A method for rapid gain-of-function studies in the mouse embryonic nervoussystem

Abstract

We used ultrasound image-guided injections of high-titer retroviral vectorsto obtain widespread introduction of genes into the mouse nervous system in utero as early as embryonic day 8.5 (E8.5). The vectors used includedinternal promoters that substantially improved proviral gene expression inthe ventricular zone of the brain. To demonstrate the utility of this system,we extended our previous work in vitro by infecting the telencephalon in vivo as early as E8.5 with a virus expressing Sonic Hedgehog. Infectedembryos showed gross morphological brain defects, as well as ectopic expressionof ventral telencephalic markers characteristic of either the medial or lateralganglionic eminences.

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Figure 1: Schematic representations of the retroviral constructs.
Figure 2: P21 phenotype of cells infected with PLAP-expressing viruses at E9.5.
Figure 3: Internal promoters improved vector expression within the ventricularzone.
Figure 4: Comparison of neuron distribution in samples infected with CLIA(G)and CLC(G).
Figure 5: Injection of retroviral stocks at E8.5 resulted in widespread infection.
Figure 6: Morphological defects in embryos infected with a Shh-expressing virus(CLES).
Figure 7: Dorsolateral Shh expression resulted in ectopic expression of ventralmarkers.

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Acknowledgements

We thank Rusty Lansford for bringing the CA regulatory element to our attentionand Alex Langston and Robin Kimmel for input at the start of this work. Wealso thank Maria McCarthy and Connie Cepko for discussions, Michelle Starz-Gaianofor reading the manuscript, Ulf Eriksson for providing the anti-CRBP antibodyand K. Mahon for the dlx2 in situ probe. This work was supported by NIH grantsNS32993 (G.F.), NS38461 (D.H.T.) and HL62334 (D.H.T.). Additional supportwas provided by March of Dimes Grant 6-FY99-634 (G.F.). N.G. is supportedby a postdoctoral fellowship from the American Cancer Society (PF4473).

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Correspondence to Daniel H. Turnbull or Gord Fishell.

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Gaiano, N., Kohtz, J., Turnbull, D. et al. A method for rapid gain-of-function studies in the mouse embryonic nervoussystem. Nat Neurosci 2, 812–819 (1999). https://doi.org/10.1038/12186

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