Human 22q11.2 locus and corresponding mouse models that have overlapping congenital features along with RTOC procedures. (A) Cartoon diagram illustrating the chromosomal organization of human 22q11.2DS along with several key genes interspersed among the first four of eight LCRs. One key gene is TBX1. Most patients with 22q11.2DS have one allele containing deletions between LCR A–LCR D, which spans ∼3 Mb. TBX1 becomes haploinsufficient. (B) An orthologous region exists on murine chromosome 16, with the conspicuous absence of LCRs. CRISPR/Cas9 was used to create a deletion on chromosome 16, matching the 3-Mb deletion in humans. (C) A distinct 22q.11.2DS mouse model with a highly penetrant thymic hypoplasia is the Tbx1^neo2/neo2 line. In this line, the neomycin gene in inserted in an inverse reading frame within intron 5 of Tbx1. Tbx1^+/neo2 adult mice are bred, yielding embryos with Tbx1^+/+, Tbx1^+/neo2, and Tbx1^neo2/neo2 alleles. (D) Depiction of the RTOC assay modified to include flow sorted cells. Single-cell suspensions from E13–13.5 fetal thymic lobes were prepared, and mesenchymal cells (Pdgfra⁺), TECs (EpCam⁺), and the remaining unstained cells (Pdgfra⁻EpCAM⁻, which includes ETPs, other hematopoietic cells, and endothelial cells) are sorted by flow cytometry. The three subgroups were reaggregated at cell ratios established with control fetal thymuses and placed onto membranes and cultured. A minimum of 20,000 cells/aggregate is needed to sustain RTOC growth with normal cells. The aggregates appear as a small dot in the yellow circled area of a single well of a 6-well tissue culture plate.