Fragile X Syndrome (FXS) In Vitro Modeling Service

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and a leading monogenic cause of autism spectrum disorder (ASD). It arises from a CGG trinucleotide repeat expansion in the FMR1 gene, leading to its transcriptional silencing and loss of the Fragile X Mental Retardation Protein (FMRP). FMRP is a critical RNA-binding protein that regulates synaptic plasticity, mRNA translation, and neuronal development. Its absence disrupts neuronal signaling, dendritic spine maturation, and network connectivity, resulting in cognitive, behavioral, and sensory impairments.

In vitro models of FXS have become essential tools for dissecting molecular mechanisms, identifying therapeutic targets, and validating drug candidates. The models from Creative Biolabs leverage patient-derived cells, CRISPR-engineered isogenic controls, and advanced neuronal differentiation protocols to recapitulate FXS-specific pathology.

Core Pathological Hallmarks & Cellular Models

FXS primarily affects neurons due to FMRP's role in regulating synaptic protein synthesis. However, glial cells (astrocytes and microglia) also contribute to disease progression by modulating neuronal support and inflammatory responses. Key cellular hallmarks in FXS include:

• Aberrant dendritic spine morphology: Immature, elongated spines in cortical and hippocampal neurons.
• Dysregulated synaptic plasticity: Excessive metabotropic glutamate receptor (mGluR)-dependent signaling.
• Mitochondrial dysfunction: Impaired energy metabolism in neurons.

Our in vitro models focus on:

• Human iPSC-Derived Neurons: We generate patient-specific iPSCs from FXS patients with full CGG repeats (>200), enabling the study of FMR1 silencing and FMRP loss in human neurons. We also create isogenic controls using CRISPR/Cas9 correction of FMR1 in patient iPSCs, providing genetically matched controls critical for validating disease-specific phenotypes. Our differentiation protocols generate various neuronal subtypes, including cortical neurons via dual SMAD inhibition and GABAergic neurons induced by ASCL1 overexpression.
• Immortalized Cell Lines: We utilize neuroblastoma lines (e.g., SH-SY5Y) engineered to knock down FMR1 using shRNA, mimicking FMRP loss for high-throughput drug screening. We also offer astrocyte lines transfected with FMR1 siRNA to study glial contributions to synaptic deficits.
• Primary Rodent Neurons: We provide primary cortical neurons derived from Fmr1 knockout mice, a gold standard for studying dendritic spine defects and mGluR-LTD.

Key Assays for Drug Discovery

• High-Throughput Screening (HTS) Assays: We offer HTS assays for drug screening and FMR1 reactivation. Our drug screening assays use iPSC-derived neurons or immortalized cell lines to screen compounds that rescue exaggerated protein synthesis or dendritic spine defects. High-content imaging or luminescence-based readouts enable rapid screening. Our FMR1 reactivation assays test small molecules or CRISPRa (activation) strategies for their ability to restore FMRP expression in FXS patient-derived cells.
• Synaptic Function and Plasticity Assays: We perform electrophysiology (patch-clamp recording) to measure synaptic transmission and plasticity in iPSC-derived neurons or primary rodent neurons. This technique can quantify exaggerated mGluR-dependent long-term depression (mGluR-LTD), a hallmark of FXS. We also use multi-electrode arrays (MEA) to record spontaneous and evoked electrical activity in neuronal networks, assessing the hyperexcitability and altered network synchrony often observed in FXS models.
• Dendritic Spine Analysis: We use high-content imaging of neurons transfected with GFP or labeled with DiI to quantify dendritic spine density, length, and morphology. FXS neurons typically exhibit immature, elongated spines, which can be rescued by therapeutic interventions. We also perform time-lapse imaging to monitor dynamic changes in spine morphology and turnover in live neurons, providing insights into synaptic stability and plasticity.
• Protein Synthesis and FMRP Function Assays: We conduct puromycin incorporation assays to measure global protein synthesis rates. FXS neurons often show elevated protein synthesis due to the loss of FMRP-mediated translational repression. We also quantify FMRP levels using Western blot or immunofluorescence staining to confirm the absence of FMRP in FXS patient-derived neurons or the restoration of FMRP expression following therapeutic interventions.
• Mitochondrial Function Assays: We utilize a Seahorse Metabolic Analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to assess mitochondrial respiration and glycolysis. FXS neurons often exhibit mitochondrial dysfunction, which can be quantified using this assay.
• Neuronal-Glial Interaction Assays: We perform astrocyte-conditioned media assays to test the effects of astrocyte-secreted factors on neuronal survival, spine maturation, and synaptic function.

Why Choose Our In Vitro Models?

• Comprehensive Model Portfolio: We offer a wide range of in vitro models, from human iPSC-derived neurons to rodent primary neurons, to meet your specific research needs.
• Advanced Techniques: We utilize cutting-edge technologies, including CRISPR/Cas9 gene editing and high-content imaging, to ensure the quality and reliability of our models.
• Customization: We can tailor our models and assays to your specific research questions and drug discovery goals.
• Expertise: Our team comprises experienced scientists with a deep understanding of FXS and in vitro modeling.

Contact us today to discuss your FXS research needs and explore how our in vitro models can accelerate your drug discovery efforts.

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Reference

1. Lee, Azalea et al. "Across Dimensions: Developing 2D and 3D Human iPSC-Based Models of Fragile X Syndrome." Cells. 2022;11(11):1725. Published 2022 May 24. Distributed under Open Access License CC BY 4.0 without modification.