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. 2007 Mar 13;104(11):4455-60.
doi: 10.1073/pnas.0609277104. Epub 2007 Mar 2.

Developmental and adult phenotyping directly from mutant embryonic stem cells

Sophia H L George  1 , Marina Gertsenstein, Kristina Vintersten, Ella Korets-Smith, John Murphy, Mary E Stevens, Jody J Haigh, Andras Nagy
Affiliations

Developmental and adult phenotyping directly from mutant embryonic stem cells

Sophia H L George et al. Proc Natl Acad Sci U S A. .

Abstract

Tetraploid embryo complementation assay has shown that mouse ES cells alone are capable of supporting embryonic development and adult life of mice. Newly established F(1) hybrid ES cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell-based genetics feasible. Here we report the establishment and characterization of 12 new F(1) hybrid ES cell lines and the use of one of the best (G4) in a gain- and loss-of-function genetic study, where the in vivo phenotypes were assessed directly from ES cell-derived embryos. We found the generation of G4 ES cell-derived animals to be very efficient. Furthermore, even after two consecutive rounds of genetic modifications, the majority of transgenic lines retained the original potential of the parental lines; with 10-40% of chimeras producing ES cell-derived animals/embryos. Using these genetically altered ES cells, this success rate, in most cases, permitted the derivation of a sufficient number of mutants for initial phenotypic analyses only a few weeks after the establishment of the cell lines. Although the experimental design has to take into account a moderate level of uncontrolled damage on ES cell lines, our proof-of-principle experiment provides useful data to assist future designs harnessing the power of this technology to accelerate our understanding of gene function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The efficiency of generating completely ES cell-derived newborns and adult mice from different F1 hybrid ES cell lines with tetraploid embryo complementation assay. The genetic background first shows the maternal component. P < 0.05. The YV1 line contains an overall expressed yellow fluorescent protein transgene; Tg(ACTB-EYFP) 2 Nagy. m, male; f, female.
Fig. 2.
Fig. 2.
The performance of single electroporated (parental) and double electroporated (neutral transgenic) ES cell lines in the tetraploid embryo complementation assay. p, parental; d, daughter; n, neutral.
Fig. 3.
Fig. 3.
Structure and basic properties of conditional and activated transgenes.
Fig. 4.
Fig. 4.
Phenotype of embryos from crosses between the conditional transgenic and a Cre deletor line recapitulated the phenotype observed in corresponding ES cell-derived embryos. (A and B) Single transgenic E9.5 embryo and yolk sac (wild type). (C and D) Double transgenic littermate expressing VEGF-A from the activated transgene. The yolk sacs (B and D) were stained for PECAM to visualize the organization of the endothelial cells. (E and F) Single transgenic E9.5 embryo and its cross-section at the caudal level. (G and H) Corresponding double transgenic littermate with Flt1-Fc expression activation. (I) Single transgenic “wild-type” embryo at E14.5. (J and K) A corresponding double transgenic littermate displaying sites of hemorrhage (black arrowheads) and edema (white arrowhead). (L and M) H&E section of wild-type and SCF expressor embryonic livers showing severe necrosis in the latter. (Scale bars: 100 μm.)
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