# Lecture 6c: Mouse Models **Channel:** Annelise Snyder **Source:** https://www.youtube.com/watch?v=HHXNwaNhv8k **Transcript page:** https://www.withtranscript.ai/video/HHXNwaNhv8k ## Chapters - 0:02 — Why Mice Are Research Models - 5:54 — Transgenic Mice and Their Uses - 14:01 — Knockout Mice and Global Caveats - 18:29 — Cre-LoxP Targeting and Expression - 24:52 — Mouse Models in Translational Immunology ## Transcript **[0:02]:** Welcome back, everyone. This is lecture 6C, the third and final lecture for the week six module, where we've been looking at experimental tools that are used in immunology research. This last lecture will focus on lab mice, which are one of the main model organisms used for in vivo immunology research. We will introduce different types of mouse strains, as well as genetic recombination systems that allow for greater control over the specificity of genetic perturbations. And then we will end today with a final discussion on the important contributions of mouse disease models towards translational immunology research. At the beginning of week six, we started with this slide comparing the two main categories of research models used in immunology, which are in vivo and in vitro models. We also went over a summary of the relative pros and cons of each before discussing the different types of cells that are used for in vitro experiments. **[0:54]:** So, if any of that isn't sounding familiar, then please go back and rewatch lecture 6A. Today, we will cover the other of experimental model, which is in vivo work that is performed on model organisms. Immunology labs may use different model organisms for in vivo work, depending on what their research questions are. So, most labs will use mice because they are vertebrate mammals, which means that they have both innate and adaptive immune systems. And they also have very well-characterized genomes that share a lot of sequence homology with humans. So, treatments and discoveries in mice are more likely to be translatable to human clinical at least when compared to more evolutionarily primitive metazoans, such as fish or flies. However, there are plenty of labs that study innate immunity in particular that may work with other models like the zebrafish or the fruit fly, since innate immunity is much more conserved between humans and these species. **[1:49]:** Indeed, toll-like receptors or TLRs of the innate immune system were actually originally discovered in Drosophila studies back in the 1980s. So, these and other models are still very important for immunology research. For the sake of brevity, today's lecture will focus on mice, since they are probably the most commonly used model organism for immunology research with translational relevance to human immunology. Again, these are live animals that need to be treated respectfully and in accordance with very strict ethical regulations. But, as we will discuss today, their contributions to immunology research is truly valuable enough to really justify their usage in experiments. One criterion for model organisms such as mice is that each individual from within one strain has to be genetically identical to one another. This allows researchers to perform controlled experiments where you manipulate only one factor at a time, such as a target gene or diet or application of some sort of drug treatment. **[2:50]:** And in the absence of genetic variability, this allows you to conclusively state that the effect of that single manipulation on an experimental readout is strictly resulting from that manipulation and nothing else. So, genetically identical individuals are created through a process called inbreeding, which is when you take mouse siblings and mate them to one another for many generations. And this is done in order to remove all of the genetic variability that you would usually observe in a natural outbred population of animals. If you remember some of your genetics background, the first generation of a sibling cross is called F1 in reference to the first filial generation that's resulting from a brother-sister mating pair. You repeat these sibling crosses for many generations, usually over about 20 generations, before you get genomic homogeneity, which means that all individuals are genetically identical or syngeneic to one another. At this point, this set of mice is then referred to as a individual mouse strain or mouse line. **[3:50]:** And when making these lines, a lot of candidate lines will die out before the 20th generation is reached. And this is due to the obvious sort of deleterious effects that accumulate over the course of inbreeding. Although, a few lines do eventually successfully arise or emerge from this process. The research community uses several well-established inbred strains for biomedical experiments, including immunology experiments. Some really common strains are the 129 mouse shown here on the top, which is a goody colored, as well as BALB/c mice, which are albino. And this one on the bottom, C57BL/6, also abbreviated as B6 or B6J, is very heavily used in immunology research since a lot of the commercially available altered strains, such as knockouts or transgenic mice, are derived from a B6 line. And you can purchase either breeding pairs or cohorts of mice from commercial vendors, such as Jackson Laboratories or Taconic Biosciences. **[4:47]:** And these are companies that maintain large mouse colonies as well as cryopreserved embryos from different mouse strains. An advantage of using inbred mice is that they have relatively similar genomes to humans, where virtually every gene in humans has a similar gene called a gene homolog in mice. Mice are also such commonly used models that each strain has extensive genetic as well as phenotypic characterizations that can be used or shared within the research community. Again, the power of using inbred mouse strains of um all genetically identical animals is that this allows you to manipulate a single condition in an experiment and have faith that any changes in response phenotypes are due to that one specific manipulation. Now, granted, this does come with the caveat that an experiment performed on a group of identical mice does obviously not um account for the genetic diversity that's present in human patient populations. **[5:40]:** So, this is just an important thing to consider when drawing conclusions from mouse experiments. Nevertheless, the genetic and disease modeling tools that are available for mouse strains makes them a real workhorse model that's used in immunology research. **[5:54]:** Let's start off by learning more about these genetic tools. We will talk about two categories of mouse lines today, which are transgenic mice and knockout mice. Each of these are genetically identical to their parental inbred strain with the exception of having been engineered to either have a modification or deletion in their genome at a single genetic locus. Let's start by looking at transgenic mice first. Transgenic mice are engineered to express a type of new or modified gene, which we call a transgene, and it's shown in this diagram here in green. Transgenic mice are often abbreviated by the name of the promoter that drives their expression once the transgene has been inserted into the genomic DNA. And this promoter name is followed by a superscript TG, which stands for transgene. These mice are created by taking a DNA vector encoding the transgene and using that vector to modify mouse embryos in vitro. **[6:45]:** And then you can implant these embryos into a pseudo pregnant female mouse. The embryos develop and then are delivered as mouse pups or progeny of that female, which can then be screened for transgene expression using a variety of techniques. Again, with transgenic mice, your goal is to get a mouse that expresses a brand new protein, shown here as the green circles, that was encoded by the transgene that you inserted into the genome. So, in summary, transgenic mice have been modified to express an extra genetic element that was purposefully introduced at a specific location in their genome. We will introduce some examples of transgenes in the upcoming slides throughout the rest of this lecture. Knockout mice accomplish something different, which is genetic deletion or inactivation of a target gene. Sometimes you will see genetic knockout abbreviated as KO or the name of the target gene followed by a superscript minus-minus. **[7:39]:** For the classic method of making a knockout mouse, you take the genomic DNA of a mouse embryo and modify it so that a target gene, shown here in blue, is excised or removed from the genome. Once these embryos have been modified, they can then be implanted into a pseudo pregnant female mouse, and you can then screen the progeny to identify individuals that lack expression of the protein product that was encoded by your target gene. This process creates a knockout strain that is genetically identical to its parental strain, except for the deletion of this one targeted gene. So, we usually refer to the knockout strain as a knockout mouse, while the parental strain is referred to as a wild type or a WT control mouse. Let's now look at some examples of transgenic or knockout mouse strains, starting with transgenic mouse lines. Again, these are mice where you've introduced expression of a new gene encoding a new protein at a specific place in the genome under the control of a specific promoter. **[8:37]:** One application of transgenic mice is to create a line that expresses a human version of a protein if you want to study its biological activity or effects in vivo. The reason why you might want to do this is because although mouse and human genomes have a lot of similarity, there are still some proteins that don't have the same structure or function shared between both species. A very relevant example of this right now is the human ACE2 expressing mouse line, which has been extremely popular recently for coronavirus research. SARS-CoV-2, the virus, um infects human cells by binding to a surface receptor called ACE2, which is an abbreviation for angiotensin-converting enzyme 2. This is the entry receptor for SARS, so it needs to bind to ACE2 in order to enter and successfully infect target cells. These are mainly epithelial cells found in organs like the lung where a lot of COVID symptoms are concentrated. **[9:29]:** However, mouse ACE2 has enough structural differences from human ACE2 that the strains of SARS-CoV-2 cannot productively infect mice, so they don't recognize the mouse version of this protein. And this means that we cannot use wild-type mouse strains to study coronavirus infection. To get around this, several years ago a coronavirus research lab made a transgenic mouse that expresses human ACE2 under the control of a promoter called K18, which stands for cytokeratin 18. The cytokeratin 18 promoter is specifically turned on in epithelial cells. So, these mice express the coronavirus entry receptor in epithelial cells in places like the lung, meaning that the SARS-CoV-2 virus can now successfully infect mice. This means that we now have a mouse model that provides an extremely important tool for studying mammalian immune responses to SARS-CoV-2 infection. As kind of a funny side note, these mice weren't super widely used outside of coronavirus labs sort of historically. **[10:27]:** But when the pandemic started in 2020, there was suddenly this massive increase in demand for these mice from all these other immunology labs that suddenly wanted to do coronavirus research. So for a while last year, there was a pretty long like 6 to 8 month wait list just for purchasing a single breeding pair of this strain since they were suddenly very popular in the research community. Another important application of transgenic mouse lines that's used by adaptive immunologists is the ability to create mice who only express a single lymphocyte antigen receptor with a single specificity. An example of this is something called a P14 mouse whose T cells are purely made up of CD8 positive T cells whose TCR is specific for an epitope expressed by a virus called lymphocytic choriomeningitis virus, which is abbreviated as LCMV. LCMV is a commonly used RNA virus infection model for studying T cell memory formation in mice and this virus expresses a glycoprotein called GP33 and it has an epitope in this protein that is recognized by the P14 TCR. **[11:31]:** To make these transgenic mice, you first knock out the endogenous TCR alpha gene in order to prevent the development of a polyclonal T cell pool as you would usually see. These mice also receive a transgene encoding TCR alpha and beta chains that form a single TCR that specifically recognizes this one epitope on the GP33 protein. And notably, this epitope is MHC class 1 restricted, so it can only be recognized by CD8 positive T cells. Therefore, compared to wild-type mice that have a polyclonal repertoire of mature T cells with many different epitope specificities, the P14 mice only develop CD8 T cells with this single to specificity. So an obvious next question is why would you ever want to make a mouse whose T cells only recognize a single epitope on a single virus? Why is this a useful tool? And the answer is that these are a very valuable tool to use in a common type of immunology experiment called an adoptive transfer. **[12:28]:** In this experimental setup, you isolate CD8s from a P14 mouse and then transfer them into a recipient mouse, such as a wild type B6 strain control mouse. If you then infect these recipient mice with LCMV, they've basically already been seeded with a large number of LCMV specific CD8s, meaning there are a bunch of virus-specific T cells that you can use to study T cell responses. And this is particularly helpful if you want to study a rarer type of T cell population like long-term memory T cells, where you're just not going to get a whole bunch of T cells in the first place. And it's also valuable if you want to manipulate the P14s with a drug treatment or genetic targeting approach prior to adoptive transfer. These are just two applications of transgenic mouse strains, but the ability to express new genes in mice can be applied to lots of other experimental strategies. **[13:19]:** You can introduce mutated versions of an existing mouse protein to see if a specific mutation affects its biological function in vivo. You can also express a fluorophore in certain cells, such as a green fluorescent protein or GFP, and this can be used to tag those cells for various applications. And in this case, we would refer to the transgenic mouse as being a reporter mouse or a reporter mouse strain. You can also express recombination enzymes like Cre in a system that we will discuss a little bit later on in this lecture. Overall, the ability to engineer transgenic mice is a very powerful tool for introducing new genes and therefore new proteins into an animal model for experimental studies. **[14:01]:** Knockout mice are a little bit more straightforward in that instead of introducing all sorts of new proteins or modified proteins into a mouse, you're really just trying to delete a specific target. This allows us to then characterize the overall response or phenotype observed in animals that are deficient in certain cell types or signaling pathways. One phenotype you can look at using knockout mice is whether a specific gene or protein product are required for normal animal development survival. A good example of this is the FoxP3 knockout mouse which lacks expression of the transcription factor FoxP3. As I'm sure everyone here remembers back to our week four lectures, FoxP3 is the master transcription factor required for differentiating CD4 positive T regulatory cells or T regs. And again, these are a very important immunosuppressive population of helper T cells. FoxP3 knockout mice that lack T regs suffer from really nasty forms of systemic autoimmunity. **[14:58]:** So this figure from a paper is a little bit grainy, but you can see a healthy wild type three-week-old mouse pup on the left which looks perfectly normal and fine. The FoxP3 knockout mouse pup on the right, however, has a lot of skin issues. So it has these raised bumps along its tail as well as a loss of fur on its legs. And it also has some deterioration of the tissue around the ear. And this is due to skin inflammation that is caused by a lack of T regs. Now, although you can't see with visual inspection, these mice also have high levels of inflammation that basically affect all organ systems throughout the body. And this autoimmunity is so bad that most of these mice only survive to about five weeks of age before they die from organ failure. And this really emphasizes the importance of T regs in maintaining immunological tolerance to self antigens. **[15:46]:** Another category of phenotype that you can look at by using knockout mice are susceptibility phenotypes. And these show how well mice respond to infections. So this graph shows a survival curve plotting percent of mouse cohort survival on the Y axis. This is plotted versus days post infection with Staphylococcus aureus on the X axis. This graph shows survival responses in a group of wild type mice in the black squares which all have um full they fully have all components of their immune system. And these are plotted in comparison to mice that are missing the key TLR adapter protein MyD88 shown in the white circles. The MyD88 knockout mice are extremely susceptible to S. aureus infection. As you can see that they all die within about a week of infection. This is in comparison to an overall mortality rate of only about 20% in the wild-type mice. **[16:36]:** This susceptibility phenotype highlights the role of TLR signaling in innate immunity in mounting successful immune responses to bacterial infection. Knockout mice are extremely useful in determining the contribution of certain cell types or signaling pathway components to types certain types of immune responses. But, there are a few potential issues to consider when using a mouse that is missing a gene in all of its cells. We refer to these as whole body or global knockout mice. One of these issues is called embryonic lethality. So, if you're studying a gene that is essential for the development of an embryo, since it may be involved in say pro-survival or growth factor pathways, knocking out that gene might mean that no embryos actually survive gestation. This by itself is an interesting phenotype that suggests your target is extremely important for development, but it obviously makes it impossible to study in live animals if none of them are actually born. **[17:31]:** Knocking out a gene or pathway can also lead to something called compensatory pathways. This is a situation where other pathways may be up-regulated in order to compensate for the missing component that you knocked out. And you see this with some metabolic pathways where a cell can adapt to deleting one form of nutrient processing by simply switching to another metabolic pathway. This confounds interpretation of results since, in addition to knocking out your target of interest, you now have activation of a new pathway that might affect your experimental readouts. So, just keep in mind that although knockout mouse strains are extremely useful, there can be a few unintended consequences or caveats that arise as a result of global genetic deficiencies. A researcher can address those by including proper controls in their experimental design to account for these caveats, or by using something called Cre-LoxP technology to increase the specificity of genetic targeting. **[18:29]:** Cre-LoxP systems enable better tissue or cell-specific deletion of knockout genes as well as expression of transgenes. And these are used extensively for both in vitro as well as in vivo genetic engineering. So, keep in mind that these principles can also be used to manipulate gene expression in cultured cells. Though today we will mostly talk about this in the context of mouse models. Now, Cre-LoxP technology works through two components. So, you have an enzyme called cyclic recombinase that's abbreviated as Cre shown here in orange. And transgenic expression of these LoxP sites shown here as the red triangles which flank a target gene in blue. When LoxP sites are located on either side of a target gene, we describe that gene as being floxed which is usually abbreviated as this superscript fl/fl. Now that we understand these two components, let's first look at this in the simplest situation where you just want to delete a target gene. **[19:22]:** In this situation, you take a mouse line that expresses Cre as a transgene under the control of a cell-specific promoter so that Cre is only expressed in that cell type where you want your target gene deleted. When Cre protein is expressed, it recognizes sites in the genome that contain two of these LoxP motifs. And once bound to the LoxP sites, Cre then exerts its enzymatic activity which is responsible for genetic recombination. Here, Cre basically cuts the DNA at the LoxP sites in a way that removes the target gene from the genome. So, following Cre expression and activation, you end up with two results. One is a loop of excised DNA here on the top and this contains the target gene along with one of the LoxP sites. Because this is a extrachromosomal loop of DNA, it is lost or degraded once the cells start dividing. **[20:11]:** The second and more important or relevant result is that within the genomic DNA, the the locus that used to contain the target gene now only has a single LoxP site and this is kind of a scar resulting from the recombination event. The Cre-LoxP system allows for genomic deletion of floxed target proteins. And importantly, this only happens in cells that express Cre. So, by encoding Cre using a tissue-specific promoter, you can restrict your genetic knockout only within cells or tissues where that promoter is activated and Cre is expressed. This enhanced tissue or cell specificity of genetic knockouts is really helpful. And it means that Cre-LoxP systems can be used to circumvent some of the issues associated with using global or whole-body knockout mice that we touched that we discussed earlier. Immunologists use a lot of different promoters to drive Cre expression, and these are summarized in this table. **[21:07]:** So, CD4 Cre targets both CD4 and CD8 T cells since both subsets go through the double-positive stage where they all express CD4 during their development. LCK Cre is driven off of the LCK kinase that is involved in TCR signaling, so this is also a T cell-restricted Cre. CD19 is specific to B cells since this is a part of the B cell receptor complex. I'm not going to go through each of these one by one, but just appreciate that there are a lot of different transgenic mouse lines expressing Cre under a variety of promoters with different expression patterns across cells or tissues. So, I've listed these all as being cell or tissue-specific promoters, but keep in mind that even though Cre expression may be predominantly restricted to one area, there is usually some promoter activation and subsequent Cre expression in other off-target tissues. So, while these Cres do provide enhanced tissue specificity compared to global knockouts, these are still usually not 100% clean, and you'll still have some expression in other cell types. **[22:07]:** This is just a caveat of working with complex animal models, and we can address this by validating and testing mouse lines to make sure that a given response phenotype is not due to genetic targeting of other cells besides your intended target. This last point plays into a running theme that I've touched on a few times during this week's discussions of experimental tools used in research. And this is that there will always be some drawbacks or caveats to certain models. And this is just kind of an inevitable reality when doing research. And it's okay as long as you include proper controls in your experiments and take these caveats into consideration when both when you're designing experiments as well as when you're drawing conclusions from your results. Besides genetic knockouts in specific tissues, Cre-Lox P systems can also be used to drive tissue-specific expression of a transgene. **[22:58]:** And this is done by expressing a construct called a loxP stop loxP cassette upstream of a certain transgene that you want to have expressed only in certain tissues. This could be a fluorophore like green fluorescent protein or GFP to tag certain cells shown in the top here in green. This could also be a target gene that you would like to overexpress in certain cells and this is shown in dark blue in the middle or something called diphtheria toxin receptor or DTR which can be used for cell-specific depletions and this is shown in light blue in the bottom row. In each of these cases, the transgene of interest is located downstream of this loxP stop loxP cassette. Here, in the absence of Cre, transcription of the transgene is inhibited because you have a stop codon placed immediately before the transgene's expression. However, when you express Cre in these cells, again under a tissue or cell-specific promoter, this stop cassette is excised out and the downstream transgene can now be expressed. **[23:54]:** LoxP stop loxP cassettes can therefore be used in combination with specific Cre's to drive transgene expression that's restricted to specific cells instead of being expressed globally. In these examples, this would include things like GFP expression as a reporter for a single type of cell. This can also be used to constitutively overexpress a protein of interest such as a model antigen or a chemokine receptor. Lastly, expression of diphtheria toxin receptor or DTR within certain cell types allows you to deplete those cell types. And this is because diphtheria toxin can only kill cells that bind to it and internalize it through a DTR. And importantly, wild-type mice do not express a receptor for diphtheria toxin. So, if you express DTR downstream of a lox-stop-lox cassette, and then one specific cells turn on Cre expression, you can then administer diphtheria toxin to that mouse in order to deplete that cell type. **[24:52]:** I want to end this lecture by emphasizing that the genetic tools available for use in mice enables researchers to conduct in vivo experiments that advance our understanding of the mammalian immune response. This means that we can not only make new mouse strains that do a better job at modeling aspects of human disease, but by being able to genetically manipulate individual cells and pathways, mice allow us to really drill into the mechanism of an immune response on the level of an entire organism. Given that immune responses require a complex coordination of multiple cells and signaling pathways across multiple locations in the body, these in vivo experimental models are an incredibly valuable tool to use in immunology research. Transgenic mice can be used to model all sorts of pathologies, including viral infections that are of public health interest, such as SARS-CoV-2. Generating things like the transgenic human ACE2 mice that we went over earlier allows researchers to characterize key components of the immune response to this virus. **[25:52]:** This includes things like T and B cell memory responses, the formation of protective antibodies, innate immune pathways that are activated early on in infection, as well as immunopathology resulting from infection. Once these baseline responses are defined in transgenic mice, we can then delve into the mechanisms involved in the immune response to coronavirus infection. And this is really a stage where we start to understand exactly how different aspects of the immune system contribute to host health. We can do things like compare experimental readouts between parental human ACE2 strains to a strain whose T cells lack a certain gene, which would allow us to ask how deficiency in that aspect of its T cell response influences the mammalian immune response to coronavirus. **[26:37]:** Transgenic mice can also be used to model different cancers such as this mouse which is abbreviated as a KC mouse and this develops pancreatic cancer called PDAC. If we take these PDAC mice and treat them with a immunotherapy such as an antibody which we can call therapy A in this example, we can compare experimental readouts like the quality of antigen presenting cells in the tumor, whether T cells are killing tumor cells, and whether this therapy results in tumor regression. If an interesting response phenotype is observed in these experiments, we can then move into mechanistic studies where we do things like cross the transgenic PDAC mice onto a strain that is deficient in some target protein like enzyme B in this example. And this would allow us to find out whether the activity of that enzyme in host cells contributes to any tumor responses that we previously observed in response to therapy A. **[27:26]:** Like with the coronavirus example above, these types of mechanistic studies provide critical information regarding exactly how a underlying immune response may function. And the genetic tools available in mice allow us to do this in a way that would be obviously impossible to study in humans. The examples I've introduced today just scratch the surface of the number and types of mouse models out there and since lab mice are such an important component of immunology research, there are strains and treatments that can be used to model basically any type of human pathology extending from the examples like infection and cancer that we talked about today to other pathologies like allergic reactions, autoimmunity, and transplant rejection. Although mice don't perfectly reflect responses seen in genetically diverse populations of humans because obviously humans are influenced by all other sorts of environmental factors as well, we can still pair these basic research findings in mice along with clinical **[28:21]:** data from human samples. This allows us to ask how well our mouse models and experimental findings may recapitulate the mechanisms of an immune response in humans. This allows us to ultimately determine how conclusions drawn from mouse research can inform therapeutic strategies for drug development that could be taken into clinical trials in humans. I hope that today's lecture has helped solidify why inbred mouse strains are such a valuable model organism that immunologists rely on heavily for studying mammalian immune responses in vivo. And that this work is all done with the ultimate goal of being able to translate basic research findings from mice into humans. This doesn't pan out 100% of the time because humans are obviously not the same as inbred mouse strains, but mice are still one of the best model organisms in terms of faithfully representing many aspects of the mammalian immune response. We've gone over the generation of inbred mouse strains today and how both transgenic and knockout mice are created as well as the different purposes that we use each of these models for. **[29:25]:** We've also discussed improving upon global transgene or knockout models by trying to get cell or tissue-specific genetic targeting, which is commonly done using Cre-LoxP technology. Taken together, these tools can be applied to all sorts of disease models in order to characterize not only general phenotypic responses to things like infection or therapy, but to define the immunological mechanisms underlying certain responses. This is the last lecture for week six. If you would like any more clarification on these topics, please see the corresponding reading assignment that's been posted to Canvas. Again, week six and week seven will cover similar overall topics, so this reading can also wait until next week if you need to focus on your midterm exam this week. This is also your last reminder that there are no discussion or extra credit quiz assignments this week, so please check your announcements in Canvas for more information about the midterm. **[30:18]:** Good luck with your exam and see you next week.