Cre-loxP System Confers Cell Lineage-Specific Expression of a Reporter Gene in Murine Preimplantation Development.

Abstract

The Cre-loxP site-specific recombination system is useful for tracing the fate of cells via transient expression of the Cre recombinase gene directly introduced into the cells with a vector carrying loxP sites. In this study, we used this system to examine whether cell lineage-specific expression of a reporter gene occurs in murine preimplantation embryos. An expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken β-actin promoter (CAG), a portion of the rabbit β-globin gene (including the 2nd intron, 3rd exon and 3' noncoding region), loxP-flanked DNA sequence [containing enhanced green fluorescent protein (EGFP) cDNA and chloramphenicol acetyltransferase gene (CAT)], and lacZ gene encoding E. coli β-galactosidase (β-gal), was constructed. When pCETZ-17 DNA was microinjected into the pronuclei of fertilized eggs, 30.0% (6/20) of the surviving 2-cell embryos exhibited distinct fluorescence in both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for β-gal. When both plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, all (12/12) embryos exhibited low or no fluorescence, but 66.7% (8/12) exhibited positive staining for β-gal activity in one or both blastomeres. This indicates that transient expression of Cre 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 2-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing 2-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 2 days up to 8-cell stage. When the developing 8-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for β-gal activity was observed in all (7/7) embryos. These findings indicate that this Cre-loxP system, which is based on transient expression of the Cre gene directly introduced into nuclei of embryonic cells by microinjection, is useful for tracing cell lineage as well as for expressing a gene of interest in a lineage-specific manner in early embryogenesis