Make friends with transgenes
From Larkum Lab
How to introduce transgenes into an organism
AAV versus Lentiviruses
AAV is commonly used in optogenetics experiments. These viruses are preferred over lentiviruses because they remain primarily episomal, while lentiviruses integrate into the genome. This is important because local chromatin structure at the site of genome integration can affect the expression of transgenes, as well as the expression of neighboring genes. The short coding sequences of channelrhodopsins, halorhodopsins, and other optogenetic genes enables them to be packaged in AAVs. Different serotypes of AAVs have also different cell/tissue specificity.
Lentiviruses carrying transgenes integrate into the genome upon infection, and so permit stable expression in both dividing and non-dividing cells.
Elements of a transgene
- Non-specific promoters: CMV (cytomegalovirus ): high transcription rate. The CAG (CMV-Actin-Globin) promoter is one of its derivatives that can have a higher expression rate.
- Syn (Synapsis I): Neuron specific (excitatory and inhibitory)
- CAMKII (calcium/calmodulin-dependent protein kinase II): excitatory-specific
Other types of promoters (e.g. PV, Sim-1, ChAT) are commonly used to restrict the expression of the gene to a certain set of cell populations. They are created / used either via:
- carefully dissecting out the genome to look for the region region responsible for population-specific expression (e.g. PV, Syn, CamKII)
- targeted introduction of Cre (or EGFP) gene into an artificial chromosome (BAC), normally followed by a careful examination of proper expression (e.g. ChAT, TH).
- randomly introducing the Cre or EGFP gene into an artificial chromosome (BAC), and then showing to display population specificity of expression. The promoter is named after the gene that was located around the site of Cre/EGFP insertion (e.g. Sim-1, Tlx-5).
Note that the expression pattern of the downstream gene(s) does not necessarily correspond to the endogenous expression pattern of that gene.
WPRE: Woodchuck Hepatitis Virus (WHP) Post-transcriptional Regulatory Element (WPRE) is a DNA sequence that, when transcribed creates a tertiary structure enhancing expression. Commonly used in molecular biology to increase expression of genes delivered by viral vectors.
Flex (or “flip-excision”) switches were designed as a genetic tool for researchers to conditionally manipulate gene expression in vivo using site-specific recombination (e.g. Cre).
- DiO (Double-floxed inverse Orientation): "Cre-On" meaning that your gene of interest starts in the inverse/antisense "off" position and is flipped to the sense (on) orientation in the presence of Cre.
- DO (Double-floxed Orientation): A"Cre-off" i.e. your gene of interest starts in the sense orientation and Cre is used to flip it to "off".
- Lines for in vivo spine imaging: B6.Cg-Tg(Thy1-YFP)16Jrs/J (https://www.jax.org/strain/003709).