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Cells transduced with lentiviral vectors are individually marked by a highly characteristic pattern of insertion sites inherited by all their progeny. We have recently extended this principle of clonal cell marking by introducing the method of RGB marking, which makes use of the simultaneous transduction of target cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. In accordance with the additive color model, individual RGB-marked cells display a large variety of unique and highly specific colors. Color codes remain stable after cell division and can thus be used for clonal tracking in vivo and in vitro. Our protocol for efficient RGB marking is based on established methods of lentiviral vector production (3-4 d) and titration (3 d). The final RGB-marking step requires concurrent transduction with the three RGB vectors at equalized multiplicities of infection (1-12 h). The initial efficiency of RGB marking can be assessed after 2-4 d by flow cytometry and/or fluorescence microscopy.

Improvements in surgical technique and the introduction of several new immunosuppressive medications mean that outcome after orthotopic liver transplantation (OLT) has improved continuously over the past 15 years. Given the increasing longevity of patients after OLT, the recognition and prevention of long-term complications after transplantation have become ever more important. With respect to graft function, physicians responsible for the everyday care of patients following transplantation should be particularly aware of the risk of late and chronic rejection episodes and of recurrence of the underlying liver disease. The major challenge of post-transplant care is, however, how best to prevent and manage the long-term adverse effects caused by the immunosuppressive medications prescribed. Screening investigations for early diagnosis of malignancy, strict control of cardiovascular risk factors, preservation of renal function, and prevention of infections are, therefore, fundamental. This Review suggests guidelines for the management of OLT recipients to improve long-term survival, overall outcome and health-related quality of life.