Effects of lipid shell microbubble on ultrasound mediated EGFP gene delivery to transplanted tumors:initial experience

Objective： To investigate the feasibility of ultrasound （US） mediated enhanced green fluorescent protein （EGFP） gene delivery in subcutaneous transplanted tumors of human cervical carcinoma （He/a） and the contribution of lipid shell microbubble （LSMB） on gene transfection. Methods： LSMB and plasmid were injected into nude mice by tail vein followed local US irradiation （P ＋ LSMB ＋ US group）. US exposure parameter was set at 2.0 W/cm2, 2 rain, duty cycle 20%. EGFP expression was evaluated by imaging for 7 days. Nude mice undergoing plasmid injection alone （P group）, plasmid injection and US exposure （P ＋ US group）, plasmid and LSMB injection （P ＋ LSMB group） were used as controls. Frozen section and histological examinations were conducted. Expression of EGFP was scored. Kinetics of protein expression post transfection and localization in vivo were evaluated. Results： Plasmid injection with LSMB plus US exposure strongly increased gene transfer efficiency. Strong EGFP expression was mainly seen in LSMB ＋ P ＋ US group. It was significantly higher than any of the following groups, P group, US ＋ P group, or LSMB ＋ P group （P 〈 0.01）./n vivo expression level of post-US 3 days was significantly higher than any other time points （P 〈 0.01）. There was not significant expression level of EGFP in other organs or tissues regardless of US exposure. No tissue damage was seen histologically. Conclusion： The combination of LSMB and US exposure could effectively transfer plasmid DNA to transplanted tumors without causing any apparently adverse effect. LSMB could be effective as a non-viral vector system in in vivo gene delivery. It would be a safe gene delivery method and provide an alternative to current clinical gene therapy.

The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos

Primordial germ cells (PGCs), as precursors of mam-malian germ lineage, have been gaining more attention as anew resource of pluripotent stem cells, which bring a greatpossibility to study developmental events of germ cell invitro and at animal level. EG4 cells derived from 10.5 dayspost coitum (dpc) PGCs of l29/svJ strain mouse wereestablished and maintained in an undifferentiated state.With an attempt to study the differentiation capability ofEG4 cells with a reporter protein: green fluorescence pro-tein, and the possible application of EG4 cells in the re-search of germ cell development, we have generated severalEG4-GFP cell lines expressing enhanced green fluorescenceprotein (EGFP) and still maintaining typicaI characteris-tics of pluripotent stem cells. Then, the differentiation ofEG4-GFP cells in vitro as well as their developmental fatein chimeric embryos which were produced by aggregatingEG4-GFP cells to 8-cell stage embryos were studied. Theresults showed that EG4 cells carrying green fluorescencehave a potential use in the research of germ cell develop-ment and other related studies.