Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo.
Bibliographical noteFunding Information:
This work was supported in part by the ASCO Young Investigator Award, the Fanconi Anemia Research Fund, the Children’s Cancer Research Fund, NIH Grants RO1 HL55209, R01 HL49997, R01 HL52952, and R24 CA 92862 (CHC), and the Child Health Research Scholar Award (NIH 5K12-HD033692-10). The confocal microscope was purchased using NCRR shared instrumentation Grant 1 S10 RR16851. We thank Sara Benning, Kevin Tram, and Amy Nordlander for their technical assistance.
- Biophotonic imaging
- Multipotent adult progenitor cell
- Sleeping Beauty
- Stem cell homing
- Whole-body imaging