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iCell Microglia RTT (MECP2)

An Engineered Disease Model for Rett Syndrome

Description

Figure 1. Schematic of the MECP2 Gene
The structure of MECP2 is shown, along with the primary (E1) and minor, alternate (E2) transcripts. The insertion site is downstream of the start codons for both transcripts, resulting in truncation of both proteins and inactivation of the methyl binding domain

Rett Syndrome (RTT) is a devastating neurodevelopmental disorder, caused mainly by mutations in the MECP2 gene. MECP2 codes for a 486 amino acid protein that binds methylated DNA, generally downregulating transcription. MECP2 mutations are associated with a range of developmental diseases including RTT, autism, encephalopathy and X-linked intellectual disability. RTT occurs mostly in females since MeCP2 is located on the X chromosome. The MECP2 line contains an insert directly after Serine 49 that includes stop codons in all 3 reading frames, generating a non-functional truncated protein before the methyl-CpG-binding domain resulting in the protein being p.(Ala50Ter), (Fig. 1).

Characterization

Western Blot Analysis of MECP2

Figure 2. Western Blot Analysis of MECP2 Mutation
Equivalent loads of total cell protein were compared by Western blot from differentiated iCell GABANeurons were probed with an anti-MeCP2 antibody and normalized to tubulin. (Western blot data courtesy Vanderbilt University)

Total cell lysates of iCell GABANeurons from both  01279 and the isogenic harboring the MECP2 mutation were compared by Western blot analysis (Fig. 2). Blot results suggest that the methyl binding domain and downstream elements are not expressed at a detectable level in the MECP2-modified line.*Note: MECP2 is ubiquitously expressed, including in neurons. Inquire about availability of iCell GABANeurons harboring the MECP2 mutation.

Comparative Marker Expression
iCell Microglia were labeled for the presence of cell surface (CD45, CD11b, CD11c, TREM2 and, CD33) and intracellular (P2RY12, TMEM119, CX3CR1, IBA1) antigens by flow cytometry (Fig. 3). The specific staining is compared against matched isotype controls. Expressed marker levels are similar to the control line across range of markers tested.

Figure 3. Comparison of Marker Expression of MeCP2 and Unmodified iCell Microglia

Phagocytosis
In Figure 4, a comparison is shown between iCell Microglia and the engineered MeCP2 line. Phagocytosis of S.aureus BioParticles® was measured over time using an IncuCyte® S3 Live-Cell Analysis System. The engineered microglia exhibit phagocytic activity, but at a substantially reduced level relative to the unengineered line.

Cytokine Response
The cytokine response of stimulated iCell Microglia and the engineered MECP2 line were compared by multiplex assay (Fig. 5). iCell Microglia were plated in microglia maintenance media and allowed to recover for 72 h prior to stimulation with LPS, interferon gamma or both for 24 h. Supernatants were collected and assayed using a Luminex multiplex cytokine assay.

Figure 4. Phagocytosis of iCell Microglia and MECP2 Engineered Line

Figure 5. Cytokine Comparison of iCell Microglia and SNCA

  • Ordering InformationFor best pricing please log in or register
    Catalog # Description Quantity Price
    R1203 iCell Microglia Kit, MECP2, 01279

    Each kit contains:

    • C1138 Cryopreserved iCell Microglia MECP2, 01279
    • M1034 50ml iCell Glial Base Medium
    • M1036 0.5ml iCell Microglia Supplement A (100x)
    • M1037 0.5ml iCell Microglia Supplement B (100x)
    • M1035 2 x 1ml iCell Neural Supplement C (25X)
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $2275
    Inquire About Pricing & Availability
    C1138 iCell Microglia, MECP2, 01279

    Product contains:

    • C1138 Cryopreserved iCell Microglia MECP2, 01279
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $1975
    Inquire About Pricing & Availability
    R1131 iCell Microglia Kit, 01279

    Each kit contains:

    • C1110 Cryopreserved iCell Microglia, 01279
    • M1034 50ml iCell Glial Base Medium
    • M1036 0.5ml iCell Microglia Supplement A (100x)
    • M1037 0.5ml iCell Microglia Supplement B (100x)
    • M1035 2 x 1ml iCell Neural Supplement C (25X)
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $1650
    Inquire About Pricing & Availability
    C1110 iCell Microglia, 01279

    Product contains:

    • C1110 Cryopreserved iCell Microglia, 01279
    • X1022 iCell Microglia Quick Guide
    • Additional reagents required; see Quick Guide
    ≥1 x 106
    Viable Cells/Vial
    $1350
    Inquire About Pricing & Availability
    R1204 iCell Microglia Media Kit

    Each kit contains:

    • M1034 50ml iCell Glial Base Medium
    • M1036 0.5ml iCell Microglia Supplement A (100x)
    • M1037 0.5ml iCell Microglia Supplement B (100x)
    • M1035 2 x 1ml iCell Neural Supplement C (25X)
    • X1022 iCell Microglia Quick Guide
    1 Kit $365
    Inquire About Pricing & Availability
    019-19741 Anti IBA1 Polyclonal Antibody
    • Rabbit polyclonal IgG
    • 50μg supplied at 0.5mg/ml in TBS
    • Protocol included
    • Store at -20° C
    50ug $355
    Inquire About Pricing & Availability