Experiments in primary cortical neurons
We can test hypotheses about brain development using neurons taken from developing mice.
Molecular Mechanisms of Cell Identity
A recurring goal of my research is to determine the mechanisms by which transcription factors confer cell identity. While computational predictions can point to thousands of potential target genes and regulatory elements for a single transcription factor, such predictions must be validated in a developmental model.
The function of a gene or regulatory element, for example, can be tested by modulating its expression in primary cells cultured in a dish. Though not as clear as in an in vivo model, results are generated much faster, and the range of testable targets is much larger.
Such tests include reporter assays, in which a region of the genome is queried for its ability to increase or decrease the levels of a target gene, knockdown experiments using siRNAs to a target transcript, and over-expression of genes cloned into viral vectors.
Example paper:
Luciferase Reporter Assays
A segment of coelacanth DNA similar to a sequence found in the mouse genome drives expression of a luciferase reporter introduced into mouse neurons, suggesting that some of the genome's rules for brain development are conserved between mammals and fish.
siRNAs targeted to noncoding RNAs
We use siRNA designed to target a noncoding RNA (ncRNA) to knock down levels of the ncRNA in cortical neurons. The photo shows a qPCR of transcript levels of the ncRNA in control and knockdown cells.
Photo Credit: Shao Ji