The Role of DEAD-box Helicase DDX53 in Autism
Luke Inoue
Research Advisor:
Dr. Stephen Scherer

Abstract
Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental condition affecting
approximately 1–2% of individuals worldwide. Recent genomic research has identified DEAD-box
helicase 53 (DDX53) as a novel susceptibility gene for ASD. Notably, DDX53 has no known ortholog in
the mouse genome, necessitating the generation of a humanized transgenic mouse model. This project
aims to investigate the developmental expression of human DDX53 in mouse brain tissues across
embryonic and postnatal stages. Using techniques such as droplet digital PCR (ddPCR), western blotting,
and fluorescence RNA in situ hybridization (f-RISH), the study will quantify mRNA and protein
expression levels and determine the spatial localization of DDX53 in various brain regions. The findings
will be compared with human gene expression databases to construct a comprehensive developmental
expression profile of DDX53. This research will provide foundational data for future studies examining
the gene's role in neuronal processes and ASD pathophysiology.
Introduction
This project stems from a strong academic interest in genetics, neuroscience, and the underlying
molecular mechanisms of neurodevelopmental disorders. ASD's complexity and the recent identification
of DDX53 as an ASD susceptibility gene make it a compelling subject of study. Through this research, I
aim to contribute to the growing field of ASD genomics while developing core skills in molecular
biology, data analysis, and translational research. Key goals include improving my proficiency with
experimental techniques such as ddPCR and western blotting, gaining experience with bioinformatics
tools, and learning from experts in the Scherer Lab. Most importantly, this research will provide a
hands-on opportunity to understand how foundational science can inform clinical insights. 

Methodology
The project will be conducted in the Scherer Lab at the Hospital for Sick Children.
1. Sample Acquisition, RNA and Protein Extraction
Sample Acquisition:
Embryonic (E10, E18) and postnatal (P7, P14, P35, P65) DDX53 transgenic mouse brains will be
collected from the established colony under appropriate ethical and institutional guidelines. Brains will be
dissected under a stereomicroscope to isolate specific regions: whole brain (at early embryonic stages),
and neocortex, hippocampus, striatum, and cerebellum at later stages. Each sample will be labeled
accurately by age and region and stored on dry ice or at −80°C until processing.
RNA Extraction:
RNA will be isolated from each dissected brain region using either TRIzol reagent or a silica
membrane-based column protocol, depending on sample throughput and purity requirements. Following
isolation, samples will undergo DNase treatment to eliminate genomic DNA contamination. RNA
concentration and integrity will be assessed using a Nanodrop spectrophotometer and an Agilent
Bioanalyzer, respectively. Only samples with RIN values above 7.0 will be included in downstream
analysis.
Protein Extraction:
Protein will be extracted from parallel tissue aliquots using RIPA lysis buffer supplemented with protease
and phosphatase inhibitors. Samples will be homogenized on ice using a mechanical homogenizer,
incubated for 30 minutes, and centrifuged to pellet debris. Supernatants will be collected, quantified via
BCA or Bradford assay, and aliquoted for storage at −80°C.

2. Quantification of mRNA Expression via ddPCR
Primer/Probe Design:
Custom primers and hydrolysis probes (e.g., FAM/BHQ-labeled) targeting human DDX53 will be
designed based on exon-spanning regions to prevent amplification of genomic DNA. Housekeeping genes
such as GAPDH and ACTB will be used for normalization. All primer/probe sets will be validated for
specificity and amplification efficiency using standard curves and control reactions.
ddPCR Analysis:
RNA will be reverse transcribed into cDNA using high-capacity reverse transcriptase. ddPCR reactions
will be prepared and partitioned into droplets using a droplet generator. Following thermal cycling,
fluorescence signals will be read using a droplet reader. Results will be analyzed with QuantaSoft
software to quantify absolute copy number of DDX53 mRNA per ng of input RNA. Expression levels will
be compared across brain regions and developmental timepoints. 

3. Protein Expression Analysis via Western Blot
SDS-PAGE and Transfer:
Equal amounts of protein (20–30 µg per sample) will be loaded onto SDS-PAGE gels (10–12%) and
separated by electrophoresis. Proteins will be transferred onto PVDF membranes using wet or semi-dry
transfer methods.
Immunoblotting:
Membranes will be blocked in 5% BSA or non-fat dry milk in TBST, then incubated overnight at 4°C
with validated primary antibodies against human DDX53. Housekeeping proteins (e.g., β-actin, GAPDH)
will serve as loading controls. HRP- or fluorescent-conjugated secondary antibodies will be used for
detection.
Data Analysis:
Bands will be visualized using chemiluminescence (e.g., ECL substrate) or infrared imaging systems.
Signal intensities will be quantified using ImageJ or LI-COR Image Studio, normalized to housekeeping
proteins, and plotted across timepoints and regions to assess developmental trends in protein expression.

4. Cellular Localization via Fluorescence RNA In Situ Hybridization (f-RISH)
Tissue Preparation:
Frozen brain tissue will be cryosectioned into 10–20 µm slices and mounted onto SuperFrost Plus slides
under RNase-free conditions. Sections will be fixed in paraformaldehyde and dehydrated through graded
ethanol series.
Hybridization and Imaging:
f-RISH will be performed using RNAscope or a comparable single-molecule detection system, with
probes targeting DDX53 transcripts. Sections will be co-stained with nuclear dyes (e.g., DAPI) and, if
time permits, cell-type markers (e.g., NeuN for neurons, GFAP for astrocytes).
Data Interpretation:
Imaging will be conducted using confocal or widefield fluorescence microscopy. Images will be analyzed
to identify which cell types and brain regions express DDX53, and how localization changes across
developmental stages. 

5. Computational and Database Analysis
Comparative Analysis:
DDX53 expression data from this study will be compared to publicly available resources such as the
Allen Brain Atlas, BrainSpan, Ensembl, and GTEx to validate developmental patterns and explore
human-mouse expression alignment.
Gene Network Analysis:
Bioinformatic tools like STRING, GeneMANIA, or Cytoscape will be used to identify genes
co-expressed with DDX53 or involved in similar neuronal pathways, particularly those linked to synaptic
function, RNA metabolism, or ASD-related phenotypes.
Data Integration:
Results from mRNA, protein, and localization analyses will be integrated into a developmental
expression atlas of DDX53, supported by figures and statistical comparisons across regions and stages. 

6. Reporting and Dissemination
Catch-up and Flexibility:
This final week provides a buffer to repeat any experiments needing optimization, complete delayed
procedures (e.g., imaging or data analysis), and tie up any loose ends.
Documentation:
Experimental results and observations will be compiled in an organized lab notebook and weekly
progress reports.

Potential Impact
This research will enhance our understanding of the temporal and spatial expression of DDX53, laying the
groundwork for future functional studies. Findings may identify critical windows of neurodevelopment
influenced by DDX53, helping to clarify its role in ASD. Ultimately, this work may contribute to
biomarker discovery or therapeutic targeting of RNA-binding proteins in neurodevelopmental disorders.