Jun 23, 2025
in vivo AAV MPRA
This protocol is a draft, published without a DOI.
- Katherine Degner1,
- Hyejung Won1
- 1University of North Carolina at Chapel Hill
- in vivo MPRA
Protocol Citation: Katherine Degner, Hyejung Won 2025. in vivo AAV MPRA. protocols.io https://dx.doi.org/
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 have optimized each step of this protocol to produce sufficient reproducibility for MPRA analysis.
Created: June 22, 2025
Last Modified: June 23, 2025
Protocol Integer ID: 220696
Keywords: in vivo, MPRA, bp enhancer sequence with each snp, vivo aav mpra, nucleotide barcode sequence, vivo aav, bp enhancer sequence, single nucleotide polymorphism, viral dna, genetic variant, genome, reporter gene, cmv promoter, aav9 capsid, in vivo mpra, aav, steps of an in vivo mpra, luciferase as the reporter gene, unique barcode sequence, murine neonates at p0, nucleic acid, snp, mpra library, parallel reporter assay, other for cannabis use, regulatory activity, sequencing, dna, cannabis use, gene
Funders Acknowledgements:
National Human Genome Research Institute and the Impact of Genomic Variation on Function Consortium
Grant ID: 1UM1HG012003
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Abstract
The massively parallel reporter assay (MPRA) was designed to interrogate the regulatory activity of thousands of noncoding, genetic variants simultaneously. In this protocol, we generally describe the steps of an in vivo MPRA in neonatal C57Bl/6J mice to target the brain. First, we designed and generated an MPRA library that encodes single nucleotide polymorphisms (SNPs) from two genome-wide association studies, one for tobacco use and alcohol consumption and the other for cannabis use. This library, along with random, 20 nucleotide barcode sequences, are cloned into an adeno-associated viral (AAV) vector. The final construct contains a 150 bp enhancer sequence with each SNP spatially centered, the CMV promoter, luciferase as the reporter gene, and a unique barcode sequence. These constructs are then packaged into AAV9 capsids and administered to murine neonates at P0 via intracerebroventricular injection. After a 3-week incubation period, the tissues of interest are harvested, homogenized, and the nucleic acids from each are isolated then prepared for next generation sequencing (NGS). The in vivo AAV-MPRA protocol takes at least 4 months for a cohort of 20 animals (cortex & subcortex from each), not including the turnaround time for NGS, nanopore sequencing of the viral DNA, or data analysis.
MPRA Library Construction
2w
Amplify library
Add random barcode sequences via PCR
Ligate digested oligo into AAV vector
Electroporate ligation mix
Clone and purify plasmid DNA
Amplify CMV-Luciferase sequence
Digest plasmid containing library and barcodes
Ligate CMV-Luciferase fragment and digested plasmid
Electroporate ligation mix
Clone and purify plasmid DNA - final construct
AAV Packaging
2w
Send purified plasmid DNA to Stanford Gene Vector and Viral Core for production of AAV9
Extract viral DNA from a portion of each batch of virus
Amplify fragment of viral DNA from upstream of library to downstream of barcode
Oxford nanopore sequence this amplified viral DNA for element-barcode mapping
Introduction of MPRA Libraries into Murine Brain
3w
Inject a total of ~3uL of undiluted AAV9 into lateral ventricles of each P0 C57Bl/6 pup
After 3 weeks, harvest brains and separate cortex from subcortical regions
Tissue Homogenization
2d
Homogenize each tissue using a bead mill and 15mL conical tubes filled with plastic beads and Buffer RLT Plus
Run lysates through Qiashredder columns
Freeze lysates and store in -80 C for up to 3 months
Nucleic Acid Isolation & Library Generation
8w
Isolate RNA and DNA simultaneously from each lysate sample using Qiagen AllPrep DNA/RNA Mini Kit
Treat isolated RNA with TURBO DNA-free Kit protocol
Bead clean treated RNA with RNAClean XP beads
Reverse transcribe treated and cleaned RNA using SuperScript IV Reverse Transcriptase
Treat isolated DNA with RNase A
Bead clean treated DNA with AMPure XP beads
Amplify barcode sequences from cDNA and DNA
Add P5/P7 adapter sequences and demultiplexing indices to both the cDNA and DNA libraries by PCR using primers
Pool cDNA and DNA libraries at a 2:1 ratio
Next Generation Sequencing
Sequence libraries with NovaSeq X 25B using custom cycles of 35x8x0 with 10% PhiX and custom sequencing primers
Analysis
Process fastq files
Align NGS reads to barcodes from Nanopore barcode map
Determine reproducibility of element activity between biological replicates
Identify variants with enhancer activity and allelic regulatory activity