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New approach to cell lineage analysis in mammals using the Cre-loxP system.
Mol Reprod Dev. 2000 May; 56(1):34-44.MR

Abstract

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 beta-actin promoter (CAG), a portion 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 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 beta-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 beta-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 beta-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.

Authors+Show Affiliations

Department of Molecular and Developmental Science, Molecular Medicine Research Center, The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa, Japan. massasato@is.icc.u-tokai.ac.jpNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10737965

Citation

Sato, M, et al. "New Approach to Cell Lineage Analysis in Mammals Using the Cre-loxP System." Molecular Reproduction and Development, vol. 56, no. 1, 2000, pp. 34-44.
Sato M, Yasuoka Y, Kodama H, et al. New approach to cell lineage analysis in mammals using the Cre-loxP system. Mol Reprod Dev. 2000;56(1):34-44.
Sato, M., Yasuoka, Y., Kodama, H., Watanabe, T., Miyazaki, J. I., & Kimura, M. (2000). New approach to cell lineage analysis in mammals using the Cre-loxP system. Molecular Reproduction and Development, 56(1), 34-44.
Sato M, et al. New Approach to Cell Lineage Analysis in Mammals Using the Cre-loxP System. Mol Reprod Dev. 2000;56(1):34-44. PubMed PMID: 10737965.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - New approach to cell lineage analysis in mammals using the Cre-loxP system. AU - Sato,M, AU - Yasuoka,Y, AU - Kodama,H, AU - Watanabe,T, AU - Miyazaki,J I, AU - Kimura,M, PY - 2000/3/29/pubmed PY - 2000/7/6/medline PY - 2000/3/29/entrez SP - 34 EP - 44 JF - Molecular reproduction and development JO - Mol Reprod Dev VL - 56 IS - 1 N2 - 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 beta-actin promoter (CAG), a portion 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 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 beta-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 beta-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 beta-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. SN - 1040-452X UR - https://www.unboundmedicine.com/medline/citation/10737965/New_approach_to_cell_lineage_analysis_in_mammals_using_the_Cre_loxP_system_ L2 - https://doi.org/10.1002/(SICI)1098-2795(200005)56:1<34::AID-MRD5>3.0.CO;2-M DB - PRIME DP - Unbound Medicine ER -