9-expressing plasmid pLv-EF1α-Cas9m1.1-Puro, and single-cell clones were selected for culture and expansion. After extracting the total protein, Western blot verified the expression level of Cas9; Infected with fluorescent protein expression plasmids pLv-EF1α-EGFP, pLv-EF1α-mCherry, pLv-EF1α-tdTomato, pLv-EF1α-Luc2-tdT, and selected single cell clones stably expressing fluorescent proteins were cultured and amplified under fluorescence microscope. Cas9 stable expression cell line was selected to be infected with pLv-EF1α-Luc2-tdT, and the monoclonal culture of stable expression of fluorescent proteins was selected for expansion under fluorescence microscope. One of the cell lines were selected to be infected with Lv-EF1a-mCherry, and the mCherry-positive cells were sorted out by flow cytometry, and then the guide RNA of mCherry gene was then infected by lentivirus to target the mCherry gene, and after cell expansion, mCherry knockdown was detected by fluorescence microscope observation and flow cytometry; 5 BALB/c Nude mice were subcutaneously inoculated with MIA PaCa-2-Luc2-tdT cells (1.0×10⁷/cells each), and imaged in vivo after 36 days. Results 48 human pancreatic cancer cell strains with stable Cas9 expression were screened(including 23 cells expressing Cas9m1.1, 25 cells expressing Cas9m1.1-Luc2-tdT),33 pancreatic cancer cell strains with stable expression of fluorescent proteins were screened (8 cells expressing EGFP, 7 expressing mCherry, and 9 each expressing Luc2-tdT and tdTomato). Cells expressing mCherry and Cas9 were infected with mCherry gRNA and mCherry was knocked down. In vivo imaging showed that both bioluminescence and fluorescence luminescence were present in MIA PaCa-2 cells expressing Luc2-tdT. Conclusions 33 pancreatic cancer cell strains with stable expression of fluorescent proteins are successfully established, in which the Luc2-tdT-expressing cell strains have luciferase activity; 48 pancreatic cancer cell strains with stable expression of Cas9 are successfully established, and the Cas9 protein has gene editing activity, gene editing activity varies depending on the original cell strains.

Key words: Cas9, luciferase, pancreatic cancer, living image, gene editing