Establishing a Murine Tumor Cell Line In Vitro Using a Virus-Cas9 System

Begin with primary murine epithelial cells carrying a Cre-dependent Cas9 gene silenced by a stop sequence.
Take viral particles containing DNA encoding Cre recombinase, guide RNAs targeting the cancer-associated genes—Kras, p53, and Apc, along with a repair template carrying a cancer-driving mutation.
Add the viral particles to the cells and incubate.
The particles enter the cells and release their DNA, triggering expression of Cre recombinase, guide RNAs, and the repair template.
Cre recombinase removes the stop sequence, enabling Cas9 expression.
Guide RNAs bind Cas9 and direct it to target genes, where Cas9 cleaves the DNA.
An error-prone repair introduces mutations and inactivates p53 and Apc, while the repair template repairs the Kras cut and introduces a cancer-driving mutation.
Replace the medium with fresh medium. Incubate.
Cells without the mutation fail to become immortal and die.
Meanwhile, cells with the mutation proliferate, establishing a tumorigenic cell line.

On day 10 of culture, use 0.25% trypsin in 0.2% EDTA for three to five minutes at 37 degrees Celsius to harvest the cells from the cell suspension or cell aggregate cultures, and seed two times 10 to the fifth primary cells in two milliliters of medium per well into each well of the 6-well plate.

The next day, transduce the cells with one times 10 to the 12 viral genome per milliliter and incubate the cells in the viral particle-containing medium for 48 hours at 37 degrees Celsius.

At the end of the incubation, replace the culture supernatants with two milliliters of fresh complete medium per well and return the cells to the cell culture incubator.

After two weeks of culture expansion, seed the cells for validation and in vivo tumorigenic experiments.