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Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos

Abstract

Using ultrasound-guided in utero infections of fluorescently traceable lentiviruses carrying RNAi or Cre recombinase into mouse embryos, we have demonstrated noninvasive, highly efficient selective transduction of surface epithelium, in which progenitors stably incorporate and propagate the desired genetic alterations. We achieved epidermal-specific infection using small generic promoters of existing lentiviral short hairpin RNA libraries, thus enabling rapid assessment of gene function as well as complex genetic interactions in skin morphogenesis and disease in vivo. We adapted this technology to devise a new quantitative method for ascertaining whether a gene confers a growth advantage or disadvantage in skin tumorigenesis. Using α1-catenin as a model, we uncover new insights into its role as a widely expressed tumor suppressor and reveal physiological interactions between Ctnna1 and the Hras1-Mapk3 and Trp53 gene pathways in regulating skin cell proliferation and apoptosis. Our study illustrates the strategy and its broad applicability for investigations of tissue morphogenesis, lineage specification and cancers.

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Figure 1: Intra-amniotic injection of lentivirus at E9.5 results in noninvasive, high-efficiency, stable and epidermally restricted transduction.
Figure 2: Epidermal infection depends on viral titer and permits delivery of multiple viral constructs.
Figure 3: Rapid assay for measuring an epidermal growth advantage or disadvantage conferred by a gene mutation reveals an unexpected growth disadvantage following α1-catenin loss despite hyperproliferation.
Figure 4: Efficient epidermal-specific lentivirus RNAi-mediated knockdown of Ctnna1 faithfully recapitulates phenotypic abnormalities shown by K14-Cre conditional and LV-Cre induced knockout counterparts.
Figure 5: Use of RNAi knockdown in vivo to functionally dissect why loss of α1-catenin results in hyperproliferation but a growth disadvantage to the epidermis.

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Acknowledgements

We thank M. Takeichi (RIKEN CDB) for antibodies and reagents; N. Stokes and Rockefeller Comparative Bioscience Center staff for expert care of mice; M. Schober and M. Perez-Moreno for helpful discussions; A. North and Rockefeller Bioimaging Resource Center staff for assistance with image acquisition and analysis; and S. Mazel and Rockefeller Flow Cytometry Resource Center staff for assistance with FACS. S.B. is supported by the International Human Frontier Science Program Organization. S.W. is an American Cancer Society Postdoctoral Fellow. This work was supported by a grant from the US National Institutes of Health (R01-AR27883). E.F. is an investigator with the Howard Hughes Medical Institute.

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S.B., G.L. and S.W. designed and performed the experiments and analyzed the raw data. S.B. and E.F. wrote the manuscript. E.F. supervised the project.

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Correspondence to Elaine Fuchs.

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The authors declare no competing financial interests.

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Beronja, S., Livshits, G., Williams, S. et al. Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nat Med 16, 821–827 (2010). https://doi.org/10.1038/nm.2167

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