I am curious to know, how do optogenetics offer cell type specific delivery of opsin gene into tissues? I'll be much obliged if you take your time to respond to me.
Article Cell type–specific genetic and optogenetic tools reveal hipp...
In Drosophila, people normally use a binary expression system (like GAL4/UAS or Q-system) to direct expression of transgenes to a particular subset of cells. For example, let's say you want to express channelrhodopsin in all neurons in a fly. Then you will use a driver line that drives expression in all neurons (like elav-Gal4 or nsyb-Gal4) and cross these driver flies to the flies that carry a UAS-channelrhodopsin transgene. Some or all of the progeny of this cross will have both the driver transgene (elav-Gal4) and a reporter transgene (UAS-....), and the channelrhodopsin will then be expressed in all neurons. If you want to affect only some neurons, let's say olfactory receptor neurons only, you will use a different driver line, for instance Orco-Gal4, and the same UAS-... line.
So basically you make cell-specific expression by using genetics. And obviously the same approach works not only for optogenetics, but for any gene that you want to express, like GFP, RFP, Toxins, etc.
I am new to optogenetics. Can you please also explain binary expression system and driver line? Also, can we use them for in vivo targeted expression? Thanks a zillion.
Meha, sorry, I have only just noticed that you referred to a particular paper in your original question, and that paper is definitely not about Drosophila. Genetics in mice is done differently, so I'm not sure how useful my explanations are.
But, since you asked, I'll write a bit more about flies. So, the most straightforward way to create a transgenic fly that expresses GFP (or any other gene) in all neurons is to take a promoter of a native fly gene that is active in all neurons, and place that promoter in a plasmid right before that GFP gene. Let's say this gene, active in all neurons, is called elav. So we take the elav_promoter, and place it in our plasmid right before GFP. Next, the plasmid is injected into fly embryos, and through a sequence of standard steps we can get transgenic flies that carry our transgene: elav_promoter - GFP. In these flies GFP will be expressed in all neurons.
This approach is good, but has two problems. First, often the expressed GFP will be very weak. Second, what if you want to express RFP instead? or YFP? or CFP? Or express GFP only in olfactory neurons? You'd have to make new transgenic lines every time. This is not very convenient.
To overcome these problems, people have figured that instead of using the promoter and the gene (GFP) in one plasmid, we can split them. We will make two plasmids, one will carry the elav_promoter upstream of a transcription factor that comes from yeast, and doesn't activate any of the native fly genes. This transcription factor is called GAL4. Our second plasmid will have the sequence from yeast that GAL4 binds to (it is called UAS) right upstream of the GFP gene. We inject these plasmids in different fly embryos and make two transgenic lines, one with elav-GAL4, and the other one with UAS-GFP. Since there are two components now, we call it a binary system. elav-GAL4 is called a driver line, since it determines where GFP will be expressed (in other words, it "drives" GFP).
Our binary system contains an intrinsic amplification step, because one GAL4 molecule may initiate transcription of the GFP gene many times, and so will produce many more GFP molecules than the direct elav-GFP construct. Also, binary system is more flexible, since we can combine different GAL4 lines with different UAS lines.
For mice though, this all is not exactly true :)
This approach can definitely be used in vivo. Animals will be running around with GFP glowing in their neurons (or whereever it is expressed). If too much GFP is expressed (aka driver is too strong), and animal may become unhealthy. So people usually avoid that.
That was a nice explanation. I now got the idea of binary system. I was asked that how optogenetics can be cell type specific since there is a problem with gene therapy (in which we deliver the gene in the cells or organs as in optogenetics). Gene therapy is not specific. So this question was also a comparison of specificity of optogentics to gene therapy. Your answer is valuable. Thankyou.
If working with mice, the most closest ones to get cell type specific expression is to use the Cre-LoxP system. There are many cell type specific cre lines available, such as GENSAT or from Allen brain institute, then you can inject viral vector containing Cre dependent opsin into the target area to achieve cell-type specificity.
Or you can creating transgenic mice through BAC library to have opsin expressed driven under cell type specific promotor.
Injecting viral vectors containing cell-type specific promotors in WT mice might achieve the same results, but it is not very reliable.