Developmental Neuroscience Techniques

Electroporation

Purpose: to introduce DNA into other cells for labeling or inducing changes

Method: pulse of electricity disrupts the cell membrane, allowing the DNA (with an attached reporter gene) to traverse the lipid bilayer; GFP (green fluorescent protein) is a good reporter gene since it makes the cells with the inserted DNA become green.

Single-Cell Lineage Tracing

Purpose: to analyze the transcriptome of a cell; to see a cell's mRNA transcript (its 'signature')

Method: group cells with similar signatures together; if a cell begins expressing a new mRNA transcript, you can infer that it is transitioning into to another cell type

Viruses

Purpose: to introduce DNA into other cells for labeling or inducing changes without electricity; some viruses have a tropism, meaning that they target certain cells preferentially

Method: incorporate DNA (with a reporter gene, like GFP) into a virus, which is then injected into a cell; the viral machinery for DNA transmission is left intact, but the harmful effects are removed/rendered inert to minimize cell damage

Birthdating

Purpose: to determine when different cells are produced and regions are formed during development

Method: introduce a label (commonly 3H-thymidine or BrdU, which are both detected by antibodies) when a parent cell produces its daughter cells; then, you can keep track of those marked cells as development occurs

Primary Antibodies

Purpose: to identify proteins

Method: inject protein A into a host animal & let the animal’s body creates antibodies against that protein; then, we can isolate the antibodies from the host’s blood, label them with a fluorophore, & use them to recognize and bind to protein A in tissue

Secondary Antibodies

Purpose: to identify primary antibodies in some cases

Method: label the secondary antibody with a fluorophore and let it bind to the primary antibody

Carbon Dating

Purpose: to determine the birth date of an item

Method: use the 14C:13C ratio to estimate the age of a cell; 14C decays at a predictable rate, so high 14C=young age

Knockouts

Purpose: to test the necessity of a gene

Method: either remove a gene globally or conditionally (gene is knocked out at a certain point in development or in certain cells)

Xenografts (transplanted tissue)

Purpose: to determine the function of a specific tissue or to test therapeutic outcomes

Method: transplant tissue into a new host and see what happens

Fate-Mapping

Purpose: to determine what a cell becomes over time

Method: label a cell with a marker (could be fluorescent) and determine what it has become at a later point in development