Background In vivo research is important for bridging the gap between preclinical and clinical research of pediatric high-grade gliomas (pHGGs). In utero electroporation (IUE) is a technique used in animal models that integrates arbitrary somatic variants into the brains of mice in utero, better approximating the development of pediatric somatic mutations. However, affordable and rapid methods to validate and quantify tumor growth in these genetically engineered models are lacking.
Objective This study explores the feasibility of using real-time sequencing to rapidly and cost-effectively validate somatic variants, copy number events, and estimate tumor cell density for novel genetically engineered models.
Methods In utero electroporation (IUE) was performed on anesthetized pregnant mice (CD1) at E13.5 (cortex), using established methodology (Neyazi,2025). Uterine horns were exposed, and individual embryos were digitally manipulated into the correct orientation. A capillary needle was loaded with a mix of plasmid (composed of PDGFRA D842V, TP53, and H3.3 K27M variants) and Fast Green fluorescent dye (0.05%, Sigma), which was injected into the lateral ventricle of the embryos. The mixture was then transfected into cortical neural progenitors of the embryos via electroporation (Figure 1.. Upon birth, successful tumor development was detected in vivo via bioluminescent imaging (Figure 2). Brain tissue was harvested from a naive control mouse and moribund mice at multiple locations (tumor, tumor-adjacent, and necrotic center) and DNA was extracted. We then performed PCR amplification of mouse PDGFRA exon 18 using a single assay that captured both endogenous exons and plasmid-induced exons with the PDGFRA D842V variant. Amplicons were sequenced using a MinION device (Oxford Nanopore) (Figure 3, 4). Reads were aligned to the mouse genome (minimap2/mm9) and the PDGFRA D842V allelic fraction was determined.
In Utero Electroporation (IUE) Plasmid transfection procedure into cortical neural progenitors of an embryo.
Diagram of in utero electroporation showing injection of a DNA plasmid mixed with dye into the lateral ventricle of a mouse embryo. Electrodes apply pulses to target the cortex, transfecting neural progenitor cells with the plasmid.
BLI+ signal of a transfected pup
Bioluminescence imaging (BLI) result showing a transfected mouse pup with localized luminescent signal indicating tumor growth.
Real-Time Sequencing (Oxford Nanopore Technologies) Molecular view of ligation-based library prep and loading into the Nanopore device.
Same-day real-time sequencing protocol Informatics can be obtained within ~4–6 hours.
Diagram illustrating ligation-based library preparation for Oxford Nanopore sequencing, including adapter attachment and loading onto a flow cell.
Flowchart showing the Oxford Nanopore sequencing library prep process:
DNA extraction from tissue.
DNA fragmentation (if needed).
End repair and dA tailing.
Adapter ligation to DNA ends.
Cleanup and quality check.
Loading prepared DNA into the Nanopore flow cell.
Real-time sequencing begins, data output streams to analysis software.
Tumor tissue collection.
DNA extraction.
Library preparation using Oxford Nanopore protocol.
Sequencing run initiated.
Base calling and alignment to the reference genome.
Variant calling and quantification of PDGFRA D842V allele fraction.
Total processing time: ~4–6 hours.
Results
Sequencing results confirmed the presence of PDGFRA D842V in tumor tissue in various quantities. Mutant fraction varied between samples, with pure tumor tissue having the highest proportion of variants, followed by necrotic tissue. Tumor adjacent tissue and wildtype tissue both produced 0–1.1% variant fractions, suggesting a limit of detection around 2%. Sequencing of pure tumor indicates that the clonal population of successfully transfected cells in utero contains about 3 copies of mutant PDGFRA per cell (Figure 5). Of note, processing and analysis of tumor tissue took about 6 hours and cost an estimated $125/sample (~$22/sample when processing a maximum of 24 samples).
Estimating Copy Number Events Visual comparison of a healthy versus mutant cell, post-transfection, with respective wild type/mutant copy number and percentage of Mutant Allele Frequency.
Illustration comparing a normal cell with two wild-type PDGFRA copies to a mutant cell with three copies of PDGFRA D842V. Includes visual percentages showing increased mutant allele frequency in transfected cells.
Conclusions and future work
Real-time sequencing allows us to validate successful transfection and estimate copy number events in novel genetically engineered models. Ongoing work entails (1) correlation of copy number estimations with quantitative fluorescent imaging to accurately estimate cancer cell infiltration in various parts of the brain, (2) comparison of copy number events and transfection efficacy for an array of additional plasmids and (3) validation of novel models currently being cloned, each carrying unique variants that we see in pHGGs.
Citations
Neyazi, Sina; Mayr, Lisa (2025), “Effective targeting of PDGFRA-altered high-grade glioma with avapritinib”, Mendeley Data, V1, doi: 10.17632/487kh7fkj6.110.17632/487kh7fkj6.1