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

Abstract

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 conijunction with fluorescence sorting to disrupt developmentally regulated genes in mice.

Selective Disruption of Genes Transiently Induced in Differentiating Mouse Embryonic Stem Cells by Using Gene Trap Mutagenesis and Site-Specific Recombination

“…Five inducible U3Cre integrations were selected during ES-cell differentiation, two of which were passed to the germ line. Since U3Cre expression was also induced during embryonic development, the results confirm previous studies showing that genes regulated in vitro are also regulated in vivo (18,20,22,27,30,32).…”

“…By first eliminating integrations into active genes, it is possible to screen large pools of recombinant clones for integrations into regulated genes. This circumvents the laborious analysis of individual clones (6,20,35,39).…”

“…The ppgklxLacZ vector was derived from ppgklxtkneoIL3 (24) by replacing the genes encoding tkneo and interleukin 3 (IL-3) with coding sequences for Neo and LacZ, respectively. Briefly, a loxPLacZ fragment was assembled in pGEM30 by blunt-end ligation of the LacZ gene (derived from U3LacZ [20]) into the EcoRI site of the loxP flanking polylinker (24). loxPLacZ was then cloned as a SalI/XhoI fragment into the XhoI site of pSBC-2 (3) upstream of the simian virus 40 polyadenylation site.…”

“…The 5Ј gene codes for neomycin phosphotransferase and is flanked by two loxP recombination targets in identical orientation. The 3Ј gene codes for ␤-galactosidase (␤-Gal) (20) and terminates in a simian virus 40 polyadenylation sequence. To suppress LacZ translation from dicistronic transcripts, two tandem copies of bovine growth hormone polyadenylation sequence were inserted downstream of the neo gene.…”

A rapid and non‐invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP)

“…Reddy et al introduced the LacZ gene into embryonic stem cells and separated the transgenic cells by fluorescence-activated cell sorting [17]. Matsumoto et al used a firefly luciferase and detected enzyme activity at the 2-cell stage of mouse embryo without killing cells [18].…”