Gene transfer into nerve and muscle by isolated limb perfusion or during replantation

In this study, the authors tested the feasibility of adenovirus vectors transferring functional genetic material into relevent soft-tissue structures during replantation of mouse hindlimbs. An adenovirus vector was constructed encoding the marker gene LacZ and CMV promoter and titered by plaque forming assay to 5 x 10(9) particles/ml. C3H mouse hindlimbs were divided into three groups. In Group 1 (n = 9), the femoral neurovascular bundle was divided and re-anastomosed . Group 2 (n = 9) hindlimbs were transected at mid-femur, perfused with adenovirus, and replanted. Group 3 limbs (n = 4) were perfused with saline only, followed by replantation. After 48 hr, morbidity and mortality were assessed, and the replanted limbs were assayed for gene transfer by histochemistry and polymerase chain reaction. 12/18 limbs were viable after 48 hr. Histochemical staining for adenovirus-mediated LacZ expression was positive within skeletal muscle, femoral nerve, and capillaries adjacent to the anastomoses. Distal muscle was also gene transfer positive. PCR analysis confirmed adenovirus-mediated gene transfer within the femoral nerve and skeletal muscle. This study confirms that viral-mediated gene transfer can be accomplished into the soft tissues of a replanted extremity.