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Structure-function studies in mouse embryonic stem cells using recombinase-mediated cassette exchange

Tim Pieters (UGent) , Lieven Haenebalcke (UGent) , Kenneth Bruneel (UGent) , Niels Vandamme (UGent) , Tino Hochepied (UGent) , Jolanda van Hengel (UGent) , Dagmar Wirth, Geert Berx (UGent) , Jody J Haigh, Frans Van Roy (UGent) , et al.
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Abstract
Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.
Keywords
SELF-RENEWAL, ES CELLS, MICE, GENERATION, GENOME, DERIVATION, CATENIN, CLONING, GENES, Developmental Biology, Issue 122, structure-function, mouse embryonic, stem cells, stem cell isolation, pluripotin, recombination-mediated, cassette exchange, ROSA26 targeting, knock-out, ROSA26 locus, embryoid, bodies, rescue constructs

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Citation

Please use this url to cite or link to this publication:

Chicago
Pieters, Tim, Lieven Haenebalcke, Kenneth Bruneel, Niels Vandamme, Tino Hochepied, Jolanda van Hengel, Dagmar Wirth, et al. 2017. “Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange.” Jove-journal of Visualized Experiments (122).
APA
Pieters, Tim, Haenebalcke, L., Bruneel, K., Vandamme, N., Hochepied, T., van Hengel, J., Wirth, D., et al. (2017). Structure-function studies in mouse embryonic stem cells using recombinase-mediated cassette exchange. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, (122).
Vancouver
1.
Pieters T, Haenebalcke L, Bruneel K, Vandamme N, Hochepied T, van Hengel J, et al. Structure-function studies in mouse embryonic stem cells using recombinase-mediated cassette exchange. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS. 2017;(122).
MLA
Pieters, Tim, Lieven Haenebalcke, Kenneth Bruneel, et al. “Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange.” JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 122 (2017): n. pag. Print.
@article{8524055,
  abstract     = {Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100\% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.},
  articleno    = {e55575},
  author       = {Pieters, Tim and Haenebalcke, Lieven and Bruneel, Kenneth and Vandamme, Niels and Hochepied, Tino and van Hengel, Jolanda and Wirth, Dagmar and Berx, Geert and Haigh, Jody J and Van Roy, Frans and Goossens, Steven},
  issn         = {1940-087X},
  journal      = {JOVE-JOURNAL OF VISUALIZED EXPERIMENTS},
  language     = {eng},
  number       = {122},
  pages        = {10},
  title        = {Structure-function studies in mouse embryonic stem cells using recombinase-mediated cassette exchange},
  url          = {http://dx.doi.org/10.3791/55575},
  year         = {2017},
}

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