To generate new beta-cells after birth is a key focus of regenerative medicine that could greatly aid the major health burden of diabetes. Beta-cell regeneration has been described using four different approaches: 1) the development of beta-cells from putative precursor cells of the adult pancreas termed neogenesis, 2) replication of existing beta-cells, 3) differentiation from embryonic or induced pluripotent stem cells, and 4) reprogramming of non-beta to beta-cells. Studies from our laboratory have shown that beta-cell reprogramming can be achieved by transduction of adult pancreatic tissues with viral constructs containing the three developmentally important transcription factors Pdx1, Ngn3, and MafA. This protocol outlines the generation of a polycistronic construct containing the three transcription factors, the expansion and purification of the polycistronic virus and in vivo transduction for acinar to beta-cell reprogramming in adult mice. The ultimate goal is to generate beta-like cells that resemble endogenous beta-cells in phenotype and function as closely as possible for potential translational applications.