Fluorescence-Activated Sorting of Totipotent Embryonic Stem Cells Expressing Developmentally Regulated lacZ Fusion Genes

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 15; pp. 6721 - 6725
• Main Authors: Reddy, Sita, Rayburn, Helen, von Melchner, Harald, Ruley, H. Earl
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01-08-1992; National Acad Sciences; National Academy of Sciences
• Subjects: 3T3 cells; Animals; beta-Galactosidase - genetics; beta-Galactosidase - metabolism; Biological and medical sciences; Blastocyst - cytology; Blastocyst - physiology; Cell Differentiation; Cell division; Cell Line; Cell lines; Chimera; Clone Cells; Embryo Implantation; Embryology: invertebrates and vertebrates. Teratology; Embryonic stem cells; Embryos; Female; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; Genes; Genetic engineering; Germ cells; Male; Medical research; Mice; Mice, Inbred C57BL; Molecular embryology; NIH 3T3 cells; Nucleic Acid Hybridization; Proviruses; Recombinant Fusion Proteins - metabolism; Restriction Mapping; Stem cells; Stem Cells - cytology; Totipotent stem cells; Transfection; Viruses; Flow cytometry; Embryo; Stem cell; Rodentia; Repression; Cell differentiation; Gene expression; Vertebrata; Regulation(control); Mammalia; Reporter gene; Mouse; Pluripotent cell; Development; Hybrid gene
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.15.6721

Murine embryonic stem (ES) cells were infected with a retrovirus promoter trap vector, and clones expressing lacZ fusion genes (LacZ+) were isolated by fluorescence-activated cell sorting (FACS). Of 12 fusion genes tested, 1 was repressed when ES cells were allowed to differentiate in vitro. Two of three lacZ fusion genes tested were passed into the germ line, indicating that FACS does not significantly affect stem cell totipotency. The pattern of lacZ expression observed in vivo was consistent with that seen in vitro. Both fusion genes were expressed in preimplantation blastulas. However, a fusion gene whose expression was unaffected by in vitro differentiation was ubiquitously expressed in day-10 embryos, while the other, which showed regulated expression in vitro, was restricted to cells located along the posterior neural fold, the optic chiasm, and within the fourth ventricle. These results demonstrate the utility of using promoter trap vectors in conjunction with fluorescence sorting to disrupt developmentally regulated genes in mice.