New approach to cell lineage analysis in mammals using the cre-loxP system

The Cre‐loxP site‐specific recombination system was used for cell lineage analysis in mammals. We constructed an expression plasmid, pCETZ‐17, which consists of cytomegalovirus enhancer/chicken β‐actin promoter (CAG), a portion of the rabbit β‐globin gene, loxP‐flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E. coli β‐galactosidase (β‐gal). When circular pCETZ‐17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two‐cell stage, 62.8% (59/94) of the two‐cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X‐Gal, a substrate for β‐gal. When both circular plasmids, pCETZ‐17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co‐injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for β‐gal activity. This indicates that transient expression of the Cre recombinase gene removed the loxP‐flanked DNA sequence in pCETZ‐17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ‐17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two‐cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP‐expressing two‐cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four‐cell stage. When the developing four‐cell embryos were subjected to staining with X‐Gal, cell lineage‐related staining pattern for β‐gal activity was observed in most (77.8%, 7/9) embryos. These findings were further confirmed using two‐cell embryos derived from a transgenic mouse line carrying CETZ‐17 transgene. Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals. Mol. Reprod. Dev. 56:34–44, 2000. © 2000 Wiley‐Liss, Inc.