Transcriptome analysis
To identify candidate genes, we use a comparative transcriptomics approach in which we analyze and compare the transcriptomes of cNPCs from different primate species. We use both classical and single-cell transcriptome analyses performed on brain organoids and tissue samples. Our aim is to find genes that are specifically active in certain primates or that only exist in these primates. Particular attention is paid to genes that are exclusive to humans and absent in other non-human primates - so-called human-specific genes.
Electroporation of brain organoids
As a model to test the function of these candidate genes, we use brain organoids from different primate species, which we produce using a standardized protocol. These candidate genes are expressed in the brain organoids by electroporation (JOVE). These genes are first incorporated into ring-shaped DNA molecules, so-called plasmids. These plasmids are then injected into the fluid-filled spaces of the brain organoids, the so-called ventricle-like structures. Short electrical pulses are applied, causing the cells of the brain organoids to take up the plasmids. The genes located on the plasmids are then activated in the cells and we can study how the behavior of the cells changes in response to these genes.
Analysis of electroporated brain organoids
An altered behavior of the progenitor cells is shown, for example, in an altered cell division activity or in the production of other cell types (such as different progenitor cells or neurons). We can detect this altered behavior using immunofluorescence staining with various markers (e.g. cell division and neuronal markers). For this purpose, the brain organoids are either cut into thin sections of 20 micrometer thickness or made transparent as a whole (so-called clearing) and then stained with antibodies against the corresponding markers. The stained organoids or organoid sections can then be analyzed under a fluorescence microscope. Other methods we use to characterize the cells are transcriptome analysis, quantitative real-time PCR, western blot and other molecular biology techniques.
Methods to analyze electroporated brain organoids





