Mesenchymal stem cells (MSCs) are pluripotent cells that reside primarily in the mammalian bone marrow. They possess self-renewal capacity and the ability to differentiate in vivo and in vitro into a number of mesodermal derivatives, including fat, bone and cartilage. These features along with their vast proliferative potential make MSCs an excellent tool for cell-based gene therapy strategies. Development of methods for improved gene delivery in MSCs and appropriate large animal models for testing various therapeutic protocols are needed. Therefore, we have designed experiments to: a) isolate pig MSCs (pMSCs) from bone marrow and identify appropriate in vitro culture conditions for these cells; b) investigate non-viral and viral approaches for transient and stable gene delivery into pMSCs; c) develop methods for optimal gene delivery in pMSCs using adenoviral vectors complexed with GeneJammer, a commercial polyamine-based transfection reagent; and d) study the effect of GeneJammer on adenoviral transduction of human cell lines. The results demonstrate that MSCs can be readily obtained from the bone marrow of pigs. Porcine MSCs grew well in a basic media and their proliferation was enhanced when cultured in low oxygen atmosphere. Transient and stable genetic modification of pMSCs was obtained by non-viral and viral vectors. Presence of GeneJammer during adenoviral vector transduction enhanced vector-encoded transgene expression in pMSCs. Interestingly, GeneJammer transduced cells retained multipotential differentiation capability in vitro. Similarly, GeneJammer improved adenoviral gene delivery efficiency in human MSCs, human mononuclear peripheral blood cells, and rodent cells lines. Human and murine cell lines infected with adenovectors carrying the gene for bone morphogenetic protein 2 (BMP2) in presence of GeneJammer achieved higher levels of BMP2 expression in vitro and in vivo. In conclusion, we have been able to established adult pMSC lines from live animals using a minimally invasive BM aspiration technique. These adult stem cells can undergo transient and stable genetic modification with non-viral and viral vectors. The use of GeneJammer as described in these studies leads to high transgene expression levels in porcine and human MSCs and rodent cells lines. These results will facilitate future use of adenoviral vectors in MSC-mediated gene therapy models and therapies.