Étiquette : experiment

Domestic Foxes and Selective Breeding

Le par julienforsciencedans Interesting Discoveries / Les découvertes intéressantesLaisser un commentaire

For today’s article, I wanted to talk about an interesting experiment started by a Russian scientist named Belyaev. This scientist was very interested in understanding how dogs came to be domesticated. Dogs are indeed curious creatures: they are able to love us , as proven by MRI studies, and can be really affectionate. But we still don’t know how dogs became what they are nowadays. We know that they must stem from wolves with specific attributes, such as being able to digest our food or being less aggressive towards humans. Other than that, little is known about their change from wild wolves to domestic dogs. One of the big reason why is the timeline. Genetic and archeological studies hypothesized that dog domestication happened 20,000 to 40,000 years ago, and has continuously evolved until today. Due to the large timeline and even larger changes in human behaviours, it is hard to understand or replicate dog domestication nowadays. However, Belyaev tried to do so about 60 years ago [source / source / source].

Instead of wolves however, Belyaev used foxes. He simply took wild foxes, and looked at the behaviours of their pups. He then chose the tamest and nicest pups that were warmer to human contact, and only bred these. And after 60 years of this, we have « domesticated » foxes. These foxes are able to live with humans without fear and don’t hide when strangers are presented. This is a stark difference from the wild foxes, which usually either hide or become aggressive towards humans. These foxes still exist nowadays, some are even in Canada, and can become pets if you have lots of money to spend. [source / source].

While these foxes may act more friendly towards human, we are far from having them fully domesticated. Simply look at our relationships with dogs: when they’re scared, they will come to you, they are happy to see you when you come home, and will ask to play with you when bored. These foxes do not do that at all. While they bear the presence of humans and some account that when afraid they will come to you, the relationship is more similar to that of a fish in a bowl: the fox is here because you feed it but to our knowledge does not feel any way towards you. Now it is unfair to compare these foxes to dogs; after all, they did not have the 40,000 years dog had to become what they are. But Belyaev was on the right path: the first « domesticated » wolves probably were more like these foxes than our dogs today. Therefore if we give another 1000 year or two, maybe we’ll have truly domesticated foxes! [source / source]

This experiment is also a good preview to an age-old practice: selective breeding. Everyone has been shown a picture of corn or watermelon before it was properly cultivated. Corn barely showed any kernel, while the watermelon had more shell than anything else, and it was full of seeds. Compare it to our current corn and watermelon, the changes are stark, and almost solely due to selective breeding. Selective breeding is the process of choosing the best offspring for our purposes. So for our crops, people only planted the corn that had the most kernel, or the watermelon with the least amount of seed, and discarded the rest. Over time, only these were produced, and the old-fashioned corn or watermelon does not exist anymore. We can explain this genetically: the amount of seed, or kernel is due to specific alleles that are expressed, and thus by only planting these crops, only the alleles you want will be expressed, and the ones you don’t like are discarded [source / source].

Now, selective breeding is quite easy in plants. In fact, it is so easy that now we do not have to wait for decades to get the crops we want. We now have genetic engineering to do it for us. Since we now know which allele produces the most kernel, we can modify our seeds so that they express this allele and not any other and get only corn with lots of kernel. This is a genetically modified organism (GMO). While some are more complicated than that, the basis of the GMO is to get the best yield out of our crops, and not lose the decades it would have taken if we were to use selective breeding instead. However, in our fox experiment, selective breeding is harder, and that is because animal behaviour is not solely genetic. Consider this hypothesis: let’s say 20,000 years ago, some wolves were tamer towards humans and some were not. The tamer wolves were more likely to get food from humans. Let’s now say that at this time, there was a contagious disease in poultry, which is a food for both humans and wolves. Humans, by cooking their food, would not get sick, but wolves eating raw poultry would get sick. However, tamed wolves would likely get the cooked food and survive, while wilder wolves would stick to raw poultry and die, which increased the amount of « docility allele », making more overall tamed wolves. While this is just a hypothesis I invented and as such is unlikely to be true, it shows how the environment and the species relationships can be crucial for domestication. Our fox experiment, being scientifically controlled, is stripped from these random social factors that could greatly influence domestication [source / source].

Overall, the fox experiment is a step towards the understanding of domestication. While not perfect, the experiment allows us to see that selective breeding must have had a role in the development of dogs. Furthermore, it is nowadays possible to buy these specific foxes, and thus remove them from the strict scientific controlled environment. Maybe these foxes will be able to tell us more on the subject of domestication.

Understanding Science: The Antibodies

Le par julienforsciencedans Understanding Science / Comprendre la scienceLaisser un commentaire

Today is the start of a series about scientific experiments. Oftentimes in the public media, scientific discoveries are presented without explaining how scientists got those results. Understanding how experiments work is a great way to understand science in general, and also be able to spot problems or misunderstandings spread by the lay media. Today, I will focus on the use of antibodies in labs. Antibodies are essential for any lab that studies proteins. We will talk about protein visualization through the use of immunohistochemistry, protein quantification via Western blots, and finally, we will see cell identification with flow cytometry.

Before going into the experiments, one question must be answered: why antibodies? As we saw in our series on the immune system, the antibodies are able to bing a specific antigen during an immune attack. An antigen is simply a specific spot on a pathogenic protein. So scientists thought of making antibodies to be specific to a protein of interest, and nowadays, we can get antibodies for virtually any protein in any animal that we want. Another advantage of antibodies is their specificity. Theoretically, they will only bind to the protein they recognize and nothing else. While this is technically not true, as macrophage can bind antibodies for example, any other binding can be prevented during the experiment so that they will only bind the protein of interest [source].

Immunohistochemistry: how scientist see the proteins

Immunohistochemistry (IHC) is a technique that most lab will use. I will explain it through a mock experiment. I want to know where the protein adrenaline is expressed the most in a mouse brain. To do so, I will slice the brain in thin slices, and block them. Blocking is the technique that prevents any non-wanted interaction with your antibody. Then I will put my antibody against adrenaline. Now, to visualize the protein, we need a secondary antibody that is specific to the first one. Thus we have a brain slice with our primary antibody binding adrenaline, and our secondary antibody binding the primary one. Our secondary antibody was modified so that it has a fluorescent tag on it. It’s this tag that will allow us to see the protein. And now the experiment is done, we can go to the fluorescent microscope. On there, we will be able to see the proteins (in the form of coloured dots) [source].

There are many advantages to IHC, and the main one is localization. for example, in our adrenaline experiment, I will be able to see in which specific parts of the brain there is more adrenaline, and which have less. Next, it is a very easy and relatively cheap experiment. Further, we can also quantify how much protein there is by counting the dots, although this is not the best way to quantify proteins. However, the quantification can be region-specific, which is good for some proteins. IHC can also be used in more clinical applications. For example, taking tissue samples of patients, we can use IHC to determine the cancer stage for example. However, IHC also has some limitations. First, we can only use a limited number of antibodies, so we can visualize only about 4 proteins in one tissue. Further, the tissue is dead, thus we lose the protein movements. Lastly, the experiment is very sensitive to light (due to the fluorescent tag). If exposed to light for too long, then the tag will not work anymore and the experiment will be lost [source / source].

Western Blots: how scientists count proteins

Western blots are also a staple in scientific labs. I will explain it through this experiment: I want to know if stressing a mouse will increase the production of adrenaline. To do so, I will stress one mouse, and then take its tissues. I will take the tissues of another mouse that has not been stressed as a control. I will take the tissues and break them down so that we only have the proteins. Now I will separate the proteins based on size. To do so, we will perform a gel electrophoresis. We will create a special membrane in which we will put our samples. Then we will run an electric current through the membrane. Proteins are negatively charged and like a magnet, they are attracted to positive charge. This will allow the proteins to travel down the membrane. However, our membrane makes it harder for heavy proteins to travel. Thus in the end, our membrane will have all of our proteins, with the lighter ones at the bottom of the membrane and the heavier ones at the top. Next, we have to transfer our proteins on another membrane that is easier to work with and that we can keep for a longer time. This is also done with electricity. We will then block the membrane (similar to a IHC), and add the primary antibody against adrenaline, and our secondary antibody. This time however, our secondary does not have a fluorescent tag, instead it has another protein called horseradish peroxidase (HRP). This protein, when in contact with another protein called HRP substrate, will allow us to visualize our protein of interest as a black band. To do so, the substrate will be applied on the membrane, and then a film will be placed on the membrane. The film will then be exposed in a special machine so that the bands will be visible [source / source].

The biggest advantage of Western blotting is the ability to quantify. Basically, the bigger the black band, the more proteins we have. In our adrenaline experiment, the stressed mouse will have a bigger band than the non-stressed mouse. Western blots are also extremely sensitive: they can detect a very small amount of protein, which allows us to study rarer proteins more easily. However, Western blots also come with their disadvantages. The main one is its difficulty. As you may have guessed after reading the protocol, this experiment is very hard to do, and a lot of things can go wrong. It is also a very long experiment (it usually takes me two to three days to finish a blot), and it’s quite expensive as well. Another problem is the difficulty of analyzing the results. Western blots are prone to show wrong or misleading results, either because the experiment was not performed well, or because the primary antibody was not as specific as we thought. Unfortunately, there is no way of knowing what went wrong until the very end of the experiment, making this experiment very demanding [source / source].

Flow Cytometry: how scientist can identify cells

Flow cytometry is a rarer experiment. It is mostly used in immunological labs but other disciplines can also use it. It also has a big use in hospitals. Flow cytometry has many uses, but the most interesting one is to identify cells. Let’s go through it with another experiment: I want to know if immune cells are able to produce adrenaline, and if they are, which one of them are able to do so. For this, I will take a mouse and take some of their blood. I will treat the blood so that only the cells are in the sample. Then I will block it and add my primary antibodies. Here I will add antibodies for adrenaline, CD4, CD8, and B cell receptor. Then I add my secondary antibodies with my fluorescent tag. Each primary antibody has its own secondary antibody, and each secondary antibody has a different fluorescent tag. Then I can go to a machine called a cytometer. This machine will analyze the cells one by one and shine lasers on them to identify which antibody they have. We can then see which cell expresses which antibody [source].

Analyzing a flow cytometry experiment is hard. In our mock experiment, I will separate my cells into three groups: the cells that have the CD4 antibody will be CD4 T lymphocytes, the cells that have the CD8 antibody will be CD8 T lymphocytes, and the cells with the B cell receptor antibody will be B cells. Then, within each group, I can see if they express the adrenaline antibody, and how many cells within each group express it. Finally, the cytometer can allow me to take my sample back for further analysis, but if I want to, I can take only the cells that express both CD4 and adrenaline antibody, and discard the rest. As you can see, many things can be done with flow cytometry: we can identify cells, see which proteins express a specific cell, sort our cell so that our sample only contain specific cells, etc… Flow cytometry has other advantages: with the right cytometer, you can have more than 10 different antibodies, allowing you to have very specific subset of cells. Since the cytometer analyzes the cells one at a time, it is extremely specific. However, just like any experiment, there are some flaws. The biggest one is the difficulty: the experiment is very hard to perform, and the cytometer is extremely hard to use. It is also an extremely expensive experiment. Another problem is that we lose the integrity of the tissue, thus unlike IHC, we lose some of the localization [source / source].