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11. SUPER RESOLUTION MICROSCOPY but the main difference is the way they integrate the mathematical model used to reconstruct the image acquired....

11. SUPER RESOLUTION MICROSCOPY but the main difference is the way they integrate the mathematical model used to reconstruct the image acquired. It is a different way of collecting light from uorescent There are many variations in the way to reconstruct the sample. markers you can have a superior resolution than in regular Common points of different modalities: all are super-resolution techniques. Microscope resolution is the shorter distance that based. They allow to see changes in the sample with a lot of allows the microscope to see 2 objects next to each other rather precision and with a lot of labels all at once. You can use them than a single blurred point. to look at different colors of labels in your sample. They are all based on the fact that you can use speci c uorophore labels In regular techniques: you have a ring of different that can be converted from one stay to the other, based on the uorophores, you shine the light to excite them, and you energy that you shine at them. There are a lot of dyes that can collect the light emitted by the uorophores à if uorophores be switched on and off: there is a series of molecules of are very close to each other, the signal you get is going to different colors that can be activated by shining certain overlap, and you will get a general single signal. Most wavelength at and then they will return to the “dark state” microscope have not such a resolution to see individual dots. losing the activation. These labels can be activated or de- activated depending on the wavelength of light that you shine With super resolution microscope: instead of illuminating all at them. You can use the light of the microscope at a certain the objects at once, it illuminates some of points in the sample wavelength to activate a set of molecules present in the sample and collect data from genes and acquire their positions, then and so you can see them; then you can let them de- activate to illuminates others require that position minimise the overlap the “dark state” and so you don’t see them anymore. In this between the different dots, because it illuminates some points way you have a control of what you see at any moment, far apart from each other and then illuminate others. depending on whether you activate or not the labels. You have a clearer view of each and every one of them, because it progressively changes the angle of image You can locate every single coordinate and spot that you see. acquisition. In this way you can recreate the signals coming After taken the image, you can bleach your sample switch of from the sample by adding all the information. the labels to look at something else: you can just wait some The information is more reliable, because you don’t get the time, because the activation doesn’t last long or you can use a overlap of signals from objects close to each other. You can different wavelength to bleach the sample, and it becomes have a sharper view and you don’t get artefacts. again non-visible (because some molecule can respond to different wavelengths to activate or de-activate) With this microscope you can do much better than with regular The sample is now ready to be activated again. In this way you limit of detection of most microscopes. It is important because can do cycles and record images only from a set of spots at it allows to see where every single signal is coming from each time, then you reconstruct the whole sample. This allows within the sample with high precision (example: if you are to see different labels on the same sample. working with very thin bres which are really close to each other). This technique is very powerful because: 1) you can see at a very high resolution 2) you can look at multiple labels in the same sample. The machine needs to switch between activating and de- activating uorophores. During activation the light shines you have the con guration when you need to activate, and them when you need to de-activated you use the deactivation. When you have nished with the excitation lights, there is a sharper that comes in and blocks the laser. Then there is the readout laser that scan and reads what is in the sample. Example of image taken with super-resolution microscope: the membrane is labelled with green and the nucleus in blue. You An example of the type of image you can get: Actin is labelled can see nanoparticles that had been absorbed by the cell. You in green and tubulin in red. It is possible to actually see the can look at nanometres size objects within the cell and you can individual bres within the cell, which you wouldn’t be able to discriminate them. see at this level of sharpness and precision by using a regular Microscope You can distinguish nano metric parts into the cell. It has been developed different variants of super-resolution. We are not going into the details to see what makes them different, Pagina 80 fl fl fi. fi fi fi fl fi fl fl fl  12. RAMAN MICROSCOPY will be able to phenotype the cells, form different lineages, because they will show a different trace in the Raman This is another type of imagining which is crossing between spectrum. different elds. It’s a modality that combines: You can identify speci c in the spectrum that are different from -Microscopy, which is looking at the light that re ects from a one cell type to another. Then you can interrogate what is that sample chemical entity that is different. -Raman spectroscopy, which is looking at signals coming from chemical bonds in a sample. Conclusion: there are different ways of illuminating a sample and collecting information. In the wide eld modality: the beam of light that hits the With this combination you get a picture of your sample with sample is wide extra quality. The Raman technique is based on the fact that In the confocal: it gives a much ne beam light and so you different chemical entities will react differently to being hit can scan the sample going through it with a speci c wavelength. Depending on the chemical In the confocal scanning: it shines a very narrow beam at composition of a sample, the chemical bonds react and interact different levels of the sample and then you can acquire and differently with energy that is shining from a laser. Then it reconstruct the whole sample. Ii’s made at multiple levels to collects the energy from these chemical bonds that are hit by a have 3D imag wave of energy. Every chemical entity has a speci c pattern of In the light sheet: the beam comes not from the same axis of reacting to this wave of energy, based on the chemical bonds the observation point, but it comes from a 90° angle. You that are present within the molecule. It gives an “identity card” image what is coming from the whole sheet of light that of every single type of molecule. Raman modality allows to get scans the sample, not from a single point. It is much faster information of the chemical composition of a sample. If you combine it with a microscope, it means you are able to not just This summaries the types of distances that you can image and see a sample but to see and read the chemical composition at the type of information you can get. the same time. You don’t get just a morphological image, but The wide eld imaging look at everything is coming you also get a chemical image. from the sample The principle is that the light shines through (common to The confocal illuminates and collect speci c layers microscopy and Raman excitation) and then you can collect an The TIRF modality looks only at the bottom part of the image of what you see and the chemical information. sample. It can be useful if trying to determine the composition of a cell Depending on what you need to look at, you will choose the based on the chemical entities. Each component of the cell has modality that is the best to answer your question. This shows a speci c chemical composition, for example DNA, lipids, the different types of resolution and the different objects you collage, all have different composition and so they will emit can see with the different modalities we have seen. different Raman signals in this process. The microscope is able to overlay the chemical information There are modalities that can image whole animals in vivo. with the physical image. For every single point in the sample, you can interrogate the chemical composition. You can get a lot of biological and biochemical information out of the sample. For every spot you can see what it is made of. You can also compare different cell types by comparing their chemical composition. This is also a way of phenotype the cells. if you read the chemical composition of the cells, you Pagina 81 fi fi fi e fi fi fi fi. fi fi fl.  OPTICAL TWEEZERS TECHNOLOGY Allows to analyze samples that are in suspension and non attached. It allows to see tissues and cells with a high precision It had merged recently (2018). The Holographic OPTICAL we can observe for instance neurons in brain. TRAPPING it is not purely microscopy, but it uses microscopy to do something with the cells. The principle based on using a focused beam of light to strap physically and manipulate objects. The principle is using light as a force, force produced by the beam’s photons striking the object and a force produced by a gradient of eld intensity that keeps the particle at the centre of the trap. It creates an “optical trap” —> it is a point underneath the microscope where the cell will hold place and will be prevented from moving away. By playing with different type of lenses and changing the way that the light hits the sample, you can create a point in the microscope stage where the energy is pulling the cell back to the centre in the focus of the beam. It is called “optical trap” because it is using optics to trap the cell. This technique can be used in vitro and in vivo. If you have cells in vitro you can use the tweezers to dictate where you want the cells to be without touching them. It works in vivo as well to block the cells like red blood cells in a capillary, see the video). Why is it useful? Cell resistance: you attach two beads to the cell (ex. Erythrocytes), and you can prove the resistance and elasticity of the cell Cell-cell adhesion: you can put two cells (even different cell types, like cancer cell and T-cell), you let them bind to each other and then you try to pull one away from the other and measure how much force is needed to detach the cells, so how strong the interaction is. The system is able to record the force you need to apply. MULTI-IMMERSION IMAGIN It was produced starting from how the anatomy of the eye of the mollusk works. The lens has a round shape edge that is able to re ect all the light of the sample and it send the light hitting the mirror back. Pagina 82 fl fi G  CELL ENGINEERING TECHNOLOGIES Modify the sequence of a gene by introducing a mutation either to study a mutation phenotype or to These are some techniques used to manipulate cells in terms of understand the relation between the gene and its gene expression and to modify gene expression. function. Principle: in order to be expressed, a gene needs to be downstream of a promoter and the promoter’s sequence Gene expression always require the introduction and the dictates the sequence of the gene that is going to be expressed. delivery in the intracellular space of exogenous sequences. The professor wants to give us an idea of how expression can This can be achieved in: be manipulated and what are the main applications and contexts. (No details of genetics) TRANSIENT: you want to express the gene in a There are different approaches to engineer gene expression in transient manner, for a short period of time to see cells. we are going to look at the way to deliver the construct to what happens. modify the cell and different tools to actually modify the genetic content of the cell. STABLE: you want to permanently change the sequences that are expressed in the cell. Genome The genetic modi cation of cell and different technologies integration or stable inheritance of non genomic DNA. used: the story started not so long ago. This will require different tools. 1. Modifying bacteria (model of choice for genetics) to introduce speci c gene and get them expressed and Transgene can be expressed in a constitutive way so always translated into proteins. This started to be done in a expressed or in an inducible way, it can be expressed only in quite ef cient way in the 1960. certain conditions. In a selective inducible manner you want to 2. 1970: Discoveries on how DNA recombination works be able to switch on or off once the gene is inside the cell. in cell: mechanism used to promote integration of foreign DNA -Transiet manner: you can use a plasmid that is delivered into 3. 1980: Virus as delivery methods for speci c construct the cell with the coding sequence as vector, in this case the to force the expression of a foreign gene in a cell plasmid doesn’t need to be integrated into the DNA of the cell. 4. 1990: Gene therapy in human based on the attempt to The expression last a limited time. To code the gene is modify gene expression (applied for a speci c necessary a promoter: sequence of DNA that regulates the immunode ciency in 1990) expression of the gene. The promoter sequence itself is not 5. 1994: Commercial genetic modi ed tomato: it was able expressed, but it needs to be there for the machinery to binds to resist the rotting for long period of time and transcripts the gene. There will be a peak of expression 6. 1996: Cloning of Dolly the sheep: it was made by after the delivery and then It will decrease changing the whole nucleus from one cell to another. The whole genetic information of a nucleus was -Stable manner: so if you want to make a permanent change in transferred from one cell to an enucleated cell, and it the gene content of the cell, you have to use a different started to develop again. strategy. You need to force the incorporation of the new gene 7. 2013: Fine gene editing: systems that allow to target into the genome. In this way it will duplicate in the way the where in the genome you want to make a change and to cell duplicates the rest of its genome and you will have a long- decide what kind of change with a precision of base term presence of the trans gene. It is useful for looking at long- pair (Crisper). term effects. This works by using a LINEAR piece of DNA: in 8. 2017: gene therapy order to get it incorporating into the cell, you need to linearise the DNA (it works very bad with plasmids). You need to cut the plasmid using an enzyme so that it will become a strand of DNA that can incorporate into the genomic DNA The application of genetic modi cations: - Induce gene expression: you want to introduce a piece of foreign DNA with a gene of interest into a cell, see if it expresses and what it does to the cell interrogate the function of the gene. - Switch-off a gene: you modify the expression by switching of a gene that is present and see what the effect is on the Types of manipulation in research and therapy: phenotype of the cell. This is something useful in terms of Induce or increase gene expression (switch on) modelling disease Repress gene expression (switch off) Pagina 83 fi fi fi. fi fi fi.. fi. fi  - Trigger a mutation in a gene: If you are working on genetic Overexpression: You can use transfection to force the disease with a speci c mutation, you may want to know what expression of something that is not just a colour indicator, but the effect of that mutation on a healthy cell is. You can trigger it is a protein of interest. a mutation and see what the outcome is and whether the A typical example is: you are working on a gene and you want pathology is developed in the cell. to know what happens if you over-express it in a cell; so it may be a gene that is already expressed in the cell, but you want to INDUCING GENE EXPRESSIO see what happens to the cell if you increase the level of its expression. If you either want to trigger the expression of An example is trying to express in the cell a protein that in not something that isn’t in the cell or increase the level of normally present in mammalian cells —> Green Fluorescent something that may be already in the cell, you have to use a Protein (GFP), which is one of the most common approaches construct with a very strong promoter that will recruit the in genetic modi cation. This gene is present in a speci c type transcription machinery at a very high level. This is where the of jelly sh. It is used a lot in biotechnology, because it allows over expression comes from, because you are arti cially to introduce uorescent marker in cell that is not normally increasing the amount of transcription of a certain gene. uorescent, like mammalian cells. You can make the cells glow by forcing the expression of that protein. It can be done transiently because for example you want to image them. You can use the transient con guration: plasmid with a promoter able to drive the expression of the trans gene. Usually, it is used a strong promoter that will actively recruit the transcription machinery and so there will be high levels of There are thousands of examples of this strategy, where you expression. want to see what happens to the phenotype of a cell when you over express a gene. The strategy is the same, you just need to choose a promoter that is constantly on at a very high level and that will result in a very high level of transcription and a high level of protein produced in the cell, so that you can study what this means. The capacity of producing a very high level of protein in the cell is something that is used in terms of protein production, not just to study the effect CELL FACTORY = you can make the cell produce a huge You can choose the make the experiment in another way, not amount of a protein of interest. Then you can extract it to study just make the cells glow but use the GFP to study a promoter: or use it for medical interest. In order to do that, you need to you want to see if a promoter of a certain gene is strong or provide the cell the construct which contains the coding weak. You can put the promoter of interest upstream of the sequence of the protein, then you feed the cell and you also GFP, that in this case is considered as a “reporter gene” = it is propagate the cell, making large number of these producing reporting the activity of the promoter cells. Each cell will produce a huge amount of protein. Then You will see if the promoter is strong —> the cell becomes you can lysate all the cells and extract the protein produced. very green; no expression or weak expression of GFP —> the promoter is not active. There is not only the jelly sh green protein, but there are other sequences that can be used as reporters. One of the famous is dsRed and it has got a uorescent red colour. By using the coding sequence for this particular protein, which is foreign for normal mammalian cells, it is possible to label the cells. it works in the same way, you can use the same plasmid, you The cell factory is used for some of the treatment that require need to switch the sequence of the gene and it will get a proteins instead of small molecule. This are pharmaceutical different colour. approaches that are not based on chemical molecules, but are based on proteins Pagina 84   fl       fi fl fi. fi fl fi fi N. fi fi  REPRESSING GENE EXPRESSIO With the “knock down” approach you reduce the amount of transcript by degrading it, with the “knock out” you stop the 1)Gene silencing (knock down production of mRNA. You may want to down-regulate a speci c gene (called gene The general principle is based on a normal endogenous silencing) to see what the effect is. If you don’t know the mechanism of cell —> homologous recombination = it is the function of a gene, one of the easiest concepts is to take it process by which cells are able to repair damaging in DNA by away/to switch it off and so you can see what is missing. using the second strand as template to reconstitute a functional In particular for disease, this is diagnostic in terms of sequence. A group of scientists developed the technique to understanding the pathology and what leads to it. If you have a switch-off the expression of a gene by using homologous speci c gene that is suspected of causing a pathology, you can recombination, that means using the capacity of the cell to silence it and see what the phenotype becomes. recognize region of homology and bind it and integrate it into the genome. The process developed is that: if you have a speci c gene in the genome and you provide an exogenous set of DNA that contain the sequence that you want to integrate into the genome and it has region of homology to the actual genome —> you have a way of forcing the cell to integrate it You provide to the cell a piece of foreign DNA that contains region of high homology to the actual genome, then the cell will effectively be able to integrate this piece of DNA in exchange of the target existing region Knock down = to decrease the level of expression of a gene. In In this way is possible to take of a whole gene or a part of a order to do that, you have to interfere with the normal gene, by tricking the cell to incorporate a different sequence of transcription of the cell. DNA to cancel the gene. Doing it for whole is quite dif cult —> the longer the sequence, the more dif cult the technique One way of down-regulate the expression of a gene is by using is. You can do it for a part of the gene. RNA interference (RNAi). The way it works experimentally is It will work at low frequencies spontaneously, but you can that you have the cell that is expressing a speci c RNA that make it more ef cient you want to silence. You can use synthetic small interfering RNAs (siRNA), these are short molecule of double-stranded RNA introduced in the cell and their sequence needs to match the gene you want to target. It triggers an interaction in the cell with a RISC complex that is a natural mechanism of the cell against dsRNA that are typically of viruses. The RISC separates the two strands and the one that is complementary and hybridized to the target gene that is so degraded. MODIFYING THE SEQUENCE OF A GENE (MUTATION At this point the target mRNA becomes without function and so it can’t no longer be used to produce protein Another example is the idea of creating mutation This mechanism targets the non-self RNAs for degradation, so In medical eld when studying a speci c disease, very often it diminishes the negative effect of foreign RNAs that might the genetic disease is related to a speci c mutation in the invade the cell. By using siRNA, you trigger this defence patient. Lot of pre-clinical research are focused in trying to mechanism, but because you would design what it is going to understand the mutation and the phenotype, to understand why recognise, you target this degradation mechanism only against a change in the base pairs is causing a diseased phenotype. mRNA you are interested in knocking down. You interfere with The aim for the genetic modi cation is to introduce a mutation cell transcription at the level of one speci c transcript by in a genome that is healthy so that you can see the effect of that designing the siRNA to match the sequence that you want to mutation. down-regulate It is possible to This is done to interrogate the effect of a speci c transcript by Create a point mutation: to change one base pair in the switching it off. sequence of a gene 2)Gene inactivation (knock out There is another way of also repressing the expression of a gene, by totally inactivating it. Pagina 85   fi fi fi. : fi.. fi ) ) N. fi fi fi fi fi. fi. fi fi ).  It is possible to do it with homologous recombination mechanism: you know the sequences around the region where you want to introduce the stop codon in, and you use an insert that contains the base pair that encode for the stop codon you introduce arti cially a stop codon into the sequence of the gene. This means that every time the RNA will be transcribed, it will contain the sequence for the stop codon and so the ribosome will stop prematurely, and you will not get the If you have a gene, it will get a speci c mRNA that will get functional protein translated into a set of amino acids that give the protein. If you create a point mutation in the DNA sequence, depending in what kind of mutation you trigger, you might get a change of a single base pair that will cause a missence mutation —> in this case the changed base pair will have an effect in the protein sequence that is one different amino acid You can also get the case of introducing a stop codon —> you have an early termination for the sequence of amino acids of In this way you have knocked out the functional protein the protein, so that you have only a part of the protein formed. because it doesn’t get produced anymore. In effect in the gene sequence there is only one base pair Typically, the kind of the experiments that are done with this changed, but this is causing the protein to end prematurely. technique are: you take cells, either healthy where you want to There are different ways of designing the genetic introduce a mutation or patient’s cell where you want to correct modi cations, depending on what you want to achieve. It may a mutation. Then you study the effect of the mutation that have a dramatic effect on the outcome of the experiment. inactivates or activates a gene. For everything that relates to introducing or forcing the expression of a gene, you can decide the promoter The promoter will be the tool that will make the gene visible to the transcriptional machinery and it will dictate the level of expression of the trans gene. So, you need to know which promoter to use in the design of the experiment. You can also have the case of frame-shift mutation —> you don’t have a change in base pair, but you lose or gain a base pair in the sequence. That means that it will make a shift in the normal three base pair that are needed to de ne each amino acid. The whole protein sequence will shift, because the sequence of the mRNA that is read it is now affected by the fact that there is one less or extra base pair.It will change the amino acids of the protein. The protein might be part normal and part totally different. You can also cause the premature stop of the protein by trigger a change in the coding sequence for the protein. The aim is to inactivate the protein. You can introduce a modi cation at genetic level by introducing a stop codon. If you have a stop codon at the moment when the amino acids are being assembled, the ribose will stop, and it will be the end of the Constitutive promoter: as soon as the construct gets in the cell, protein. You can stop the production at a very early stage and the trans gene will be expressed. The promoter is constantly on in this way you get rid of the functional protein. and recruits the transcriptional machinery. One example of stable promoter is the CMV promoter. This promoter typically drives a very strong expression of the trans gene that is behind. That is a strategy used when you need to label the cells with GFP at high level. The cells will be glowing green at a stable way. Selective promoter: you may want to label only some cells, depending on particular characteristics. For example, you may Pagina 86       fi. fi fi. fi. fi  want to use GFO to label only one particular cell type in the there is no tetracycline you have the binding of the element sample. You deliver the GFP in the cells, but only the cells that that activates transcription, so if you don’t give the drug then have the phenotype you are interested in will be labelled. you will get the expression. If you start to give the drug, it will You can choose a promoter that is selective for a speci c cell inactivate the capacity of the element of activating type. Even though all the cells will receive the construct, only transcription and so it is switched off. the cells in which the promoter is normally active will switch the gene on, while the others will remain with the trans gene You can do both the systems, that mean that by adding or switched off. removing tetracycline you can be able to switch on or off the expression of a gene. You can have control of the timing of expression, because you can decide when to start. You can also control the amount of transcript you are going to produce, depending how longer you maintain the tetracycline Example: label only the brain cells with the GFP. You will choose a promoter that is active in brain cells but is not active in other cell types. When the trans gene will be present in brain cells, the brain promoter is active and so the cells will be green. If the construct ends up in a cell where the promoter is not normally expressed, then nothing will happen. By selecting the promoter used, you can dictate whereabout the Use a stimulus to trigger transcription (light) expression will be activated or not. You can use a model where a certain wavelength of light is Inducible transgene activation: you can decide when the able to activate or inactivate transcription. This is a fancy way promoter is active or not active in the cells, by switching it on of regulating gene transcription trough a light-activated protein or off—> using a drug-responsive expression system (LAP). In this case you have the gene of interest and a light- You can use inducible systems to drive the expression of a responsive element that is the promoter, which is the docking trans gene by using an inducible promoter so that you can place for a set of factors that will not be able to bind in the control its activity. One of the famous systems is called normal state. If you shine the right wavelength of light, this tetracycline on system. It relays in the fact that you give will change the conformation of the factors that then will be tetracycline to the cells that are been transfected to activate or able to bind to the light-responsive element and so the inactivate the promoter you used. In this case you have a trans transcription is activated. In this case you don’t use a drug to gene (ex. GFP) and upstream there is a promoter that contains change the conformation, but a wavelength to make the a speci c sequence called “tetracycline response element” that transcription active. Because it is light controlled, this means will function as a docking point for this element (rtTA) which that you can do it in a localised way. If you have the cells on a is able to activate the transcription of the downstream trans dish you can decide to illuminate only part of the dish and gene. When tetracycline is not included in the medium, the activate the transcription of the trans gene only in part of the cells that are transfected will not express the trans gene cells. The nice thing is that when you take the light off, the If you provide the drug, the tetracycline binds to the element system switches off again. So you can have a very ne control and it makes changes in conformation to make it able to bind and a lot of bene ts instead of using a promoter which is to the tetracycline response element and that will activate the always on at the same level transcription. By giving the drug you can switch one the transcription of the target gene. If you want to stop the expression of the trans gene, you need to take away the medium with tetracycline and add a medium without tetracycline, in this way the transcription switches off again. This is called “tetracycline on system” because when you give the drug the transcription is on This particular approach is something that is relatively new, but it is being used for neuroscience because in this eld the timing There is also the tetracycline off system, which is the opposite. for expression of a certain set of genes is critical to affect the There is a different element that is regulated. In this case when neurones. The spatial control is also very important in Pagina 87     fi fi.. fi fi.. fi.  neuroscience, because depending on which cell in which part study the effect and the role of speci c genes. This techniques of the brain is activated, it will get different effects. requires the injection of the transgene into the embryo These are examples of manipulation of speci c subsets of (pronuclear microinjection) or inside the blastocyst stade. neurones So the technology works very well and it allows you, as in this particular example, to make mice, for example, that would express in every single cell the GFP (Green Fluorescent Protein) as supposed to the control. That means that mouse will look different from any other mouse. If you shine and you’ll be light on, you can see differences, so you can see that this animal carries a speci c piece of current DNA (the one that encodes for the GFP) and that transcript is expressed in all of This is a silly veri cation of how it works. It is an in vitro test the cells so in all of the organs of the mouse that glows. for this system: you have a dish of cells that has been transfected with the construct (light-response element + GFP). It means that the expression in this case is controlled by a In this test they have made a mask with a shape to put on the strong universal promoter because you have a lot of signal and dish so that the light will only penetrate in the area where there because the promoter works in any cell type and all the tissues are gaps while the rest is masked by the lter. Then they are marked. So that corresponds to the strategy of using a activated with the right wavelength and then they take way the stable promoter that will work regardless of the cell type and mask. The result is exactly following what the lter is like, that will always be on, so that means that cells are constantly showing the activation of the GFP. Only the cells that had marked. received the light are become positive for the GFP, the ones around have the construct, but the protein is not present. This The strategy tends to be, for in vivo studies, a little bit more looks very silly, but it is actually testing whether the system re ned because very often we want to study the effect of works. It works in vitro. The study then looked at the brain, by particular set genes in a set cell type, because you are looking illuminating a speci c part of the brain. If there is no light then at a particular disease or particular physiological process. So there is no signal, but if they illuminate a certain part of the very often you come across studies where the transgene (in this brain that has been transfected by a speci c construct then you case GFP) is controlled by a tissue speci c promoter that can see activation. So it works also on tissue. would be on only in the cell type that you are studying, so that you can look at the effect of the gene in the context of the speci c cell type or organ or tissue that you are looking at and not in everything, The GFP (just as shown in the example), is particularly applied if you are looking at a functional gene for a speci c physiological process and you’ll be looking on a GALT tissue or if you want to study the effect of a speci c gene on galt physiology or galt cells and you want to look the expression of that gene in the tissue. But if you start overexpressing that gene in another tissue where it is not supposed to be expected, you are going to create some problems, so the animal might be spoiled not because of the galt phenotype you are looking at, but because you start express genes that are normally expressed only in the galt, you start expressing it in all the cells and probably the phenotype Transgenesi will be not related to what you are studying. Consist in producing transgenic animals particularly mouse So the system is much more precise and physiologically lines by genetic modi cation. As you know, you can make a relevant if you are using a tissue-speci c promoter to study a transgenic animal, that means you can make an animal that will speci c disease in a tissue of interest. express either in all its cells or in some tissues the transgene that you are interested in and that’s been the basis for a So you have to decide the strategy to create the transgenic massive progress in terms of studying genetics and studying animal that carries the genetic modi cation. different genes because it has allowed and it still allows To create a transgenic animal, you need to modify the embryo scientists to test the function of speci c genes in vivo and to because if you want all the cells to carry the transgene, you Pagina 88 fi fi fi. s fi fi fi fi fi fi fi fi fi fi fi fi fi fi fi  need to repeat all of the cells with the transgene, so it means because otherwise, your cell will die, instead of incorporating that you have to have to intervene at the start of development. the transgene. There are different ways to do it. Delivery of exogenous sequences to the intracellular space: Pronuclear microinjection: direct DNA microinjection into fertilized oocytes. One way is to deliver the transgene inside Calcium Phosphate Transfection the fertilized egg before you start getting the division of the egg itself. That means that you distribute your target and then (stochastically, it is not something that works 100%of the time) you will get the proportion of your foreign sequence. Then you need to screen the embryos or to screen the animals once they are born to see which ones have incorporated your transgene of interest. This technique, usually used to deliver your foreign construct The historical way to do this is using Calcium Phosphate to the embryo, is the pronuclear microinjection because the transfection because, in this case, you use the calcium embryo is large enough so that you can see it under the phosphate to coat your DNA all around, so it makes like an microscope and also you can hold it in place by this kind of aggregate with all the calcium phosphate ions around. holding pipettes. Because of the charges, that are negative on the DNA and So the idea of microinjecting is to inject physically with a positive on the Calcium ions, when you mix your calcium sharp needle the DNA directly inside the embryo. phosphate with your DNA that contains your transgene, they will bind to each other to make a physical precipitate, so they Blastocyst injection: injection of transgenic ES cells into the will make nanoparticles. blastocyst. Another way of injecting and creating a transgenic This is making the transition through the membrane more animal and it involves injecting not so much of the transgene ef cient because basically, these aggregates can stop attaching directly in the egg but involves creating transgenic stem cells. to the membrane and then they get picked up by under cycles You have to modify in vitro the embryonic stem cells and then inside the cell, so basically, the cell absorbs these aggregates reinject the stem cells into the blastocyst where they normally that you have between the DNA and calcium phosphate ions. come from and there they would be modi ed; then you have to implant the blastocyst, that now contains the stem cell with the So the system works by making a precipitate between the mutation of the transgene, into the pseudo pregnant female that calcium phosphate ions and the DNA that you have: ions are will carry the embryo and that will develop the embryo to turn. coming around the DNA because of the negative charge of In this way you have created transgenic animals as well. DNA and the positive charge of calcium ions so they have made this precipitate and particles are formed by putting them How can you get a transgene into the target cell? together. When they get in contact with the membrane, the membrane As shown, with the embryo the way to make the construct goes through endocytosis to internalize these aggregates, and enter the cell, so it can actually get incorporated into the DNA, this is the way the DNA gets into the cytoplasm (so you helped is physically with the micropipettes. For somatic cell types, this crossing by wrapping DNA with calcium phosphate ions). which are not as big as an embryo, how do you do it? So that was the original form of transfection for cells, it works well because it is a physical mechanism, quite universal in the Somatic cells are very much smaller than fertilized eggs, so it way it works. is dif cult to see them under the microscope for injection, for this reason, they can’t be injected physically, so in this case, You can make it more ef cient by using competent cells this way doesn’t work very well and it is totally inef cient. because what would make the process easier to achieve is if the There are other ways for somatic cells, even for embryonic membrane was a bit less dense than it normally is. So if you cells that are not eggs, to facilitate the crossing of the can make the membrane a bit looser, it has a better chance of membrane for the transgene to get inside the cell and get the letting things through like the precipitate. This can be done in transgene to be expressed. different ways. To be able to deliver a piece of DNA inside the cell you can’t just mix your cell with the DNA because it’s not going to get in One of the ways used to improve the ef ciency of calcium passively, so you need to facilitate this step. phosphate is through using ultrasounds, so to expose cells to ultrasounds probes because this looseness of membrane got What you need to do is somehow nd a way to get your damaging it too much; transgene to cross the membrane without damaging it too much Pagina 89 fi fi fi fi fi fi fi  but you can use a mild energy to make the membrane more and, by going there, at some point, it will be touching one of exible and more likely to let this kind of deposit and that the cells in suspension, so you can making a ow of DNA in improves the ef ciency and this is normally refers to one direction and at the same time you are making the cell sonoporation because the ultrasounds create pores in the membrane weaker, so you are favoring the entry of DNA in membrane. cells as well. So it works without it, but it is more ef cient if you add ultrasound trick to the calcium phosphate method. Electroporatio Representation of what happens in other terms: at the start the membrane is intact and you have the DNA around the cell that is not getting in by itself, then you apply the electric pulse so you start to get some pores in the membrane (disorganization) and at the same time you see that the DNA (charge negatively) can move towards the plus side, so it’s kind of coming across If we are trying to make pores in the membrane and your sample, so it is increasing the chance of getting inside. disorganize it, there is another physical technique to disrupt the The pulse needs to be short and mild so that the cell can membrane temporarily, that is electroporation. ( the same idea recover, so the membrane after the pulse will get back to its as in sonoporation). In sonoporation you use ultrasounds, integrity, so it is closing again, and that means that the instead in this case you use electric current with pulses for a transgene has caught inside the cell, and that way it can start to short period of time, they need to be short and not too high, so get expressed. you need to make short pulses of electric current to the cells to disorganize the membrane for a bit and during this time of It’s the same as doing with bacteria: when you try to get DNA disorganization, the cell has got some pores in the membrane in bacteria you need to disorganize the membrane so that the and the DNA can get in much more ef ciently. DNA can get in. Cationic Lipid Transfectio Electroporation is one of the most widely used techniques for transgenic experiments because it works quite well and it is a cheap method because all you need is this kind of cuvette with 2 metal plates on either side and you put the cell suspension in the middle. In effect, when you are going to connect each plate to the plus and minus part of your power back, you allow the current to go The 3rd method of getting your transgene inside the cell is through and, doing that, will disrupt the membrane and it will based on trying to create something that is like a liposome, help the DNA to get in into 2 different ways: which is basically a bubble with a membrane around, that is 1. the rst is the disorganization of the membrane so the made out of lipids, but that means that you can actually have a bilayer is going to be invaded by the check of the current cargo in and the fact that you have lipids in the outside means passing through, so it becomes looser and that will make pores that the af nity for the membrane and the fusion with the in the membrane. membrane will be assisted because you have lipids on the cell 2. the second is by applying the current, you also effectively membrane as well. So you can use typically for this a speci c act on the DNA itself because the DNA is charged, so it will kind of cationic lipids that are very ef cient in doing that. also be affected when the cell suspension with the DNA is exposed to the current. The DNA will also be able to migrate So you have your liposome, you need to mix the two and get based on the current because of its negative charge. the DNA inside the liposome, then you have your DNA which is trapped in this kind of vesicle and the vesicle will then get in So that means that the DNA will be moving from the minus contact in solution with the cell membrane and the actual side to the plus side so it will effectively go through the cuvette Pagina 90 fl fi fi n fi n fi fi fi fl fi  uptake of the vesicle is favorable because you have lipids on and incorporated, and therefore they don’t get duplicated when the outside, which is similar to the lipids on the membrane, and the cell divides and duplicates its own genome. you have a charge on the outside, which is a plus side because The advantage is that it doesn’t leave any footprint in the you are using cationic lipids. This means that the af nity for genome of the cell that you want to study. the membrane is going to be favorable and what happens is that this is getting internalized again by the cell and the DNA The alternative is to use lentiviruses, they are involved a lot in gets released inside the cell. So it is assisting the entry by using gene therapy because in gene therapy you want to leave this kind of liposome. something permanent to correct, for example, a mutation permanently. Therefore, you need a virus that will integrate There are different commercial products to do that with into the host genome and that will have a stable effect over different compositions in terms of lipids and you can also have time. So this is a different type of action because, once you get some polymers that do exactly the same. The important is to the infection and you get the DNA that is carried within the protect DNA inside something that is going to be accurately virus, this is then able to integrate within the host genome, and internalized by the cell membrane. therefore it remains there for how many generations and So you can have different lipid-based, liposomes or vesicles divisions you get for the cell. Depending on the application similar to liposomes that are made by different kinds of you will choose one of the other. chemistry but they do the same. The imagine on top is for the calcium phosphate transfection and on the bottom the lipid transfection, we see that there are different ef ciency depending on the cell. Viral Transduction So again, for adenoviruses, the cargo that is inside is DNA so One of the ways to get a piece of foreign DNA inside the cell is the transgene would be the coding sequence in dsDNA form. It the normal way of acting of viruses. You can use the machinery will be a transient effect because it doesn’t integrate within the that viruses normally use to get inside the cell and deliver the genome and remains as an episome (remains as a separate content which would be the transgene that you have created. entity that does not integrate within the genome). This happens using different types of viruses, the 2 main types As opposed to the lentivirus, which has an RNA cargo, in this of viruses typically used for genetic modi cations like this are case, if you want to express a gene you need to provide the adenoviruses and lentiviruses. RNA copy, not the DNA copy, inside the virus, but this would be a stable modi cation because it will be integrated within the The adenovirus is a DNA virus (the content of the virus is host cell genome and therefore it will remain for the lifetime of typically DNA) so it can deliver DNA inside the cell; the the cell. We can compare them to other non-viral transfection advantage of this type of viruses is that it has a transient effect, methods as electroporation, the calcium phosphate method, and and this effect consists of the fact that it doesn’t integrate the liposome method. In this case, you decide whether it is permanently in the genome. So it can infect the cell and deliver transient or stable depending on the design that you use. inside the cell the DNA content, and then the DNA will use the host cell machinery to get expression without physically These methods can have different ef ciencies because the integrating choice is based on what you want to achieve and if you want a stable or transient transfection. It also depends on the cell type you are working with because not all cell types will accept foreign DNA with the same ef ciency, some cell types are particularly dif cult to transfect and in particular, it is related to the proliferative capacity of your cells. Primary cells, that you extract from tissues and that are typically slowly dividing, are much more dif cult to transfect than a cell line that has been established for a long time that grows very quickly or cancer cells that proliferate very quickly, they tend to be much easier to transfect. So you need to adapt It is the same thing with plasmids: you can deliver them in the the method also based on the necessities of your target cell. cell, they don’t integrate into the genome but they will use the machinery to get the transcription of the transgene. At a certain