New genetic techniques like fluorescent reporter genes, optogenetics and DREADD allow scientist to manipulate the functions of individual cells in live animals.
Many of my scientists colleagues are thrilled about the new transgenic techniques. Transgenic methods are sophisticated molecular biology techniques that consist of moving genes from one species to another, or in creating artificial genes and expressing them in a living organism. They represent the convergence of decades of work in areas as diverse as molecular biology, protein chemistry, cellular biology, neuroscience, microbiology, virology and animal behavior.
Transgenic techniques are likely to be applied to humans in the near future and this will dramatically change medicine, hopefully bringing cures for diseases that we cannot treat today. And yet, the general public appears to be completely unaware of them. Maybe because they are done in animals, and animal research is a modern taboo. Genetic modification is also politically incorrect, so these scientists tend to be shy about explaining what they are doing, even though is one of the most remarkable advances of modern science.
It is also true that these techniques are difficult to explain. So, let me give it a shot.
What are knock-in genes?
The techniques that I describe here are known as genetic knock-in.
As you probably know, genes are sequences of DNA that get translated into proteins, which, in turn, are like little machines that carry out most of the cell’s functions. Therefore, by changing a gene we can change the protein that it makes and the functioning of that cell, which eventually shows up as a change in the whole animal.
Translation of a gene into protein is initiated by a sort sequence of DNA located just before the gene, called the promoter.
Gene knock-out is to manipulate a gene so that it cannot be translated into protein - it becomes non-functional.
Gene knock-in is the opposite: making an organism express genes from another organism, or artificial genes that encode novel proteins.
In biomedical research, gene knock-in techniques are applied to a few animal species: the worm C. elegans, the fly Drosophila, zebrafish and mice. However, mice are the most relevant for translation to humans because their physiology, nervous systems, and genes are closer to humans than those of flies and fish. Mice are studied instead of rats or other mammals because it is easier to insert genes in their embryos.
Germ line and somatic line insertion of the transgenes
Gene knock-in can be done in two fundamentally different ways
Germ line insertion. The new genes are inserted in the cells used for reproduction, so that they will be present in the sperm and the oocytes when they combine to produce a new animal. Then, the new genes will be present in every cell of the offspring. Since this includes the germ-line cells of the offspring, they will also be present in the descendant of that animal. However, they will not be expressed - translated into protein - in cells that do not activate the promoter of the new gene.
Somatic line insertion. The news genes are inserted only into some cells of an animal using a viral vector. For example, the gene may be targeted to particular type of neurons in the brain of the animal. Viral vectors are viruses that have been emptied of their genetic material (DNA or RNA), so they cannot produce a disease. Their genetic material is replaced by the transgenes. The viral vector can still infect the cells that it usually target - for example, a rabies virus vector will infect neurons. However, it will inject them with the transgene instead of the genetic material to make new viruses.
Transgenic techniques often combine germ-line and somatic line insertions to administer two genes. Only the cells where the two genes find each other will express the new protein that changes their function.
The difference between germ line and somatic line insertion is fundamental for ethical reasons. It is okay to do somatic line insertion in humans, because the genetic change will not be passed to the descendants of that person. However, germ line insertions will be passed to new generations, permanently altering the human genome. That’s a Pandora's Box that scientists are reluctant to open.
Cell-selective gene knock-out
An important feature of these transgenic methods is that the expression of the artificial proteins is cell-selective, that is, it can be targeted to a particular population of cells inside a tissue, even if they are mixed up with other cells types.
This is done by taking advantage of the fact that a particular cell type expresses genes that are not expressed in other cell types. This is because each cell type activates a different set of promoters in their DNA. The selection of which promoters are activated is part of the cell differentiation during the development of the embryo and the fetus - the process that produces the different organs and tissues of the body.
The gene encoding the artificial protein that we want to express is put after the promoter of a gene that is exclusive of that cell type.
More specificity is achieved by using a technique called Cre-lox recombination (Heldt and Ressler, 2009). Cre is an enzyme derived from a bacteriophage (a virus that infects bacteria) that recognizes portions of DNA flanked by two sequences called lox and deletes them. Hence, when Cre is expressed in a cell with a gene flanked by lox - ‘floxed’ - that gene is deleted.
The simplest way to use Cre-lox recombination is to selectively knock-out a gene in a particular cell type. Cre is expressed under the promoter of a gene exclusive of that cell type, either in the germ line or delivered with a viral vector. The gene that we want to delete is floxed. When Cre and the floxed gene meet in a cell, the floxed gene is deleted (Figure 3).
Cell-selective gene knock-in
Knocking-in a gene encoding an artificial protein requires a modification of Cre-lox recombination called DIO (Double-floxed Inverse Open reading frame). The gene encoding for the artificial protein is introduced in the DNA in reverse order, so that it cannot be translated into protein. The enzymes that transcribe DNA to messenger RNA cannot read inverted sequences, just like you cannot read cinegsnart as transgenic. That inverted gene sequence is flanked by two sets of lox - it’s double-floxed. Cutting and pasting by Cre on these two sets of lox sequences results in turning around the gene, so that now it can be read and translated into protein.
Hence, only cells that have both Cre and the double-floxed inverted gene are able to express the artificial protein. It is usually expressed together with a fluorescent protein - a reporter gene -, to let us know that the expression has been successful.
Fluorescent proteins
Fluorescent proteins occur naturally in jellyfish and corals. They make them glow - fluoresce - when they are illuminated by light of certain colors.
Scientists have extracted the genes that encode for these fluorescent proteins and inserted them in the mouse genome preceded by the promoter of a certain gene. This way, only the cells that activate that promoter to express that particular gene get loaded with the fluorescent protein. This allows them to label specific types of cells in a given tissue with bright colors. The resulting images are amazingly cool!
When used in neurons, fluorescent proteins allow the visualization of very small features, like neurons (Figure 1) axons (Figure 2) or dendritic spines (Figure 4).
By labeling neurons that activate different promoters with different fluorescent proteins, scientists were able to create the “brainbow” shown in Figure 5.
The first fluorescent protein to be used was green fluorescent protein (GFP), extracted from a jellyfish. It was followed by others that were given names of fruits representative of their colors (mCherry, mOrange, mRaspberry and tdTomato) or exotic names like Venus, Citrine, mRuby or FusionRed.
The use of fluorescent proteins has transformed the fields of physiology and anatomy by vastly improving our ability to identify and locate specific cell types. Until now, this was done using a technique called immunohistochemistry, based on the use of antibodies against specific proteins. However, fluorescent proteins provide much higher resolution and avoid many of the artifacts produced by the antibodies.
But the way fluorescent proteins are mostly used is as reporter genes. They are expressed under the same promoter as another transgene, so we can now identify the cells expressing the transgene by the bright color provided by the fluorescent protein. The brightness of the cell also serves as a measure of the level of expression of the transgene.
I have used fluorescent proteins as reporter genes in my own lab. You can see the results in some of the figures, which are images taken by me with a confocal microscope.
Humanized mice
Groups that oppose research on animal argue that animal research has no predictive value because animal physiology and human physiology are radically different, which is completely false. All mammals use the same set of proteins for the same functions.
What is true, however, is that small changes in the amino acid sequence of a protein can cause a drug that binds well to a mouse protein to perform poorly on the same protein in humans. This has slowed down the translation of discoveries from animal research to clinical application.
This problem can be solved by replacing the mouse gene with the human gene for the same protein. For example, we could replace the gene for the mouse mu-opioid receptor (which binds opioid drugs like morphine) with the human gene for the mu-opioid receptor. This does not result in monsters that are half-mouse, half-human, as in some horror movie, but in mice that are entirely normal in their looks and behavior, but in which we can test new opioid drugs for their effects on humans.
Calcium indicators
The calcium ion Ca2+ is one of the most important conveyors of information inside cells, what scientists call intracellular signals. Together with compounds like cyclic-AMP, diacylglycerol and inositol triphosphate, calcium ions are called second messengers. The first messengers are hormones and neurotransmitters that convey signals outside the cell. These signals are picked up by receptors in the cellular membrane or inside the cell and transformed into signals by the second messengers.
Concentrations of calcium inside the cells are 10,000 times lower than outside the cells. They increase 10 to 100 times by opening calcium channels in the cell membrane or in intracellular calcium stores. This conveys a signal that turns on or off different proteins, the machines that perform the various tasks inside the cell. That way, calcium ions regulate cell function.
In the 1980s, biochemist Roger Tsien invented several compounds (like Fura-2 and Fluo-3) that can be used to measure the concentration of calcium inside cells through changes in their fluorescence. These calcium indicators perform very well in cell cultures, but their use in tissue slices is problematic because cells absorb them randomly.
The solution was to fuse the gene for green fluorescent protein with the gene encoding calmodulin, a protein that binds calcium inside the cells, to create a genetically-encoded calcium indicators called GCaMP.
Like the fluorescent proteins, genetically encoded calcium indicators can be selectively expressed in particular cell types by associating their gene with a particular promoter in the DNA. This allowed scientists to observe cell activation as changes in in intracellular calcium live mice. This is done by mounting tiny microscopes on the mice that they carry while they move freely in their cages - another technological feat. These tiny microscopes have digital cameras connected to a computer.
Chemogenetics
Chemogenetics allows controlling the functioning of specific cell types in live animals. If calcium imaging lets us know how a cell is functioning, chemogenetics allows to change the function of precisely defined groups of cells.
The most popular chemogenetic method was named DREADD by its inventor, Dr. Bryan Roth, an acronym for Designer Receptor Exclusively Activated by Designer Drug (Sternson and Roth, 2014; Roth, 2016). The name caught on, perhaps because of its humorous association with the word dread, and it is frequently used instead of chemogenetics.
The Roth’s lab started with the genes for neurotransmitters receptors that inhibit cell function - like the M4 muscarinic receptor for the neurotransmitter acetylcholine - or receptors that increase cell function - like the M3 muscarinic receptor for acetylcholine. Then they altered these genes to modify binding site in the receptor protein so that it no longer bound acetylcholine, but an artificial compound, clozapine-N-oxide (CNO), that has no effect on mammals. This way, the new DREADD receptor cannot be activated by acetylcholine and to many drugs, but responds to CNO.
Inhibitory DREADDs, like the one derived from the M4 muscarinic receptors, can be triggered by CNO to decrease the firing of action potentials in neurons that express it.
Excitatory DREADDs, like the one derived from the M3 muscarinic receptors, when triggered by CNO, increase the firing of action potentials in neurons that express it.
Other DREADDs are neither excitatory nor inhibitory, but activate different second messengers pathways. This allows fine-tuning the function of any cell in the body in ways that are much more specific for the cell type and the function than any drug used in medicine today.
CNO can be given to mice with a simple subcutaneous injection. Its effect lasts more than four hours. The activation and inhibition of neurons by DREADD in mice can then be observed as changes in their behavior.
Optogenetics
Optogenetics (Kravitz and Kreitzer, 2011; Yizhar et al., 2011) is similar to chemogenetics in that an artificial protein is expressed in neurons to turn them on or off. However, instead of an artificial drug like CNO, optogenetics uses light. This was done by starting with the gene of a light-sensitive protein like channelrhodopsin (which activates cells) or halorhodopsin (which inhibits cells).
The advantage of optogenetics over chemogenetics is that its effect on cells is extremely fast.
Its main disadvantage is that it requires the use of light guides: small fiber-optic strings that have to be precisely inserted in the brain or other organs of the animal. There are now tiny light sources that can be mounted over the head of the mouse and activated by radio waves, so that the mouse doesn’t need to be tethered by cable for the experiment. Still, this is more invasive than DREADD.
How we eliminated stress in mice in my lab using DREADD
In my lab, we used DREADD to control stress in mice. We did this by manipulating the amygdala, a brain region that induces fear and distress. Some of the neurons in the amygdala produce corticotropin-releasing factor (CRF), a neuropeptide that increases stress (Andreoli et al., 2017).
We bought transgenic mice that express Cre under the CRF promoter, so that Cre would be expressed in the CRF neurons of the amygdala. This provided one of the elements for Cre-lox recombination.
The other element for Cre-lox recombination was packaged inside a viral vector, which we also bought. As I explained above, viral vectors attach to cells and inject the artificial genes into them. Inside the viral capsid there was a highly sophisticated genetic construct: a DIO (Double-floxed Inverse Open reading frame) inhibitory DREADD and a reporter gene, the red fluorescent protein mCherry.
The most difficult part of the experiment was to inject the viral vector into the amygdala of the small brain of the mouse. This was done using a stereotactic injection, a method consisting of placing a small needle inside the brain of the mouse at the coordinates of a particular region, using a three-dimensional set of micro-manipulators and a computerized atlas of the mouse brain. Then the needle is used to inject minuscule amounts of solution containing the viral vector. The vector delivers the transgenes into all the neurons of the amygdala, but only the CRF neurons that express Cre can turn the DREADD-mCherry genes around so that they can be translated into proteins.
Figure 6 shows the result: CRF neurons in the amygdala expressing the mCherry reporter gene.
But how did we know that the DREADD is working? We took the mouse and made it swim in a bucket of water for 6 minutes. It got stressed and became more sensitive to pain. We measured its pain sensitivity by gently poking its paw with nylon filaments and measuring how long it took it to withdraw the paw. Then we took the same mouse and activated the inhibitory DREADD with an injection of CNO. When be put the mouse in the bucket of water for 6 minutes, it didn’t get stressed and did not develop pain sensitivity.
Can transgenic methods be applied to humans?
The experiment in my lab that I just described indicates that it is possible to inhibit stress in humans using DREADD.
Similarly, we could use inhibitory DREADD to inhibit the neurons that transmit pain in chronic pain disorders to make the patients feel better. Or we could use excitatory DREADD to stimulate the neurons in the substantia nigra that release dopamine to counter the symptoms of Parkinson's disease.
Basically, we could pick up any population of neurons in the brain and turn them on or off to fine tune any function of the brain. And we can do the same with any cell of the body. This will create a new type of medicine that selectively targets specific cells to fine-tune their function, instead of administering drugs that affect the whole body and thus create a bunch of side effects.
Application for brain-computer interface
As for optogenetics, it may one day allow fast communication with individual neurons using fiber optics. That way, we could use light instead of electrodes to have the computer stimulate specific neurons in a brain-computer interface. This would serve to input information from the computer into the brain. To output information from the brain to the computer, we could use calcium indicators, which would monitor the activity of single neurons.
Ethical barriers
The main obstacle to apply transgenic techniques to humans is that it is currently forbidden to alter the germ line of a human being. As I explained before, any changes in the germ line will be transmitted to our descendants, permanently altering the human genome. As the genetic modifications start piling up, we would create a new human species.
We have transgenic mice, but we cannot have transgenic humans.
I don’t think that the gene knock-in modifications that have been done in mice will be harmful to humans. Transgenic mice expressing Cre or floxed genes are healthy. They behave normally until these genes are activated.
This problem can be overcome if we keep the genetic modifications to the somatic line, leaving the germ line untouched. This could be done by giving the Cre gene and the floxed genes in two separate viral vectors. We are already doing this in mice, but more animal work would be needed before we know how to do it safely in humans.
Research on animals is more necessary than ever
DREADD (Nagai et al., 2016) and optogenetics (Chernov et al., 2018) are already being used in monkeys, a necessary step to adapt these techniques to humans. However, any experiments done in monkeys are fought tooth and nail by animal liberationists.
In fact, these new transgenic techniques are exactly the opposite of what was expected from future developments in biomedical research. It was believed that research with animal would eventually become a thing of the past, a necessary evil that would eventually be eliminated by replacing lab animals with in vitro methods, cell cultures, computer models and clinical studies.
However, transgenic techniques are moving science in the opposite direction. Experiments that could only be done in vitro or in cell culture now can be done inside live animals. This means that in a single experiment now we can gather information about interlinked molecular, cellular, physiological and behavioral events, providing valuable insights into the relationships between them.
Far from replacing animal research, cell culture and in vitro methods are the ones that risk becoming obsolete. The use of transgenic animals today represents the cutting edge of science. Any country that curtails animal research with onerous and unnecessary regulations risks being left behind in the race to develop these exciting new technologies.
References
Andreoli M, Marketkar T, Dimitrov E (2017) Contribution of amygdala CRF neurons to chronic pain. Exp Neurol 298:1-12.
Chernov MM, Friedman RM, Chen G, Stoner GR, Roe AW (2018) Functionally specific optogenetic modulation in primate visual cortex. Proc Natl Acad Sci U S A 115:10505-10510.
Heldt S, Ressler K (2009) The use of lentiviral vectors and Cre/loxP to investigate the function of genes in complex behaviors. Frontiers in Molecular Neuroscience 2.
Kravitz AV, Kreitzer AC (2011) Optogenetic manipulation of neural circuitry in vivo. Curr Opin Neurobiol 21:433-439.
Nagai Y et al. (2016) PET imaging-guided chemogenetic silencing reveals a critical role of primate rostromedial caudate in reward evaluation. Nat Commun 7:13605.
Roth BL (2016) DREADDs for Neuroscientists. Neuron 89:683-694.
Sternson SM, Roth BL (2014) Chemogenetic tools to interrogate brain functions. Annu Rev Neurosci 37:387-407.
Yizhar O, Fenno LE, Davidson TJ, Mogri M, Deisseroth K (2011) Optogenetics in neural systems. Neuron 71:9-34.