May 29, 2025

Public workspaceAnimals: Nestin-Cre::DIO-SPOTlight transgenic mouse line generation

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvjb4pwgk5/v1
  • Matt Oliver1
  • 1Duke University
Matt Oliver
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjb4pwgk5/v1
Protocol Citation: Matt Oliver 2025. Animals: Nestin-Cre::DIO-SPOTlight transgenic mouse line generation. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjb4pwgk5/v1
Manuscript citation:
Matthew L Oliver, Zachary F Caffall, Callie B Eatman, Timothy D Faw, Nicole Calakos
(2025) DIO-SPOTlight Transgenic Mouse to Functionally Monitor Protein Synthesis Regulated by the Integrated Stress ResponseeLife14:RP104457https://doi.org/10.7554/eLife.104457.1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 29, 2025
Last Modified: May 29, 2025
Protocol Integer ID: 219163
Keywords: ASAPCRN, DIO-SPOTlight , Transgenic mouse line, spotlight transgenic mouse line generation this protocol, spotlight transgenic mouse line generation, spotlight transgenic mouse line, nestin
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020607
Abstract
This protocol describes the steps performed to generate Nestin-Cre::DIO-SPOTlight transgenic mouse line.
Troubleshooting
Nestin-Cre::DIO-SPOTlight transgenic mouse line generation
To produce the Rosa26-DIO-SPOTlight mouse clone the DIO-SPOTlight contruct into the Ai9 vector backbone in place of the Lox-STOP-Lox-tdTomato cassette.
Transfer the linearized Ai9-DIO-SPOTlight plasmid to an available transgenic mouse core (rodent genetic engineering services).
Integrate the the linearized Ai9-DIO-SPOTlight plasmid into the Rosa26 locus using homologous recombination and electroporate into hybrid 129S6/C57BL/6N (G4) embryonic stem cells.
To achieve pan-neuronal expression cross Rosa26-DIO-SPOTlight hemizygous transgenic founders with Nestin-Cre mice.
Acknowledgements
  • Ai9 vector backbone was a gift from Hongkui Zeng; Addgene plasmid # 22799; http://n2t.net/addgene:22799; RRID:Addgene_22799.
  • Duke Transgenic Mouse Core performed generation of Rosa26-DIO-SPOTlight hemizygous transgenic founders
  • Nestin-Cre mice were obtained from Jackson Laboratories; JAX: strain #003771, RRID: IMSR_JAX:003771 (Tronche, et al 1999)