Methodology
To identify genes, and in particular zinc finger transcription factors (ZNFs), that have a role in the differential regulation of cortical neural stem and progenitor cell (cNPC) activity and behavior between different primate species, we use multiple methods:
To identify candidate genes, we use a comparative transcriptomic approach in which we compare the transcriptomes of cNPCs from different primate species.
As a model to test the function of these candidate genes, we use brain organoids from different primate species, which we produce using a unified protocol (see here).
These candidate genes are expressed in brain organoids by electroporation. In this process, expression constructs are transfected into the cells by short electrical pulses.
Candidate genes that lead to changes in cNPC behavior and activity in the electroporation experiments will be analyzed in more detail in knock-out (KO) studies using CRISPR/Cas9.
Electroporated and KO brain organoids are analyzed with immunofluorescence stainings and confocal microscopy. Depending on the question, molecular biological methods such as Western blot and quantitative real-time PCR are also used.
In addition to these established methods, we continue to develop our brain organoid protocols (e.g., for generating vascularized organoids) and analysis protocols (e.g., for 3D reconstruction of organoids by optical clearing).