Understanding the molecular circuitry of cell lineage specification in the early mouse embryo.

Pluripotent stem cells hold great promise for cell-based therapies in regenerative medicine. However, critical to understanding and exploiting mechanisms of cell lineage specification, epigenetic reprogramming, and the optimal environment for maintaining and differentiating pluripotent stem cells is a fundamental knowledge of how these events occur in normal embryogenesis. The early mouse embryo has provided an excellent model to interrogate events crucial in cell lineage commitment and plasticity, as well as for embryo-derived lineage-specific stem cells and induced pluripotent stem (iPS) cells.

A Cre-loxP based system for studying horizontal gene transfer.

We have previously shown that DNA can be transferred to phagocytosing cells via the uptake of apoptotic cells. We report a model system that facilitates study of antigen presentation of genes transferred specifically via horizontal gene transfer. Constructs were generated encoding the LacZ gene or the influenza A nucleoprotein silenced by a STOP sequence flanked by two loxP sites.

DNase II and the Chk2 DNA damage pathway form a genetic barrier blocking replication of horizontally transferred DNA.

We have previously shown that DNA from dying tumor cells may be transferred to living cells via the uptake of apoptotic bodies and may contribute to tumor progression. DNA encoding H-ras(V12) and c-myc oncogenes may be transferred to the nucleus of the phagocyte but will only integrate and propagate in p53- and p21-deficient mouse embryonic fibroblasts, whereas normal cells are resistant to transformation. Here, we show that this protective mechanism (activation of p53 and p21 after uptake of apoptotic bodies) is dependent on DNA fragmentation, where inhibition of the caspase-activated DNase in the apoptotic cells, in conjunction with genetic ablation of lysosomal DNase II in the phagocytes, completely blocks p53 activation and consequently allows DNA replication of transferred DNA.

Loss of the p21(Cip1/Waf1) cyclin kinase inhibitor results in propagation of horizontally transferred DNA.

We have shown previously that phagocytosis of cells dying by apoptosis results in transfer of whole or fragments of chromosomes into the nucleus of the recipient cell. Although DNA transfer was detected in normal cells, stable propagation of the transferred DNA was only observed in cells deficient in p53. Here we show that mouse embryonic fibroblast cells lacking the p21 (Cip1/Waf1) cyclin-kinase inhibitor are able to propagate DNA engulfed by phagocytosis of apoptotic bodies.