Virus Resources

Genetics

  • Cre-Lox System

Breaking Down the Terminology

  • Using this construct as an example: AAV-EF1a-DIO-GCaMP6s-P2A-nls-dTomato.

    • AAV: adeno-associated virus. This is a mutated form of the adenovirus (which causes some forms of the common cold) and easily infects mammalian cells. The plasmids from the virus also do not integrate into the host genome, so should not affect normal host cell function. 

    • EF1a: This is the promoter for the EF1a protein involved in translation, which should occur in all cells. Thus all infected cells should have RNA polymerases bound to the promoter ready to transcribe the mRNA for the gene of interest. 

    • DIO: This means the expression is Cre dependent. Cre is a protein that will take two loxP sites (or variants of that) and either excise whatever is in between or invert whatever is in between, depending on the orientation of the loxP sites. In the DIO (Double-floxed Inverse Orientation) case, the gene of interest (GCaMP6s-P2A-nls-dTomato) is inverted and flanked by a loxP and a lox2722 site on each side in different orientations. If no Cre is present, the gene of interest is inverted so it is not expressed. If Cre is present, it will flip the gene of interest when performing the recombination, causing the gene to be expressed. Therefore, only cells expressing Cre will express the gene of interest. See this page for diagrams. 

    • GCaMP6s: This is the main protein of interest. If is the 6s version of the genetically-encoded calcium-dependent fluorescent indicator. When neurons fire action potentials, calcium would rush in the cell to release neurontransmitters. Thus imaging calcium is reflective of neuronal activity. 

    • P2A: This encodes a self-cleaving peptide that allows the translated protein to break into two: a GCaMP6s protein and a dTomato protein. 

    • nls: This is the nuclear-localization peptide sequence that causes the dTomato protein to be imported into the nucleus. This way the dTomato signal would not interfere with the GCaMP6s signal, which comes from the cytoplasm. 

    • dTomato: This is a fluorescent protein that allows one to determine if the gene of interest is expressed or not, even if there are no calcium signals present.

  • A summary:

    • In summary, when you inject this AAV virus into the thalamus of GAD-Cre animals, although both excitatory and inhibitory neurons will be infected and take up the plasmid, only neurons expressing GAD (which are the inhibitory neurons) will express GCaMP6s. Now if we induces oscillations electrically under the microscope, we can then probe how the inhibitory neurons respond in each cycle of the oscillation. For example, how spread out the coactive neurons are, how much synchrony there is, what percent of the population is activated at any given cycle, etc.

Virus Injections

  • A note from UVA Biosafety

  • Angled Injection Calculator