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 a nalysis in mammals. We constructed an expression plasmid, pCETZ-17, which c onsists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a po rtion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E. coli beta-galactosidase (beta-gal). When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were al lowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos e xhibited distinct fluorescence in one or both blastomeres, but never expres sed lacZ protein, as evaluated by histochemical staining with X-Gal, a subs trate for beta-gal. When both circular plasmids, pCETZ-17 and pCAG/NCre (co ntaining CAG and DNA sequences encoding nuclear location signal and Cre), w ere coinjected into fertilized eggs, almost all (87.0%, 47/54) embryos exhi bited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity. This indicates that transient expression of the Ore recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and the n caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected egg s for 1 day, and collected only two-cell embryos expressing EGFP in both bl astomeres. One blastomere of the EGFP-expressing two-cell embryos was micro injected 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 beta-gal activity was observed in most (77.8%, 7/9) embryos. These findings were fu rther 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 e mbryonic cells by microinjection, is a powerful means for cell lineage anal ysis in mammals. (C) 2000 Wiley-Liss, Inc.