Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

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 studies^1,2,3,4. 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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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 transduction^8,13,14. 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.......

Materials

Name	Company	Catalog Number	Comments
0.1-2.5 µL 1-channel pipette	Eppendorf	3123000012
2 µL pipette tips	Fisher Scientific	02-707-442
20 µL pipette tips	Fisher Scientific	02-707-432
2-20 µL 1-channel pipette	Eppendorf	3123000098
DNAZap PCR DNA Degradation Solutions	Fisher Scientific	AM9890
ECM 830 Square Porator Electroporator	BTX	45-0662
Electrode Gel	Parker Labs	PLI152CSZ
Fast Green Dye	Sigma-Aldrich	F7258-25G
Helping Hands Soldering Aid	Pro'sKit	900-015
Micro Dissecting Scissors, 4.5" Straight Sharp	Roboz	RS-5916
Mouse Strain: C57BL/6J	The Jackson Laboratory	JAX: 000664
Mouse Strain: Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J	The Jackson Laboratory	JAX: 007676
Parafilm	Grainger	16Y894
Plasmid: pCag-FlpO-2A-Cre EV	Addgene	129419
Platinum Tweezertrode, 7 mm Diameter	BTX	45-0488
Sharps container, 1-quart	Uline	S-15307
Standard Glass Capillaries, 4 in, 1 mm OD, 0.58 mm ID	World Precision Instruments	1B100F-4	Capillary pipettes need to be pulled - see reference 10 for details.
Vertical Micropipette Puller	Sutter Instruments	P-30	Heat settings: Heat #1 at 880, Heat #2 at 680; pull at 800. See reference 10 for more details on pulling.
Vimoba Tablet Solution	Quip Laboratories	VIMTAB
XenoWorks Digital Microinjector	Sutter Instruments	BRE
XenoWorks Micropipette Holder	Sutter Instruments	BR-MH