Figure 3.

Temporal modeling via three inducible systems. As human malignancies often involve several mutations, a compound inducible system may be employed to target three successive transgenes to the same cell type. For example, mtKras may be first induced through a TVA/RCAS virus system. In this system, expression of a TVA receptor is targeted to the pancreas via the elastase promoter. Upon reaching adulthood, animals can be administered a RCAS virus coding for the mtKras gene. This will interact only with cells expressing the TVA receptor, allowing for targeted and inducible expression of KRAS in the pancreas. A second mutation, such as loss of p16, can then be induced in the same cells via an elastase driven tTA that, in the presence of doxycycline, will induce expression of Cre through TRE-Cre. Combining this with a p16^flox/flox gene will allow for doxycycline-induced loss of the p16 gene, and the second genetic hit. Finally, a tamoxifen-responsive Sox9-FLP^ERT2 can target cells expressing ductal markers (including those having undergone acinar-ductal metaplasia), allowing for inducible expression of mtP53 via an FSF cassette, providing the third genetic hit as it often occurs in humans. It is important at each induction point that promoter/gene regulatory elements employed to run the next step be evaluated in the previous model. Hence, acinar-specific markers (eg., Amylase) should be assessed in pancreas following mutant Kras expression (TVA/RCAS delivery) and Sox9 antibodies should be used to demonstrate Sox9 expression in mtKras expressing pancreas with loss of p16. This would need to be done at the empirically derived time points (times provided in this figure are merely considerations) when the next induction is scheduled to begin.