rAAVs, host-cell contamination and ddPCR
In this episode of Talking Techniques, supported by Bio-Rad, we discuss a key component of many gene therapies: recombinant adeno-associated viruses (rAAVs) and their production. These viruses act as efficient, accurate delivery vesicles for the gene therapy’s plasmid.
Speaking to Associate Director of Biopharma Product Marketing at Bio-Rad Laboratories, Mark White, we take a look at the different expression systems used for their production and compare their advantages, before looking at some of the challenges involved in the production rAAVs, such as host-cell contamination.
Discover the tools that can help minimize host-cell contamination and differentiate between nuclease resistant and nuclease reactive contaminant DNA and find out about some of the most exciting developments in rAAV technologies.
- The role of rAAVs in gene therapies: 00:40-02:15
- The production of rAAVs and gene therapies: 02:15-03:30
- Why are HEK cells so popular for cell therapy production? 03:30-05:45
- HEK vs SF9 Insect cell expression systems: 05:45-06:45
- Challenges in cell therapy expression systems: 06:45-08:05
- Host DNA contamination: 08:05-10:30
- The risks of host DNA contamination: 10:30-12:45
- Key techniques to minimize host DNA contamination: 12:45-14:40
- The advantages of ddPCR in gene therapy production: 14:40-17:50
- Distinguishing between nuclease resistant and nuclease reactive host cell DNA: 17:50-19:10
- The most exciting developments in rAAV technology: 19:10-20:20
- What is one thing you would ask for to improve rAAV and gene therapy development 20:20-26:38
4. Long-read sequencing, metagenomics and the microbiome22:18In this episode, sponsored by Zymo Research, we explore the microbiome and how long-read sequencing techniques are changing our approach to the field and the exciting discoveries that they have led to. To do this we speak to Kris Locken, Molecular Biologist in the Research & Development department of Zymo Research (CA, USA), and Jeremy Wilkinson, Global Marketing Specialist for Microbial Genomics at PacBio (CA, USA).Find out how microbes compare to nuts and what this means for the challenges of metagenomic sample preparation, how long reads can stack up vs short reads for metagenome assembly and much more, all in this latest episode of Talking TechniquesContents:Intro: 00:00-01:55What is metagenomics and why is it important for microbial studies? 01:55-03:55How has long-read sequencing impacted metagenomics? 03:55-06:05Long-read vs short-read sequencing workflows: 06:05-07:15Addressing barriers to long-read sequencing: 07:15-08:45Sample preparation for long-read sequencing: 08:45-12:20Development of long-read sequencing to improve accuracy and capabilities: 12:20-14:40Best practice for assembly and analysis: 14:40-17:40Exciting examples of the benefits of long-read sequencing: 17:40-20:00What would you wish for to improve the ability long-read sequencing in microbiomics: 20:00-21:40
3. Open neuroscience and the meaning of FAIR37:53This episode, our second recorded at Neuroscience 2022 (13-19th April 2022; San Diego, CA, USA), delves into the importance of open data in neuroscience and the FAIR guidelines, which encourage researchers to make their data Findable, Accessible, Interoperable and Reusable.Sharing her considerable expertise in this area is one of the authors of the FAIR guidelines, Maryann Martone, who provides some key examples of the successes that open data practices have delivered so far and cautionary tales for how current practices are damaging the field.Listen on to find out how to implement open data practices, how they can help your lab and why Maryann sees it as our responsibility to resolve!Contents:Introduction: 00:00-02:00Introducing open neuroscience and the meaning of FAIR 02:00-03:00Exposing outdated paradigms in science: academic targets, publications, reproducibility and data accessibility: 03:00-06.30Incentivizing open data and reassigning value restructuring academia: 06:30-08.40The impact of FAIR within labs: 08:40-09:50Challenges of establishing and barriers to Open and FAIR neuroscience 09:50-12:00The reception of these guidelines in the field: 12:00-16:50Examples of the impact of open data in the spinal cord injury community: 16:50-18:10Marryann’s experience of enacting changes early in her career: 18:10-21:20Judging value in scientific research and understanding your purpose: 21:20-24:20The importance of investment: 24:20-26:30The impact of industry on lab data: 26:30-27:50Practical tips for addressing your lab data: 27:50-31:10Key tips for preparing data for an open-source repository 31:10-33.15The FAIR data principles explained: 33.15-37:50
2. Welcome to the jungle: sequencing for conservation43:22In this episode of Talking Techniques, we head to the Ecuadorian rainforest to chat about the use of sequencing in conservation with Zane Libke, a field researcher based at Sumak Kawsay In Situ (Pastaza, Ecuador). Zane discusses how he turned his fascination with nature into a career, his current work using nanopore sequencing technologies to find undescribed species and helping train future field researchers.We also explore the benefits of fighting biodiversity in a more local-centric way and using research to protect the area from exploitative companies.Contents:Intro: 00:00–01:35Zane’s work at Sumak Kawsay In Situ: 01:35–05:05Turning a fascination with nature into a career: 05:05–08:20The importance of sequencing for conservation: 08:20–12:25Technologies being used to sequence in the field: 12:25–14:15Portable nanopore sequencing with the MinION: 13:15–15:35Exporting samples means exporting opportunity: 15:35–18:40The sequencing workflow: 17:40–24:50Data processing: 24:50–27:45Using these technologies to find undescribed species: 27:45–32:20Fighting biodiversity loss in a more local-centric way: 32:20–34:55Using research to protect the area from exploitation: 34:55–38:50What the future holds: 38:50–43:22
1. Neuropixels: big data heaven or burying the lead in averages?19:30In this special episode of Talking techniques, one of two recorded at Neuroscience 2022, we speak to Tim Harris (Howard Hughes Medical Institute, VA, USA). Tim is the creator of Neuropixels, neural recording probes inserted into the brains of animal models, which allow researchers to collect signals from hundreds of individual neurons in different brain regions simultaneously over extended periods of time.Here, he shares the story behind his creation and details how they have changed the data collection landscape in Neuroscience. Tim also considers every inventor’s greatest concern: has his invention led to purely positive outcomes for the field? By enabling the collection of huge datasets have Neuropixels led to studies that bury findings in vast swathes of data or have they enabled researchers to collect enough information to discover the emphatic truth?Listen now to find out Tim’s opinions on these contentious debates in neuroscience and get key tips for utilizing neuropixels! Contents:Introduction: 00:00-03:00Tim's symposium on the capabilities of Neuropixels: 03:00-04.00Key takeaways from the symposium: 04:00-07:45The story of the development of neuropixels: 07:45-13:50Best practice tips for utilizing neuropixels: 13:50-15:15Challenges of utilizing neuropixels: 15:15-15:50Addressing the challenge of big data and sharing uncertainty: 15:50-17:30How neuropixels have accelerated neuroscience data generation: 17:30-19:30
13. Microbiome profiling techniques: from immunology to agriculture22:10In this episode of the Talking Techniques podcast, we explore microbiomes and the techniques used to profile them. Get an overview of the different techniques’ working principles, their pros and cons and the different applications that they are best suited to.Providing an expert insight into this field is Annabelle Damerum, Microbiome R&D Scientist at Zymo Research. Annabelle reveals some key tips best practice when profiling the microbiome and details the importance of an emerging aspect of the field: the vaginal microbiome.Contents: Into: 00:00-01:50Why it is important to profile microbiomes. 01:50-04:15Key applications of microbiome profiling: 04:15-06:10Techniques used to profile the microbiome and their working principles: 06:10-09:20Targeted sequencing: 06:10-08:00Shotgun metagenomics: 08:00-09:00Metatranscriptomics: 09:00-9:15The strengths and limitations of these techniques: 09:12- Targeted sequencing: 09:20-10.15Shotgun metagenomics: 10:15-11:25Metatranscriptomics: 11:25-11:50Matching techniques with applications: 11:50-13:30Tips for best practice for using these techniques: 13:30-16:25What is one thing you would ask for to improve the ability of microbiome profiling techniques: 16:55-18:10Investigating the vaginal microbiome: 18:10-20:55Conclusions and closing: 20:55-22:10
12. The gut–brain axis and addiction29:39In this special episode of Talking Techniques, brought to you from the Federation of European Neuroscience Societies Forum (FENS; 9th–13th July 2022), an expert panel discuss their research into the relationship between the gut–brain axis and addiction.The panel features Benjamin Boutrel (Lausanne University Hospital; Switzerland), Lorenzo Leggio (NIH Intramural Research Program; MD, USA) and Nathalie Delzenne (University of Louvain; Louvain-la-Neuve, Belgium), who discuss their current research into the role of the microbiota in alcohol addiction, if this work could be translated into the clinic, and if similar processes are at play in other addictions, such as food and cocaine. The discussion also features an audience Q&A, which explores the microbial relationship between mother and fetus, and social factors in addiction.Contents:Introduction: 00:00–01:35Introduction to panelists: 01:35–03:40Techniques being used to investigate the relationship between the gut and addiction: 03:40–06:55Translating this research to the clinic: 06:55–11:40Parallels between alcohol addiction and other addictions, such as food, cocaine and tobacco: 11:40–17:05Microorganisms as drivers of behavior: 17:05–18:30The hot topic of gut health: 18:30–20:30Future directions for the research, including microbiota transfers, precision medicine and avoiding alcohol dependence: 20:30–24:35Audience Q&A – microbial elements between mother and fetus, and social factors in addiction: 24:35–29:15
11. 3D cell cultures: the latest developments, applications and regulations in the field30:22In this episode, supported by BrandTech, returning guest Rob Vries, CEO of HUB organoids (Utrecht, The Netherlands), fills us in on the advancement of 3D cell cultures and organoids over the last 2 years, starting off by documenting how the pandemic impacted their development and uptake.We also discuss how the recent FDA Modernization Act, removing the requirement for drug candidates to be tested on animals, has impacted the drive for improved models, the key techniques available to analyze them and what still needs to change for 3D cell cultures to fully replace animal models in the lab.Listen today to find out which resources can help you begin to work with 3D cell cultures, their most exciting recent applications and how issues of reproducibility are currently being addressed in the field.Contents:Intro: 00:00-00:50How COVID-19 impacted the uptake and development of 3D cell cultures: 00:50-02:05Key developments in 3D cell culture technology in the last 2 years: 02:05-03:00Intestinal organoids and the investigation of irritable bowel syndrome: 03:00-5:50Evaluating assembloids: 05:50-08:35Complexity vs clinical relevance: 08:35-10:45The impact of 3D cell cultures in precision medicine in cancer: 10:45-12:40The impact of the FDA’s Modernization Act on 3D cell culture uptake: 12:40-14:45What needs to improve in organoid technologies to fully replace animal models? 14:45-16:10Changing inbuilt reliance on animal models: 16:10-18:30Key techniques to analyze organoids: 18:30-19:50Developments in imaging technology that have improved the analysis of organoids: 19:50-21:15Current challenges in 3D cell culture implementation and reproducibility: 21:15-24:15Improving access to 3D cell cultures: 24:15-25:55Resources to help people implement organoids into their work: 25:55-27:15Searching for the holy grail in 3D cell cultures: 27:15-28:00
9. HPV, epigenetics and cancer: sequencing for new insights and to correct healthcare inequality17:00In many regions of the world, HPV and its association with cervical cancer is a well-known but rarely considered issue, after the rollout of the HPV vaccine in the late 2000s. However, while this rollout significantly impacted cervical cancer rates where it was implemented, it was not universal and in many areas of the world HPV-induced cervical cancer remains a critical issue.Cervical cancer is the leading cause of death for people with a cervix living in poverty in low-resource regions and on the continent of Africa, it is estimated that in 2020 as many people with a cervix died of cervical cancer as COVID-19.In this episode of Talking techniques, supported by Oxford Nanopore, Senior Investigator Michael Dean and Post-baccalaureate Fellow Nicole Rossi, from the NIH’s National Cancer Institute (MD, USA), discuss their research into the relationship between HPV and cervical cancer. Expounding on the points mentioned above, they reveal the tremendous insights into cancer and immunology still to be gained, document the key tools used in their studies and explain how their work can be translated into more effective immunotherapies and treatments for cervical cancer.ContentsIntroduction: 00:00-01:40How big an issue does HPV present to the world? 01:40-03:30HPV’s association with cancer: 03:30-05:15Why does HPV target DNA repair mechanisms? 05:15-06:00Key goals of research into HPV: 06:00-07:15Challenges of HPV research: 07:15- 08:20Solutions available to deal with these challenges: 08:20-09:30The most exciting discoveries in HPV using long-read sequencing: 09:30-11:00HPV16- the most oncogenic variant: 11:00-11:40Translating research into clinical results for HPV: 11:40-12:45How effective is the current HPV vaccine? 12:45-13:30What would you ask for to improve research into HPV and cancer? 13:30-14:30How big an issue is vaccine rejection? 14:30-15:40Final thoughts: 15:40-17:00