Podcasts about cre loxp

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549413v1?rss=1 Authors: Sun, Y., Guo, C., Chen, Z., Lin, J., Yang, L., Zhang, Y., Wu, C., Zhao, D., Jardin, B., Pu, W., Zhao, M., Dong, E., Hu, X., Zhang, S., Guo, Y. Abstract: Aims: Recombinant adeno-associated viruses (rAAVs) are federally approved gene delivery vectors for in vivo gene supplementation therapy. Loss-of-function truncating variants of LMNA, the coding gene for Lamin-A/C, are one of the primary causes of inherited dilate cardiomyopathy (DCM). Here we aim to study whether AAV-based LMNA supplementation could treat LMNA deficiency-triggered cardiac defects. Methods and Results: We compared whole-body, cardiomyocyte-specific and genetic-mosaic mouse models that carry Lmna truncating variants at the same genetic loci and uncovered primarily a non-cell autonomous impact of Lmna on cardiomyocyte maturation. Whole-body lamin-A supplementation by rAAVs moderately rescued the cardiac defects in Lmna germline mutants. By contrast, cardiomyocyte-specific lamin-A addback failed to restore the cardiomyocyte growth defects. A Cre-loxP-based AAV vector that expresses lamin-A throughout the body but excluding the heart was able to restore cardiomyocyte growth in Lmna germline mutants. Conclusions: Lmna regulates cardiomyocyte growth non-cell autonomously. Non-myocytes are the key cell targets for a successful gene therapy for LMNA-associated cardiac defects. Translational perspective: LMNA truncating mutations are among the major causes of inherited DCM. AAV gene supplementation therapy is emerging as a promising strategy to treat genetic cardiomyopathy, but whether this strategy is suitable for LMNA cardiomyopathy remained unclear. Our study counterintuitively showed that the cardiomyocytes are not necessarily the correct therapeutic cell targets for AAV-based treatment of LMNA cardiomyopathy. By contrast, careful elucidation of cell-autonomous versus non-cell-autonomous gene functions is essential for the proper design of a gene supplementation therapy for cardiomyopathy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

This week's episode features special Guest Host Mercedes Carnethon, as she interviews author Miriam Cortese-Krott and Associate Editor Charles Lowenstein as they discuss the article "Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure." Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast, summary, and backstage pass to the journal and its editors. We're your co-host I'm Dr. Carolyn Lam, associate editor from The National Heart Center in Duke National University of Singapore. Dr. Greg Hundley: And I'm Dr. Greg Hundley, associate editor, director of the Pauley Heart Center at VCU Health in Richmond, Virginia. Dr. Carolyn Lam: Greg, today's feature paper is one of those really, really landmark papers that really advance our understanding of Nitric oxide signaling. And it's about red blood cell and Endothelial eNOS, and how they independently regulate circulating nitric oxide, metabolites, and blood pressure. A real, real must, but let's go on and look at the other papers in this issue first. Greg, you want to go first? Dr. Greg Hundley: You bet, Carolyn. Better grab a cup of coffee. And my first paper is from professor Nathan Mewton from Hôpital Louis Pradel Hospices Civils de Lyon. Carolyn, these authors hypothesized that Colchicine a potent anti-inflammatory agent may reduce infarct size in left ventricular remodeling at the acute phase of STEMI. And so to address this hypothesis, they performed a double-blind multi-center trial and randomly assigned patients admitted for a first episode of STEMI referred for primary PTCA to receive oral Colchicine two-milligram loading dose followed by 0.5 milligrams twice a day, or matching placebo from admission to day five and the primary efficacy outcome was infarct size determined by cardiovascular magnetic resonance imaging at five days. And the relative left ventricular end-diastolic volume change at three months and infarct size at three months was also assessed by cardiac MRI. And these were secondary outcomes. Dr. Carolyn Lam: Nice. Okay. So what were the results? Dr. Greg Hundley: Right, Carolyn. So 192 patients were enrolled. 101 in the Colchicine group and 91 in the controls. And as a result of this trial, the oral administration of high dose Colchicine at the time of Reperfusion. And for five days thereafter did not reduce infarct size assessed by cardiac MRI. And so Carolyn, the clinical implications of these results suggest that other studies exploring the timing, pharma kinetics, and dose-response of Colchicine, as well as other anti-inflammatory agents are needed to identify an effective method to reduce infarct size and limit remodeling in this group of patients. Dr. Carolyn Lam: Wow, it's just such a rich field done with all this about Colchicine. Well, our next paper is a pre-specified sub-analysis of the randomized EAST-AFNET 4 Trial and the sub-analysis assess the effect of systematic early rhythm control therapy that is using Antiarrhythmic drugs or catheter ablation compared to usual care, which means allowing rhythm control therapy to improve symptoms in patients with heart failure. And this was defined in the sub-analysis as the presence of heart failure symptoms of New York Heart Association status two to three or a left ventricular ejection fraction of less than 50%. Dr. Carolyn Lam: Now, the authors led by Dr. Kirchhof at University Heart and Vascular Center UKE in Hamburg, Germany included 798 patients in this sub-analysis of whom 442 had HFpEF, 211 had heart failure with mid-range ejection fraction and 132 had HF-rEF over a median of 5.1 years of follow-up the composite primary outcome of cardiovascular death stroke or hospitalization for worsening heart failure, or for acute coronary syndrome occurred less often in patients randomized to early rhythm control therapy compared with patients randomized to usual care. And this was not altered by heart failure status with an interaction P-value of 0.6. Left ventricular function, symptoms, and quality of life improved equally in both treatment strategies. Dr. Greg Hundley: Wow, Carolyn, a lot of information here. So what can we take away from this? Dr. Carolyn Lam: Well, let's remember that this is a sub-analysis, albeit pre-specified of that randomized trial of the EAST-AFNET 4 Trial, but nonetheless, the data supports a treatment strategy of rhythm control therapy with Antiarrhythmic drugs or ablation within a year of diagnosing atrial fibrillation in patients with signs and symptoms of heart failure to reduce cardiovascular outcomes. Dr. Greg Hundley: Very nice, Carolyn. So, Carolyn, my next paper pertains to Alarmin Interleukin-1 Alpha, and it comes to us from Dr. Thimoteus Speer at Saarland University. So, Carolyn, Alarmin Interleukin-1 Alpha is expressed in a variety of cell types, promoting sterile systemic inflammation. And the aim of the present study was to examine the role of Alarmin Interleukin-1 Alpha in mediating inflammation in the setting of acute myocardial infarction and chronic kidney disease. Dr. Carolyn Lam: Wow, sterile inflammation. It's a really hot topic now. So what did these authors find? Dr. Greg Hundley: Right, Carolyn. So we're going to call Alarmin Interleukin-1 Alpha. Let's just call it IL-1 Alpha and so increased IL-1 Alpha surface expression on monocytes from patients with acute myocardial infarction in patients with chronic kidney disease was found to be associated with cardiovascular events. Next, IL-1 Alphas itself served as an adhesion molecule, mediating leukocyte-endothelial adhesion, and finally, abrogation of IL-1 alpha prevented inflammation after myocardial infarction and ameliorated chronic kidney disease in Vivo. Dr. Carolyn Lam: Wow. So what does this mean clinically? Dr. Greg Hundley: Right, Carolyn, so perhaps targeted therapeutic inhibition of IL-1 Alpha might represent a novel anti-inflammatory treatment strategy in patients with myocardial infarction and in patients with chronic kidney disease. Dr. Carolyn Lam: Amazing. Thanks, Greg. Well, in today's issue, there's also an exchange of letters between doctors Lother and Filippatos on Finerenone and risk of hyperkalemia in CKD and type two diabetes. There's an On My Mind paper by Dr. Sattler on the single-cell immunology and cardiovascular METs in, do we know yet what we don't know? Dr. Greg Hundley: And then Carolyn, from the mailbag, a Research Letter from Professor Wehrens entitled “Atrial Specific LK Beta One Knockdown Represents a Novel Mouse Model of Atrial Cardiomyopathy with Spontaneous Atrial Fibrillation.” Well, Carolyn, how about we turn our attention to those red blood cells and endothelial nitric oxide synthase. Dr. Carolyn Lam: Yeah. Can't wait. Dr. Mercedes Carnethon: Well, welcome to this episode of Circulation on the Run. Our podcasts, where we have an opportunity to speak with authors of important papers that are appearing in the journal of circulation. I'm pleased to introduce myself. My name is Mercedes Carnethon, professor and vice-chair of preventive medicine at the Northwestern University Feinberg School of Medicine. And I'm pleased today to invite our guest author, Miriam Cortese-Krott, who is the faculty of the University of Duesseldorf, and a guest professor at the Karolinska Institute in Stockholm. And we have with us as well the other associate editor who handled the piece for circulation, Dr. Charlie Lowenstein from Johns Hopkins University. So welcome to each of you this morning. Miriam Cortese-krott: Thank you. Dr. Charles Lowenstein : Thanks for having me. Dr. Mercedes Carnethon: Well, thank you. I'm really excited to jump right into this piece, Miriam, can you tell me a little bit about the rationale for carrying out the study, why you pursued it? Professor Miriam Cortese-Krott: The reason is because when I was working as a post-doc, I had to isolate an enzyme from red blood cells, which is a very, very difficult. And if you know, this enzyme is endothelial nitric oxide synthase, which produce nitric oxide, and actually, the red blood cell is full of the worst enemy of nitric oxide, which is hemoglobin. So actually, when I was talking about my project, everybody was asking, "Why are you doing that?" And I was actually able to isolate the enzyme and look at activity and be sure that the enzyme was fine, but the function of this enzyme was absolutely unknown. Professor Miriam Cortese-Krott: And the only way to study proteins in red blood cells is to make modification in the bone marrow of the mice. So in the Erythroid cells, because you can not, of course, if there are cells without nucleus you don't have any chance to modify them in culture, something like that. So the only way was to generate mice with modification specifically in the red blood cells. And I had the chance to create, to generate red cell-specific eNOS knockout mice. And of course, as a control endothelial-specific eNOS knockout mice by using the Cre-loxP technology. And with this technology, I could really understand what's happening to the physiology of the mouse if you remove this protein from the red blood cells. And so this was the whole idea. Dr. Mercedes Carnethon: Thank you so much. It was really exciting for me to read this piece. We are on opposite ends of the scientific inquiry spread as I'm an epidemiologist who does things at the population level, and you're identifying things at the basic science level. I thought the paper was extremely well-written and that encouraged people to dig in, even if you're unfamiliar, and in part that's because you provided such a great explanation of how your findings are used and how they're relevant to the process. Do you mind sharing a little bit about your findings and how you expect that they will be used by our scientific community? Professor Miriam Cortese-Krott: I think the main finding of this paper is that if you remove eNOS from the red blood cells if the mice are hypertensive, have hypertension, and this is completely something that you actually will not expect, as I told you that indeed red cells are full of the enemy of nitric oxide that remove it immediately. So you can ask yourself how it is possible. But I think the key finding here in this paper was that I also generated the opposite model. So I created the model a conditional eNOS Knockout model where you can decide in which tissue you want to have your enzyme. And of course, I applied for red blood cells. And what you see in this model is that you start from a global knockout mouse with hypertension, you reintroduce the eNOS just in the red blood cells, you have normal tension. So this means, this is the main finding. You have a switch in the red blood cells, which is the enzyme eNOS, which it's behaving in a completely different way clearly as compared to the vessel wall eNOS and still regulating blood pressure. Dr. Mercedes Carnethon: Well, thank you so much. I think this is the point at which I like to turn to the associate editor who handled the piece. Charlie, you and I don't get to talk as often given the diversity of work that we each pursue, but Charlie, tell me a little bit about what excited you about this piece? Dr. Charles Lowenstein: Thanks, Mercedes. So I love this piece. I thought Miriam, your article is so great. So a couple of thoughts. One is nitric oxide and nitric oxide synthase are so important in biology and medicine, nitric oxide regulates blood pressure. It regulates neurotransmission. It regulates inflammation. And this is true, not only in the lab, looking at cells in mice, but also in the human. So genetic variance in the endothelial nitric oxide synthase gene or NOS3 are associated with risks for diseases like coronary artery disease. So eNOS is just so important in biology and medicine. And now some ancient history. When I was a cardiology fellow, about a hundred years ago, I worked in the lab that first purified nitric oxide synthase proteins, and we cloned two of the nitric oxide synthase genes that was in the lab of Dr. Solomon Snyder at Johns Hopkins back in the 1700s. Dr. Charles Lowenstein: So when we cloned the nitric oxide synthase genes, when we and others did, we made a huge mistake. We chose the names for these isoforms from the tissue where they were first isolated. So we called the brain nitric oxide synthase nNOS, because it's a neurons, macrophages MCnos we called it MCnos and in endothelial cells, we called it the nitric oxide synthase eNOS or endothelial NOS. But in the last 20 years, lots of investigators have found these isoforms are in other cells, not just the original cells at discovery. And so Miriam's question is just so important, which cells make endothelial NOS also called NOS3. That's the history. Now what Miriam has discovered is just so important. I was so fascinated by her work because as she just said, she made two amazing discoveries. One, red blood cells make endothelial nitric oxide synthase. Dr. Charles Lowenstein: And that's been a controversy for a long time. Some people have said, "Yes." Some, "No." And Miriam made the definitive answer. Yes, red blood cells make eNOS, and secondly, she has discovered so much about the physiology of ENO coming from red blood cells, the nitric oxide that's made inside red blood cells regulates blood pressure. What a magical, interesting, and important finding. That's a little bit about the history. Nitric oxide and NOS are important in medicine. The people who originally cloned and purified the nitric oxide synthase isoforms named them after the tissue in which they discovered. And Miriam has made a major discovery that it's not only endothelial cells that make nitric oxide but also red blood cells. Dr. Mercedes Carnethon: Thank you so much for that summary. And I guess, I would have thought perhaps this was something of an Elixir of youth because if you've been working in this area for 200 plus years and Miriam, you started working on this as part of your dissertation work, you both have a lot of insight and background on where we've been and what the advances are. Miriam, can you tell me a little bit about how you'd like to see these findings used by the scientific community? Professor Miriam Cortese-Krott: I think I would like that the scientific community would use my mice first because I think, as Charles has said, it's not only red cells that express eNOS and it's not only endothelial cells. There are other cells producing eNOS and the function in the other cells is not known even in leukocytes, even when they have iNOS of course, but also have eNOS. So you can use my mice since it's a flux model. You can choose whatever you want, what cell you want, and then knock it in and knock it out. So this is one thing that I think the community could really do. I cannot do everything. So I'm happy to give my mice away. Professor Miriam Cortese-Krott: And the second thing is I would like too that in particular, the clinical community would see this link between Emathology and cardiovascular disease. This is something that was started, of course, there are studies looking at anemia and cardiovascular disease, but these studies have sometimes some issues I of course cannot speak as a basic scientist. I cannot speak about huge clinical trials, but I think this link exists and exists at the molecular level and it can be a target for pharmacological therapy. So I think this is what I would like to transport with this study to the clinical community and the basic science community. Dr. Mercedes Carnethon: Yeah. I think this is the point at which Charlie, I turn it to you because you really stand at the intersection of both of those communities. What questions do you have for Miriam going forward, as you think about spreading the word on this important work? Dr. Charles Lowenstein: So Miriam's discovery is just so important and she now has the tools to help answer really, really important questions. How is nitric oxide made in red blood cells? How is it stored in red blood cells? How is it transported throughout the body in red blood cells? What is the chemistry of nitric oxide, when it is stored, when it combines with oxygen when it forms nitrite and nitrate, how is it released from red blood cells? How is it targeted from a red blood cell to the vasculature? So there're these great basic science questions that Miriam and her colleagues are now poised to answer. So there's the science part of it. Then there's the medicine part of it because Miriam's mice and her great discovery have really huge implications for medicine. And so the question is, how can we use ENO? How can we deliver it? How can we target ENO to human tissues? Dr. Charles Lowenstein: How can we turn on erythrocyte, nitric oxide synthase? How can we turn it off? Because there are all these medical diseases where too much nitric oxide is bad, like in sepsis or inadequate amounts, don't protect the vasculature like atherosclerosis. Then there are all these other interesting questions. When we transfuse red blood cells, sometimes if you transfuse aged red blood cells, it's not good. You can harm people. Maybe we can load up or activate eNOS in stored red blood cells and then help deliver more ENO to patients who need red blood cells. So there are all these fascinating medical questions that we can look at based on Miriam's really important discovery. Dr. Mercedes Carnethon: Well, thank you so much. We're coming to the end of this wonderful and informative podcast. And I guess, I'd just ask Miriam, do you have anything else you'd like our listeners to know about your work and about the findings from this study? Professor Miriam Cortese-Krott: I would like people know that hard work help a lot, and that you have to believe in what you are doing and the quality of your science at the end would bring their true discoveries. So I think it's important specifically, for the young women in science that having this message too. So the science per se must be excellent and to proceed, you need a lot of work, but then the work goes to a good end. Dr. Mercedes Carnethon: Miriam, thank you so much for that inspirational note. The hard work that scientists need, the persistence across one's career and building from earlier discoveries, and bringing those forward through one's career are always critically important. And so I hope everyone has really enjoyed this episode and this opportunity to hear from Dr. Cortese-Krott. Miriam, you've done such wonderful work, and thank you as well, Charlie, for your insights about the intersection of this work with clinical care and basic science. Professor Miriam Cortese-Krott: Thank you. Dr. Charles Lowenstein: Thank you. Dr. Mercedes Carnethon: Thank you all very much for joining us today in this episode of Circulation on the Run. Dr. Greg Hundley: This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more, visit ahajournals.org.  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.24.215160v1?rss=1 Authors: An, H., Lee, H.-L., Cho, D.-W., Hong, J., Lee, H. Y., Lee, J. M., Woo, J., Lee, J., Park, M., Yang, Y.-S., Han, S.-C., Ha, Y., Lee, C. J. Abstract: In spinal cord injury (SCI), the scar-forming reactive astrocytes with upregulated GFAP proliferate aberrantly near the injury site, allowing themselves as a prime target for transdifferentiation into neurons to replenish dead neurons. However, the conventional use of GFAP promoter to target reactive astrocytes has two inherent problems: inadvertent conversion of normal astrocytes and low efficiency due to progressive weakening of promoter activity during transdifferentiation. Here, we report that the scar-forming reactive astrocytes are selectively transdifferentiated into neurons with 87% efficiency and 96% specificity via TRANsCre-DIONE, a combination of the split-Cre system under two different promoters of GFAP and Lcn2 and a Cre-loxP-dependent inversion and expression of Neurog2 under the strong EF1 promoter. After SCI, TRANsCre-DIONE caused transdifferentiation into Isl1-positive motor neurons, reduced astrogliosis, enhanced regeneration in surrounding cells, and a significant motor recovery. Our study proposes TRANsCre-DIONE as the next-generation therapeutic approach for patients suffering from SCI. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.15.204255v1?rss=1 Authors: Contino, S., Vrancx, C., Suelves, N., Vadukul, D. M., Payen, V. L., Stanga, S., Bertrand, L., Kienlen-Campard, P. Abstract: Presenilins 1 and 2 (PS1 and PS2) are predominantly known as the catalytic subunits of the {gamma}-secretase complex which generates the amyloid-{beta} (A{beta}) peptide, the major constituent of the senile plaques found in the brain of Alzheimer's disease (AD) patients. Apart from their role in {gamma}-secretase activity, a growing number of cellular functions have been recently attributed to PSs. They are involved in synaptic transmission, endo-lysosomal function and calcium homeostasis. PSs were also found to be enriched in mitochondria-associated membranes (MAMs) where mitochondria and endoplasmic reticulum (ER) interact. PS2 was more specifically reported to regulate calcium shuttling between the ER and mitochondria by controlling the formation of functional MAMs through its interaction with the Mitofusin2 protein. We have previously demonstrated that the absence of PS2 (PS2KO) alters mitochondrial morphology and function. Indeed, a PS2KO cell line showed reduced mitochondrial respiration along with disrupted mitochondrial cristae and increased glycolysis. This phenotype is restored by the stable re-expression of human PS2. Still, all these results were obtained in immortalized Mouse Embryonic Fibroblasts (MEF) and one bottom-line question is to know whether these observations hold true for the Central Nervous System (CNS) cells, and in particular neurons and astrocytes. To that end, we carried out primary PS1KO, PS2KO and PS1/PS2KO (PSdKO) neuronal and astrocyte cultures. All the conditions were obtained in the same litter by crossing PS2 heterozygous and PS1 floxed (PS2+/-; PS1flox/flox) animals. Indeed, contrary to PS2KO mice, PS1KO are not viable and therefore require the use of the Cre-LoxP system to achieve gene deletion in vitro. Strikingly, we did not observe any mitochondrial phenotype in PS1KO, PS2KO or PSdKO primary cultures. Mitochondrial respiration and membrane potential were similar in all models, as were the glycolytic flux and NAD+/NADH ratio. We further investigated the discrepancies between these results and the ones previously reported in the MEF PS2KO cell line by analyzing PS2KO primary fibroblasts. No mitochondrial dysfunction was observed in this model, in line with observations in PS2KO primary neurons and astrocytes. These results indicate that the mitochondrial phenotype observed in immortalized PS2-deficient cell lines cannot be extrapolated to primary neurons, astrocytes and even to primary fibroblasts. The PS-dependent mitochondrial phenotype reported so far might be the consequence of a cell immortalization process and, therefore, should be critically reconsidered regarding its relevance to AD. Copy rights belong to original authors. Visit the link for more info

Die mitochondriale Thioredoxinreduktase (Txnrd2) stellt als ubiquitär exprimiertes Selenoprotein ein wichtiges redox-aktives Enzymsystem zum Schutz vor oxidativem Stress dar. In einem Txnrd2-defizienten Mausmodell stellte sich eine embryonale Letalität am Tag E13.0 heraus. Hingegen zeigte sich in einem pankreasspezifischen Txnrd2-knockout ab der vierten Woche post partum eine spontan entstehende Pankreaserkrankung, die sich innerhalb einen Jahres zu einer fibrotischen exokrinen Pankreashypotrophie entwickelte. Dieses pankreasspezifische Modell, das durch Kreuzung einer Ptf1a-Cre transgenen Linie mit einer Mauslinie, deren Txnrd2 von loxP Sequenzen flankiert ist, generiert wurde, wird derzeit umfassend untersucht. In der vorliegenden Arbeit erfolgte die Generierung und Testung zweier Targetingvektoren zur Etablierung eines Mausmodells, das die Überexpression der murinen Txnrd2 in unterschiedlichen Organsystemen erlaubt. Dies geschieht im Hinblick auf die Generierung eines genetischen rescue des Txnrd2-Phänotyps im bestehenden pankreasspezifischen knockout-Modell zur Klärung der Fragestellung, ob durch das Wiedereinschalten der Txnrd2 Veränderungen oder gar eine Reversibilität des beobachteten Phänotyps möglich sind.

Insights into the developmental processes during which the brain forms from the neuroepithelium may provide a deeper understanding how the brain works. The Rho family of small GTPases is known for its many cell biological functions such as regulation of the cytoskeleton, gene expression, cell migration, adhesion, cell polarity and the cell cycle. All of these functions are of importance during the formation of the cerebral neocortex, which consists of the generation of its different cell types, their migration to their destination and their maturation to a functional network. These roles have been mostly established in vitro using dominant negative or constitutively active constructs. Since these approaches are often not entirely specific for single pathways, this work used the Cre/loxP system to genetically delete an individual member of the Rho family, RhoA, to examine its role following a loss-of-function approach. Specifically, we examined a mouse line where part of the RhoA gene has been deleted by means of the Emx1::Cre mouse line. This idea is based on previous experiences with the deletion of Cdc42 in the developing neocortex, which leads to a loss of apical progenitors. RhoA often works as a functional antagonist to Cdc42. Using immunofluorescence, we could detect a loss of RhoA at embryonic day 12 (E12) in Emx1::Cre-positive offspring carrying the floxed RhoA-construct in both alleles (cKO). At E14, we detected an increase in mitotic cells to 160% (±25%, p

The transcription factor HNF1B, encoded by the TCF2 gene, plays an important role in the organogenesis of vertebrates. In humans, heterozygous mutations of HNF1B are associated with several diseases, such as pancreatic β-cell dysfunction leading to maturity-onset diabetes of the young (MODY5), defective kidney development, disturbed liver function, pancreas atrophy, and malformations of the genital tract. The African claw frog Xenopus laevis is an excellent model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a series of methods. In the present study, we overexpressed HNF1β mutants in the developing Xenopus embryo to assess their roles during organogenesis, particularly in the developing pronephric kidney. Towards this goal, we developed a heat-shock inducible binary Cre/loxP system with activator and effector strains. Heat-shock activation of the mutant HNF1B variants P328L329del and A263insGG resulted in malformations of various organs and the affected larvae developed large edemas. Defects in the pronephros were primarily confined to malformed proximal tubules. Furthermore, the expression of the proximal tubule marker genes tmem27 and slc3a1, both involved in amino acid transport, was affected. Both P328L329del and A263insGG downregulated expression of slc3a1. In addition, P328L329del reduced tmem27 expression while A263insGG overexpression decreased expression of the chloride channel clcnk and the transcription factor pax2. Overexpression of two mutant HNF1B derivatives resulted in distinct phenotypes reflected by either a reduction or an enlargement of pronephros size. The expression of selected pronephric marker genes was differentially affected upon overexpression of HNF1B mutations. Based on our findings, we postulate that HNF1B mutations influence gene regulation upon overexpression in specific and distinct manners. Furthermore, our study demonstrates that the newly established Cre/loxP system for Xenopus embryos is an attractive alternative to examine the gene regulatory potential of transcription factors in developing pronephric kidney as exemplified here for HNF1B.

Cytomegalovirus (CMV) is frequently transmitted by solid organ transplantation and is associated with graft failure. By forming the boundary between circulation and organ parenchyma, endothelial cells (EC) are suited for bidirectional virus spread from and to the transplant. We applied Cre/loxP-mediated green-fluorescence-tagging of EC-derived murine CMV (MCMV) to quantify the role of infected EC in transplantation-associated CMV dissemination in the mouse model. Both EC- and non-EC-derived virus originating from infected Tie2-cre(+) heart and kidney transplants were readily transmitted to MCMV-naïve recipients by primary viremia. In contrast, when a Tie2-cre(+) transplant was infected by primary viremia in an infected recipient, the recombined EC-derived virus poorly spread to recipient tissues. Similarly, in reverse direction, EC-derived virus from infected Tie2-cre(+) recipient tissues poorly spread to the transplant. These data contradict any privileged role of EC in CMV dissemination and challenge an indiscriminate applicability of the primary and secondary viremia concept of virus dissemination.

Epstein-Barr Virus (EBV) is involved in several human malignancies via its latent gene products, which interact with cellular proteins and mimic discrete functions of cellular signalling pathways. Enigmatically, more than 90% of the human population carries this human tumour virus but virus-associated tumours are relatively rare. Most studies on EBV have been carried out in vitro and ex vivo on EBV-transformed human B cells or on human biopsies. Established in vivo model systems do not reflect the main aspects of EBV-associated diseases in humans. This limited tool set is the result of EBV’s inability to infect cells of non-human origin, which lack the surface receptor for EBV. My PhD work aimed at engineering a transgenic mouse, which carries a conditionally inactivated EBV genome. This study took advantage of the well-established techniques of mouse genetics in order to stably integrate the entire EBV genome into the murine genome. This approach would not only overcome the inability of EBV to infect animal cells but it would also permit to study the complete virus in an immunocompetent and easy-to-handle living organism. I undertook two routes to establish a transgenic mouse with the complete EBV genome inserted. One route was based on the site-specific integration into the hprt locus of murine embryonic stem cells. The other route engaged pronucleus microinjection of the EBV DNA into fertilized murine oocytes. In addition, the EBV genome was genetically manipulated prior to its introduction into murine cells. On the basis of the E.coli cloned EBV strain B95.8, I constructed a novel EBV mutant with unique features. This EBV targeting construct (InvTarg) allows conditional expression of EBV’s latent genes via a Cre/loxP system. Such approach prevents potentially adverse effects of EBV’s latent genes on embryonic development but allows their expression in almost any chosen cellular compartment for which specific Cre-expressing mice are available. The InvTarg recombinant EBV genome is 185 kb in size, based on a bacterial replicon, and therefore belonging to Bacterial Artificial Chromosomes (BACs). Two genetically modified and inversely oriented loxP sites were introduced in E.coli cells at the predetermined sites of the InvTarg, and the bracketed segment was inverted by Cre recombinase, disrupting transcription of almost all viral latent genes. In transgenic animals this inversion can be reverted and the latent genes can be re-activated at will by cross-breeding with Cre-expressing mouse (re-inversion). The ability of Cre to invert the big fragment was verified in infection experiments with human primary B cells. As expected, the ‘inverted’ EBV construct, such as InvTarg, failed to transform primary B cells, when the viral latent genes were not expressed. Despite sustained efforts, both gene delivery techniques did not lead to a transgenic mouse with the entire EBV genome inserted, but resulted in the integration of only subgenomic segments of the InvTarg recombinant EBV DNA. A number of technical problems were identified during this work, indicating more specific direction for further research. On the basis of the experience gained here, the project of an EBV transgenic mouse can be carried on. In addition, the InvTarg maxi-EBV conditional vector might be employed in other experimental conditions, like different cell types or distinct stages of cell differentiation, for studies on latent EBV genes.

The hyperpolarization-activated, cyclic-nucleotide-gated cation channel (HCN) familiy comprises four members. The channels play a role in the formation of rhythmic activity of heart and brain. In contrast to the other three members of the HCN channel family, there are only a few studies of tissue distribution and electrophysiological properties of HCN3 so far. Expression has been reported at low levels, but throughout the brain, in some other tissues such as retina, olfactory epithelium and recently in the heart. In order to study the physiological relevance of HCN3 expression, we generated HCN3-deficient mouse lines by gene targeting and homologous recombination: a complete Knockout, a complete Knockout expressing the reporter gene lacZ instead of HCN3 and a conditional Knockout using the Cre-loxP system to study spatial and temporal functions of this pacemaker channel. The functional characterization included behavioural studies and heart physiology.