Fluorescence-activated sorting of totipotent embryonic stem cells expressing developmentally regulated lacZ fusion genes.

Reddy S, Rayburn H, von Melchner H, Ruley HE
Proc Natl Acad Sci U S A. 1992 89 (15): 6721-5

PMID: 1495960 · PMCID: PMC49575 · 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.

MeSH Terms (18)

Animals beta-Galactosidase Blastocyst Cell Differentiation Cell Line Chimera Clone Cells Embryo Implantation Female Flow Cytometry Male Mice Mice, Inbred C57BL Nucleic Acid Hybridization Recombinant Fusion Proteins Restriction Mapping Stem Cells Transfection

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