Podcasts about scnt

This week, DaLeyna & Deandra chat with trademark attorney and owner of The Creator's Law Firm, Ticora Davis, about building and protecting your brand, her own personal journey to entrepreneurship and the advice she gives to others with similar pursuits! Powerfully positioned as an intellectual property advocate, Ticora Davis is the attorney, author, and speaker providing culturally relevant legal representation to black women and creatives of color. As a "purpose activator," Ticora seamlessly blends her knowledge of creative business law with her passion for pushing women to achieve economic freedom. Ticora founded The Creator's Law Firm in 2017 to help experts and entrepreneurs protect their brands so they can grow their business with peace of mind. Her work has been featured on VH1, Black Enterprise, and in partnership with Facebook. The Creator's Law Firm has helped hundreds of business owners secure their brand and proudly boasts a 98% success rate for trademark registrations. Learn more about The Creator's Law Firm at creatorslawfirm.com Follow Ticora: Twitter @creatorslawyer Instagram: @ticoradavis ------------------------------------------ Check out DaLeyna's recommendations for Black-owned candle companies: SCNT: scntcandles.com Brighteyed for Treasures: brighteyedfortreasures.com Private Suite Collection: privatesuitecollection.com --- This episode is sponsored by · Anchor: The easiest way to make a podcast. https://anchor.fm/app --- Send in a voice message: https://anchor.fm/and-another-thing/message

Sonja Schrepfer Bio: Sonja Schrepfer, M.D., Ph.D., Professor of Surgery, founded the Transplant and Stem Cell Immunobiology (TSI) Lab in 2009 in Germany. In 2015, she joined the faculty of the Department of Surgery at the University of California San Francisco and was Director of the TSI Lab at UCSF. Sonja is scientific co-founder of Sana Biotechnology Inc. which she joined as SVP in 2019. Dr. Schrepfer's research career has been dedicated to making fundamental discovers in transplant and stem cell immunobiology. Pluripotent stem cell (PSC)-based approaches are effective in immunosuppressed/deficient animal models; but in humans, systemic immunosuppression cannot be justified, due to severe side effects and significant risk of infections and malignancies. So far, only a few immunological strategies have been proposed to overcome these hurdles. Work by Dr. Schrepfer is at the forefront of PSC immunobiology and paves the way for treatment of a wide range of diseases – from supporting functional recovery of failing myocardium to the derivation of other cell types to treat diabetes, blindness, cancer, lung, neurodegenerative, and related diseases. She spent many years examining in detail the fetomaternal interface for application to the envisioned cell therapy. Her work with one of the most antigenic phenotypes, antigen-presenting endothelial cells, demonstrates that hypo-immunogenic cells reliably evade immune rejection in allogeneic recipients that are entirely mismatched in their major histocompatibility complex profile, and further, these cells show long-term survival without immunosuppression in mice and humanized mice (published in Nature Biotechnology in 2019). Sonja is currently Adjunct Professor at UCSF investigating the immunobiology in “tissue chips in space”; that is sending tissue chips to the international space station (ISS). She participated in three flight missions as collaborator and was the PI on the SpaceX16 mission (December 2019). This research will provide insight into what physiological effects time in outer space might have on astronauts, with potentially important implications for future longer-term missions, and has the possibility to open the door to fascinating new discoveries that could be used in earth-bound immunology research. Tobias Deuse Bio: Tobias Deuse, M.D. is a cardiac and heart and lung transplant surgeon internationally renowned for his pioneering work in the development of minimally-invasive techniques for mitral valve repair.  Dr. Deuse graduated the University of Stuttgart (Germany) in 1994 with a BS in Physics, and in 2000 earned an M.D. from University of Wuerzburg. Dr. Deuse thereafter received advanced training in cardiothoracic surgery at the University Hospital Munich-Grosshadern and University Heart Center Hamburg-Eppendorf. After obtaining his board certification in Germany in 2007 as a heart surgeon, Dr. Deuse completed a surgical fellowship in Lung and Heart-Lung Transplantation at Stanford and joined the UCSF faculty in 2015. Dr. Deuse's laboratory at UCSF is working on the immunobiology of pluripotent stem cells. To circumvent rejection, techniques such as somatic cell nucleus transfer (SCNT) into an enucleated oocyte (formation of a SCNT stem cell), fusion of a somatic cell with an embryonic stem cell (ESC; formation of a hybrid cell), and reprograming of somatic cells using certain transcription factors (induced PSCs, iPSCs) have been used. However, his work has shown that SCNT stem cells and iPSCs may have immune incompatibilities with the nucleus or cell donor, respectively, despite having identical nuclear DNA (published in Cell Stem Cell 2014). Further, he has demonstrated that mitochondrial (mt) DNA-encoded proteins as well as mtDNA mutations and genetic instability associated with reprograming and iPSC expansion can create minor antigens, producing rejection. His work also demonstrated that even autologous iPSC derivatives are not inherently immunologically inert for autologous transplantation (published in Nature Biotechnology in 2019). This has provided an important, promising avenue for selection of optimal stem cell therapeutics for future clinical applications ¾ via identifying the most compatible starter cell line and monitoring “near match” autologous iPSC products for mtDNA mutations and single nucleotide polymorphism (SNP) enrichments during the manufacturing process. Director Sonja Schrepfer, M.D., Ph.D., and co-director Tobias Deuse, M.D., explain the lab's research towards understanding and overcoming transplant rejection. They touch on Why finding ways to reduce rejection and successfully find transplantation avenues that don't require immunosuppression drugs is so important, How their research starts with pluripotent stems cells that must be differentiated and then transplanted,  Why using a patients' specific stem cells still face rejection due to mitochondrial proteins that eventually form despite gene editing, and How the lab is working toward an "off the shelf" solution by altering proteins that trigger rejection and other means. Drs. Schrepfer and Deuse run the Transplant and Stem Cell Immunobiology Lab (TSI) at the University of California in San Francisco and specialize in heart and lung transplant issues through CRISPR, gene editing,and stem cell therapy. They begin by explaining the many complications a person taking immunosuppressant drugs faces and why their research seeks to address these issues and make for a safer system for patients. Further, they explain that patient-tailored stem cell therapy approaches are not suitable for large populations for several reasons, including the frequent need to treat a patient almost immediately for heart damage or other similar issues. They explain that while they can generate cardiac cells that don't get rejected at first, these cells can develop mutant proteins that causes rejection later. They are following a couple of approaches to address the rejections including learning how fetuses survive the mother's immune system. A big leap forward for the lab was learning how to knock out the molecule that signaled to the immune system its foreignness through CRISPR: in other words, they are learning how to make these introduced cells silent to the immune system. Finally, they describe their "off the shelf" goal of producing non-immunogenic cells ready for injection for a majority of patients and alternatively generating a hypo-immunogenic environment in the patient to prevent long-term rejection. For more, see the lab's web page at https://tsilab.ucsf.edu/

Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) features an 8-fold volumetric resolution improvement over conventional microscopy and is well established on flat, adherent cells. However, blastomeres in mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to non-adherent embryos moving during scanning and a large distance to the cover glass. The biggest challenge and achievement of this doctorate thesis was the development of a novel method to perform 3D-SIM on mammalian embryos (“3D structured illumination microscopy of mammalian embryos and spermatozoa” published in BMC Developmental Biology). The development and fine-tuning of this method took over two years due to the time-intense generation of embryos and the subsequent two day long embryo staining, embedding and scanning with steps that required novel techniques such as micromanipulation which was not associated with sample preparation prior to this protocol. Problem identification was time-intensive since each of the numerous steps necessary could negatively affect the image quality. This method was fine-tuned during three studies. The first study “Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos” (published in Nucleus) investigates the profound changes of nuclear architecture during cattle preimplantation development of embryos generated by somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Fibroblast nuclei in embryos generated by SCNT go through similar changes in nuclear architecture as embryos generated by IVF. In both embryo types the occurrence of a large, chromatin-free lacuna in the center of nuclei around major embryonic genome activation (EGA) was noted. Similarly, the chromosome territory-interchromatin compartment (CT-IC) model applied to both types of embryos, featuring a lacuna or not, with an enrichment of RNA polymerase II and H3K4me3, a histone modification for transcriptionally competent chromatin, in less concentrated chromatin and an enrichment of H3K9me3, a transcriptionally restrictive histone modification, in more concentrated chromatin. However, large, highly concentrated H3K4me3 and H3K9me3 clusters were noted in both embryo types at chromatin concentrations that did not fit to the model. The chromatin-free lacunas were highly enriched in newly synthesized mRNA. The second study “Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications” (published in PLOS ONE) presents the changes of the nuclear envelope and lamina during bovine preimplantation development. Before major EGA, chromatin-free areas of the nuclear periphery were also free of nuclear pore complexes (NPCs), whereas after major EGA, the entire nuclear periphery was equipped with at least a fine layer of chromatin and associated NPCs. Three types of nuclear invaginations were predominant at different stages. The most common invagination was lamin B and NUP153 positive and was most prominent between the 2-cell and 8-cell stages until the onset of major EGA. Lamin B positive, but NUP153 negative invaginations were most prominent during stages with large nuclear volume and surface reductions. The least common invagination was lamin B negative but NUP153 positive and occurred almost exclusively at the morula stage. RNA-Seq and 3D-SIM data showed large deposits of spliced NUP153 mRNA and cytoplasmic NUP153 protein clusters until shortly after major EGA. NUP153 association with chromatin was initiated at metaphase. The third study “Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development” (published in the Journal of Reproduction and Development) demonstrated that rabbit embryonic nuclei feature a nuclear invagination type containing a large volume of cytoplasm that provides cytoplasmic proximity to nucleoli in addition to the small volume invaginations that were previously observed in bovine nuclei. The underlying mechanism for these two invaginations must differ from each other since small volume invaginations were frequently emanating from large volume invaginations emanating from the nuclear border but large volume invaginations were never emanating from small volume invaginations emanating from the nuclear border. Abundance of import/export competent invaginations featuring NPCs peaked at the 4-cell stage, which is the last stage before a drastic nuclear volume decline and also the last stage before major EGA is initiated at the 8- to 16-cell stage. Import/export incompetent invaginations positive for lamin B but not NUP153 peaked at the 2-cell stage. This was the stage with the largest variability in nuclear volumes. This may hint at an interphase nuclear surface reduction mechanism. Additionally, previously generated but unpublished 3D-FISH data about the localization changes of a stably inserted reporter gene upon activation in cloned bovine embryos was analyzed and documented in the study “Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos” (published in Nucleus). This study showed that the stably inserted OCT-4 reporter gene “GOF” in bovine fetal fibroblasts was initially moved towards the nuclear interior in day 2 bovine embryos generated by SCNT of bovine fetal fibroblasts. However, in day 4 SCNT embryos the localization of GOF had moved towards the periphery while it was still activated. Its carrier chromosome territory did not significantly move differently compared with the non-carrier homolog. Constant proximity of GOF to its carrier chromosome territory ruled out a movement by giant loops. In cooperation with the Department of Histology and Embryology of the Ege University (Izmir, Turkey) the destructive effects of cryopreservation on blastomere integrity were analyzed in the study “Ultra-Structural Alterations in In Vitro Produced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification” (published in Anatomia, Histologia, Embryologia). The cryopreservation method slow freezing caused more damage to blastomeres and to the zona pellucida than its fast freezing alternative vitrification. This was most likely caused by ice crystal formation and the longer exposure to the toxic side effects of cryoprotectants before freezing was complete.

Guest Dr. Dieter Egli from the New York Stem Cell Foundation joins the hosts to talk about his work in using SCNT to derive new embryonic stem cell lines. They discuss his previous and current…

Background: Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency. Results: 109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring. Conclusion: Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.

Background: Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs) as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination. Results: PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs) and porcine ear fibroblasts (PEFs) could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the Nucleofector (TM) technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT. Conclusion: The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.

Because of the tremendous need for transgenic large animal models for human diseases, the process of SCNT is a crucial step in transgenic pig production. In our study, we evaluated the particular steps during the production for their impact on the efficiency of cloning transgenic pigs. For this purpose, statistical analysis was performed for all SCNT data from the years 2006 until June 2010. The RANKL transgenic osteoporosis model was chosen for an example for the production steps needed to finally achieve a disease model, to elucidate pitfalls and chances of SCNT procedure. In total 151 in vivo SCNT experiments using different transgenic cell lines were carried out, resulting in 243 piglets and fetuses. Statistical analysis revealed that donor cells treated exclusively in our laboratory had a significant better birth rate than donor cell originated of other laboratories. Furthermore, there was a significant relation between number of transferred NT embryos and later pregnancy checks, birth rate and abortion rate. The more NT embryos were transferred, the more pregnancies finished to terms. It was also elucidated that in our studies a different in vitro culture time of 24 or 48 hours had no significant impact on the outcome like pregnancy or birth rate. Seasonal changes during the years had no significant influence on pregnancy rate, birth or abortion. But there was a strong tendency that autumn showed best performance of all seasons, and most pregnancies were lost after embryo transfers during the summer. All these findings will be integrated in future in vivo SCNT experiments and embryo transfers. For the production of a transgenic osteoporosis model 17 in vivo experiments took place so far, with an outcome of 4 fetuses and 25 piglets. For gaining a controllable expression of RANKL, it was necessary to establish double transgenic pigs to sidestep harmful effects of RANKL overexpression during the fetal development. First attempts to integrate both genes, tetracycline controlled transactivator (Tet-On) and RANKL, in a single step of cell transfection and SCNT, had no satisfying result. We obtained 4 fetuses and stillborn recloned piglets carrying both genes, but they showed only expression of Tet-On and it was impossible to induce RANKL overexpression. Therefore the strategy was changed in favor to two rounds of transfection and nuclear transfer. First Tet-On transgenic piglets were established and screened for integration and expression. Piglet 9894 showed the best expression and severed as donor for next cell transfection step. These Tet-On + TARE RANKL cells were in vitro tested for their inducibility. Thereafter SCNT and embryo transfer of the best candidate were performed and they resulted in 4 pregnancies which all finished to term. One double transgenic piglet could be raised and will be kept until adulthood to establish a line of Tet-On +TARE RANKL transgenic pigs. Importantly, this founder animal showed inducible RANKL overexpression. Other constructs might be based on the existing Tet-On cell line in the future, offering an inducible system for a broad variety of different transgenes. Thus a functional Tet-On system in the pig is reported for the first time.

Similarities between the bovine female and women in terms of reproduction and fertility, such as oogenesis, folliculogenesis and reduced fertility with advanced age make the bovine a valuable model for the study of ovarian function and dysfunction as well as reproductive aging in women. The aim of the present work was to investigate the influence of donor age on follicle numbers, yield and quality of COCs obtained by repeated OPU and on the developmental competence in vitro after oocyte maturation in vitro versus in vivo. Further, the ability of oocytes from different age classes to reprogram nuclei of bovine fetal fibroblasts was studied. Since the individual is a major factor influencing parameters of fertility and results of ART in both humans and cattle, the present study used in parts the same animals to rule out inter-individual effects on the response to one or the other approach. Experiment 1 investigated the effect of donor age in non-superstimulated German Simmental heifers (n = 12, 14 months at the beginning of the experiments), young cows in their first lactation (n = 8, 2-4 years) and old cows (n = 8, 10-15 years). A total of 38 OPU sessions were performed in two experimental periods on independent sets of animals from all age classes: 5/5/5 (32 sessions) and 7/3/3 (6 sessions). In spite of a marked influence of the experimental period, a number of parameters were also significantly affected by donor age. The total number of follicles increased with age (P

Die normale Entwicklung von Embryonen nach somatischem Zellkerntransfer („somatic cell nuclear transfer“, SCNT) hängt unter anderem von der erfolgreichen Reprogrammierung und Aktivierung von Schlüsselgenen ab. Ein Beispiel ist das Gen für den Transkriptionsfaktor Oct4, der im frühen Embryo nachweisbar und als Marker für pluripotente Zellen gilt. Die in klonierten Mausembryonen häufig beobachtete abnormale Expression von Oct4 wird als eine mögliche Ursache für die hohen Verluste und schweren Fehlentwicklungen nach Kerntransfer diskutiert. Beim Rind liegt im Vergleich zur Maus und anderen Spezies die Erfolgsrate am höchsten. Daher ist die Untersuchung der Reprogrammierung von OCT4 nach SCNT in der frühen Embryogenese beim Rind von besonderem Interesse. Um Fragen der epigenetischen Reprogrammierung des Rindergenoms und der Rolle von OCT4 nach SCNT nachzugehen, wurden bovine fetale Fibroblasten stabil mit einem Oct4-EGFP-Reportergenkonstrukt transfiziert. In den unilokulär stabil transfizierten Zellen mit unauffälligem weiblichen Karyotyp war in Analogie zur Inaktivität des endogenen OCT4-Gens in differenzierten Zellklonen keine EGFP-Fluoreszenz nachweisbar. Das Anschalten der Oct4-EGFP-Expression nach SCNT entsprach weitgehend der nach in vitro Fertilisation beobachteten Aktivierung des endogenen OCT4-Gens. In SCNT-Embryonen mit weniger als neun Zellkernen wurde keine EGFP-Fluoreszenz nachgewiesen. In allen Embryonen mit mindestens 17 Zellkernen war das Oct4-EGFP-Reportergenkonstrukt aktiv, was darauf hindeutet, dass Blastomeren nach der vierten Zellteilung den Oct4-Promotor aktivierten. Mittels konfokaler Laser Scanning Mikroskopie wurde an zentralen optischen Schnitten die Intensität der EGFP-Fluoreszenz jedes Embryos gemessen. Im Vergleich mit Tag 4 SCNT-Embryonen war die EGFP-Fluoreszenz in Tag 6 Embryonen deutlich stärker mit erheblichen Unterschieden in der Expressionshöhe zwischen einzelnen Embryonen. Dabei zeigten Embryonen mit einer niedrigeren EGFP-Fluoreszenz im Vergleich zu Embryonen mit stärkerer EGFP-Fluoreszenz einen erheblich höheren Anteil an Zellkernuntergängen (kondensierte und fragmentierte Zellkerne). 34 Tage nach dem Transfer von EGFP-exprimierenden Embryonen auf Empfängertiere wurden drei lebende und morphologisch unauffällige Feten gewonnen. In Fibroblasten, die aus diesen Feten isoliert wurden, war das Reportergenkonstrukt, analog zur normalen Inaktivierung des endogenen OCT4-Gens in differenzierten Zellen, inaktiviert. In SCNT-Embryonen aus den Oct4-EGFP-transgenen Fibroblasten dieser zweiten Generation („second round“ SCNT) wurde das Reportergenkonstrukt erneut regelmäßig aktiviert wie in den SCNT-Embryonen vom Ausgangszellklon („first round“ SCNT). Die Herstellung stabil transfizierter boviner fetaler Fibroblasten mit einer unilokulären Integration des Oct4-EGFP-Reportergenkonstruktes stellt eine wichtige Basis für ein breites Spektrum experimenteller Ansätze zur Aufklärung grundlegender Mechanismen nach Kerntransfer beim Rind dar.