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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

Abstract

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

Introduction

Murine brain tumor models are crucial for understanding the mechanisms of brain tumor formation and treatment. Current models typically include rapidly produced subcutaneous or orthotopic transplantations of commonly used tumor cell lines, based on a limited number of driver mutations or patient-derived xenograft models, using immunodeficient mice that hinder proper immunotherapy studies1,2,3,4. Additionally, these preclinical results can lead to false positives, in that such models can exhibit dramatic, oftentimes curative effects in respon....

Protocol

All procedures in this protocol were approved by the Cedars Sinai Medical Center Institutional Animal Care and Use Committee (IACUC). Homozygous mTmG mice were bred with C57BL/6J mice to obtain litters of mixed-sex, heterozygous mTmG mice for use in the following protocol. The animals were obtained from a commercial source (see Table of Materials). Mouse pups were electroporated between postnatal days 0 and 3 (P0-P3).

1. Surgical setup

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Representative Results

The protocol described above has been used to successfully develop both pediatric and adult brain tumor mouse models, with the former published in extensive detail in Kim et al.8. With proper technique and careful planning of plasmid design, the success for EP development of tumors is typically 100%. Histology is the quickest and easiest way to check for successful DNA plasmid insertion when a reporter protein is used. This protocol involves steps on how to develop a GBM brain tumor model with 100.......

Discussion

Electroporation-based delivery of plasmid DNA allows for the in vivo use of molecular biology, similar to that used in genetically engineered mouse models, but with the speed, localization, and efficiency of viral transduction8,13,14. With the latter, however, comes safety concerns as well as immune responses. We have shown in our modeling system using EP-delivery of plasmid DNA that minimal immune response occurs due t.......

Acknowledgements

We thank Gi Bum Kim for the immunofluorescent staining and images. We also thank Emily Hatanaka, Naomi Kobritz, and Paul Linesch for helpful advice on the protocol.

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Materials

NameCompanyCatalog NumberComments
0.1-2.5 µL 1-channel pipetteEppendorf3123000012
2 µL pipette tipsFisher Scientific02-707-442
20 µL pipette tipsFisher Scientific02-707-432
2-20 µL 1-channel pipetteEppendorf3123000098
DNAZap PCR DNA Degradation SolutionsFisher ScientificAM9890
ECM 830 Square Porator ElectroporatorBTX45-0662
Electrode GelParker LabsPLI152CSZ
Fast Green DyeSigma-AldrichF7258-25G
Helping Hands Soldering AidPro'sKit900-015
Micro Dissecting Scissors, 4.5" Straight SharpRobozRS-5916
Mouse Strain: C57BL/6JThe Jackson LaboratoryJAX: 000664
Mouse Strain: Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/JThe Jackson Laboratory JAX: 007676
ParafilmGrainger16Y894
Plasmid: pCag-FlpO-2A-Cre EV Addgene129419
Platinum Tweezertrode, 7 mm DiameterBTX45-0488
Sharps container, 1-quartUlineS-15307
Standard Glass Capillaries, 4 in, 1 mm OD, 0.58 mm IDWorld Precision Instruments1B100F-4Capillary pipettes need to be pulled - see reference 10 for details. 
Vertical Micropipette PullerSutter InstrumentsP-30Heat settings: Heat #1 at 880, Heat #2 at 680; pull at 800. See reference 10 for more details on pulling. 
Vimoba Tablet SolutionQuip LaboratoriesVIMTAB
XenoWorks Digital MicroinjectorSutter InstrumentsBRE
XenoWorks Micropipette HolderSutter InstrumentsBR-MH

References

Explore More Articles

GlioblastomaBrain TumorIn Vivo ModelingElectroporationPlasmid DNAPatient Mutation SignaturesImmunocompetent Mouse ModelTumor MicroenvironmentImmunotherapyAutochthonous Tumor GrowthPreclinical TrialsDNA PlasmidsNeural Precursor CellsMADR MethodSomatic MutagenesisDriver Mutations

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