Encyclopedia of Experiments: Cancer Research
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Transcript
Begin with an anesthetized and prepped CRISPR/Cas9 knock-in mouse model bearing a genome engineered to express Cas9 endonucleases and green fluorescent proteins, or GFPs, by a strong upstream promoter. A floxed-STOP cassette or LSL inserted immediately downstream to the promoter blocks Cas9 and GFP transcription under normal conditions.
Incise the mouse's abdominal wall to expose its anterior prostate lobe attached to the seminal vesicle. Inject the desired adenoviral suspension into the anterior lobe to facilitate targeted viral genome delivery into the cells. Place the prostate lobe back into the abdominal cavity and suture the incision.
Within virus-infected cells, the viral genome expresses Cre recombinase enzymes and guide-RNAs targeting the gene to be mutated. The Cre enzymes recognize the LSL cassette and excise the floxed transcription blocker. This step allows the promoter to drive the expression of Cas9 endonucleases and GFPs.
Subsequently, Cas9 endonucleases form complexes with virus-encoded guide-RNAs that direct these complexes to the target sequence within the gene to be mutated. This localization allows the Cas9 endonuclease to cleave the genomic DNA within the target gene, leading to genetic alteration.
An oncogenic alteration causes mutated cells to turn cancerous. The co-expression of GFP reporter proteins within mutated cells facilitates the identification and tracking of cancer progression.
