Rapid In Vivo Validation of Tumor Suppressor Gene Function in Prostate Cancer Progression
Technical Report,15 Apr 2015,14 Apr 2016
Trustees of the University Pennsylvania Philadelphia United States
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We have established a powerful system to interrogate CRISPR efficiency in a standard 3 day cell culture transfection system. Our system works exactly as anticipated and we were able to identify potent sgRNAs that target the tumor suppressors p53 and Rb. The lentiviral constructs that we designed and cloned efficiently disrupted a heterologous gene construct containing an mCherry cDNA fused to the sgRNA target. As predicted, we observed variable efficiencies between sgRNAs. Our assay facilitated the rapid identification of the best sgRNA sequences and accelerated our ability to move to the in vivo studies proposed in Aim2. Our goal was to use CRISPRCas lentiviral transduction of the adult prostate to inactivate p53 or Rb. We timed to recapitulate the effects of conditional floxed alleles of p53 and Rb and to initiate prostate cancer in the mouse after injection of lentiviral particles expressing CRISPRCas components and Cre recombinase. Our initial efforts were thwarted by the negative impact that the relatively large size of our lentiviral construct had on our ability to generate high-titer lentivirus needed for the infection. We circumvented this by re-designing the lentiviral construct to delete unnecessary viral elements and utilize alternative promoter sequences that were smaller to overall reduce the size of the lentiviral genome to be packaged. This effort allowed us to generate 10 to 50 fold higher viral titers. We have used these viral preps to infect the adult prostate of mice. Currently, we are aging mice to determine the effects of the procedure. At this time the animals are 20 weeks post infection and, based on the published latency of the model, we anticipate the onset of tumors between 35 and 60 weeks post infection.
- Medicine and Medical Research