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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 engineered iCell® Microglia, 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). The MECP2 line was engineered in the otherwise healthy background line, 01279.
The cytokine response of stimulated iCell Microglia and the engineered MECP2 line were compared by multiplex assay (Fig. 4). 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.
In Figure 3, 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.
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. 2). The specific staining is compared against matched isotype controls. Expressed marker levels are similar to the control line across the range of markers tested.
Total cell lysates of iCell GABANeurons from both 01279 and the isogenic harboring the MECP2 mutation were compared by Western blot analysis (Fig. 1). 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. 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)
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 Advantages