How do scientists transfer the specific DNA in Optogenetics?

Question

I'm a bit new to genetic modification and I was wondering in Optogenetics (a field in neuroscience) how the scientists transfered the desired DNA strand from the light-sensitive ion channel opening cell (I forgot the name) to the specific neuron that will grow these cells sensitive to green light and trigger an action potential in the neuron? (open the neuron ion channels)

I don't understand how scientists know exactly which strand of DNA to cut from the source and where to put it in the target. I mean, if scientists can do this, why not cut the legs growing part of my DNA and insert in a butterfly and boom, you have a butterfly with legs, no?

Answer 1

Well for one, just having a gene doesn't mean you can make it work as intended, and indiscriminately tweaking the genetic material can produce unwanted effects. For example, if I overexpress p53, a common tumor suppressor gene, the cell undergoes apoptosis (1). If I break/delete the same gene, the cell might undergo apoptosis or it might become cancerous. Human development is of course regulated by hundreds of genes at a time, however, so legs on a butterfly is far more complex than just a single gene.

A common approach for inserting genes into a host genome is the lentiviral transfection. Generating a lentiviral vector is detailed here. A similar strategy for transfection using HEK293 for viral production is described in the following paper. I have no knowledge of optogenetics but I tried my best to find a paper in the field that employs the method in practice. In case you have no access to the article,

Viral Production

HEK293T cells (ATCC, UK) were cultured in Iscove’s modified Dulbecco’s medium (Sigma-Aldrich, Germany) supplemented with 10% (v/v) FCS (Sigma-Aldrich) and penicillin–streptomycin–glutamine (Sigma-Aldrich) in 5 × 150 mm dishes. After 80% confluency was reached, cell were transfected in serum-free medium with the helper plasmids pRV1, pH21 and pDFΔ6 and pAAV1/2-hSyn-BLINK-IRES-eGFP or pAAV1/2-hSyn-IRES-eGFP at a molar ratio of 1:1 with CaCl2. On the next day, the medium was replaced with serum-containing medium, and 48 h after transfection cells were harvested, pelleted and resuspended in lysis solution (150 mM NaCl, 20 mM Tris, pH 8).....

Culture & Transfection

Hippocampal neuronal primary cultures were prepared from embryonic day 18–19 (E18– E19) rat hippocampi as previously described43. All the experiments were approved by the Institutional Animal Care and Use Committee of University of Milan and by the Italian Ministry of Health (#326/2015). Neurons were transfected at 7 days in vitro (DIV7) via the calcium-phosphate precipitation method with 4 μg of plasmid DNA for GFP for the experiments assessing the axonal and dendritic distribution of BLINK2 reported in Fig. 2c. Neurons were infected with AAV1/2-hSyn-BLINK2-IRES-eGFP at DIV10 and fixed at DIV12 for the immunocytochemistry assays.

There's more to the method, but in short they transfect the 293 cells with these plasmids that create a functioning virus enclosing their plasmid of interest, and then they harvest the virus and use that to further transfect their neural cells with the gene(s) of interest.

Answer 2

I think your question can be broken down into a few parts:

1. How do scientists know which DNA sequence encodes a specific protein?

It seems that you've asked this question here, which I'll answer separately. So please go read my answer there first :)

2. How do scientists transfer genes between organisms?

@CKM did a great job of explaining how lentiviral transduction works. It is a very common method for introducing genes into organisms. A few steps before that: if you know the sequence of a gene and want to cut it out, you can use restriction enzymes which recognize specific sites in the DNA and only cut there. You can then purify and amplify that DNA sequence by polymerase chain reaction (PCR).

To address one of the subpoints of your question, it is true that a lentivirus will integrate the gene of interest at a random location within the genome. It could be in a non-coding region. It could be in another gene. You would have to do some sequencing to find out.

3. How did scientists do this to invent optogenetics?

In the case of channelrhodopsin, scientists noticed that microalgae were able to detect and swim towards light. They used EST sequencing to find the responsible genes. Once the a photoreceptor gene could be isolated, scientists constructed lentiviruses containing this gene (plus a fluorescent protein gene) and infected neurons with them. The rest is history.

One final tidbit: though you can't exactly give a butterfly the genes for human legs, you can make it grow butterfly legs in weird places, thanks to a family of transcription factors called the Hox genes. People learned about Hox genes in flies by mutating the different genes and observing the wacky stuff that ensued, such as legs growing where antennae should grow. Google Antennapedia to see what I mean.