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In This Article

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

Summary

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

Abstract

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.

This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Introduction

Before the last two decades, the treatment of human cancers relied heavily on radiotherapy and chemotherapeutic agents that broadly targeted rapidly proliferating cellular populations by damaging DNA or inhibiting DNA synthesis. Although these approaches did inhibit cancer cell growth, they also had deleterious side effects on normal rapidly proliferating cell types such as intestinal epithelial cells and hair follicle cells. More recently, cancer therapy has begun to utilize chemotherapeutic agents that precisely target proteins within signaling pathways that are critically important for the growth of an individual patient's neoplasm. This approach, commonly refe....

Protocol

Prior to performing any procedures with mice, all procedures must be reviewed and approved by the Institutional Animal Care and Use Committee. The protocol described in this manuscript was approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina. This protocol was performed by properly trained personnel following MUSC's institutional animal care guidelines.

1. Generation of mice that develop MPNSTs homozygous for Erbb4 flox

Representative Results

Figure 4 illustrates a typical result obtained when transducing P0-GGFβ3;Trp53-/+;Erbb4fl/fl MPNST cells with either the Ad5CMV-eGFP adenovirus or Ad5CMV-Cre/eGFP adenovirus (Figure 4A). Cultures are viewed with fluorescence microscopy to identify eGFP-expressing cells and by phase-contrast microscopy to determine the total number of cells present in the same field at 10x (top) and 40x (bottom). The pe.......

Discussion

The detailed methods presented here were developed using a GEM model of MPNSTs. However, if the tumor tissue of interest can be dispersed into individual cells, these methodologies are easily adaptable for various tumor types arising in GEMs. It is important to ensure that the floxed allele does not result in i) decreased survival that can make it difficult to obtain sufficient mice to screen for tumors, or ii) increased tumor latency that can make it difficult to obtain enough tumor-bearing mice. If the floxed allele do.......

Acknowledgements

This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01 NS048353, R01 NS109655), the National Cancer Institute (R01 CA122804), the Department of Defense (X81XWH-09-1-0086, W81XWH-11-1-0498, W81XWH-12-1-0164, W81XWH-14-1-0073, and W81XWH-15-1-0193), and The Children's Tumor Foundation (2014-04-001 and 2015-05-007).

....

Materials

NameCompanyCatalog NumberComments
Ad5CMV-eGFPGene Transfer Vector Core, Univ of IowaVVC-U of Iowa-4
Ad5CMVCre-eGFPGene Transfer Vector Core, Univ of IowaVVC-U of Iowa-1174
alexa 568 secondary antibodyThermo/FisherGaR A11036
Bioconductor Open Source Software for BioninformaticsBioconductorhttp://www.bioconductor.orgalternative statistical analysis tool used for step 4.4
CD31Abcamab28364
Celigo Image CytometerNexcelom BioscienceN/A
Cell StripperCorning25-056-Clmixture of chelators
DAB staining kitVector LabsMP-7800
DAVID (Database for Annotation, Visualization, and Integrated Discovery)DAVIDhttps://david.ncifcrf.govfunctional enrichment analysis software used for step 4.5
DMEMCorning15-013-Cl
DreamTaq and Buffer (Genotyping PCR)Thermo/FisherEP0701 and K1072
erbB4 antibodiesSanta Cruzsc-284
erbB4 antibodiesAbcamab35374
erbB4 antibodiesMilliporeHFR1: 05-1133
FACS SorterBD BiosciencesAria II
ForskolinSigmaF6886
GenomeSpace Tools and Data SourcesGenomeSpacehttps://genomespace.org/support/tools/general resource for several types of open source bioinformatic tools for step 4.5
GlutamineCorning25-005-Cl
Gorilla Gene Ontology enRIchment anaLysis and visuaLizAtion toolGorillaN/A, http://cbl-gorilla.cs.technion.ac.ilfunctional enrichment analysis software used for step 4.5
GSEA Gene Set Enrichment AnalysisBroad InstituteN/A, https://www.gsea-msigdb.org/gsea/index.jspfunctional enrichment analysis software used for step 4.5
HSD: Athymic Nude-FOxn1nu miceEnvigo (Previously Harlan Labs)69
Illumina HiSeq2500 (next generation DNA sequencer)IlluminaHi Seq 2500DNA sequencer used for step 4.2
Lasergene: ArrayStar Gene expression and variant analysisDNAStar LaserGene softwareN/Asoftware statistical and normalization analysis used for step 4.4
Lasergene: SeqMan NGen sequence alignment assemblysoftware alignment used for step 4.3N/Asoftware alignment used for step 4.3
Matrigel, low growth factor basement membrane matrixCorning354230
NRG1-betaIn houseGenerated by SLC, also commercially available from R & D Systems(396-HB-050/CF).
Nuclear Stain Hoeschst 33342Thermo62249
Panther Gene Ontology Classification SystemPantherhttp://pantherdb.orgfunctional enrichment analysis software used for step 4.5
Partek (BWA aligner and analyzer)Partek, Ver 7N/Asoftware alignment and statistical/normalization used for step 4.3
Pen/StrepCorning30-002-Cl
Primer 1: 5′-CAAATGCTCTCTCTGTTCTTTGT
GTCTG- 3′
Eurofins GenomicsPrimer 1 + 2: 250 bp ErbB4 null product and a 350 bp Floxed ErbB4 product;
Primer 2: 5′-TTTTGCCAAGTTCTAATTCCATC
AGAAGC-3′
Eurofins GenomicsPrimer 1 + 2: 250 bp ErbB4 null product and a 350 bp Floxed ErbB4 product;

Primer 3: 5′-TATTGTGTTCATCTATCATTGCA
ACCCAG-3′

Eurofins GenomicsPrimer 1 + 3: 350 bp wild-type ErbB4 product.
Propidium IodineFisher51-351-0
Proteom Profiler Phospho-Kinase ArraysR&D SystemsARY003B
Real time gloPromegaG9712bioluminescent cell viability assay
ToppGene SuiteToppGenehttps://toppgene.cchmc.orgfunctional enrichment analysis software used for step 4.5
Trizol (acid-quanidinium-phenol and choloroform based reagent)Invitrogen15596026
Tyramide Signal Amplification KitPerkin ElmerNEL721001EA

References

  1. Ricciuti, B., et al. Antibody-drug conjugates for lung cancer in the era of personalized oncology. Seminars in Cancer Biology. 69, 268-278 (2019).
  2. Litak, J., Mazurek, M., Grochowski, C., Kamieniak, P., Rolinski, J. PD-L1/PD-1 axis in....

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