Podcast
Questions and Answers
What is the purpose of creating a random 'indel' during gene knock out?
What is the purpose of creating a random 'indel' during gene knock out?
Which strategy for gene knock out involves using two guide RNAs?
Which strategy for gene knock out involves using two guide RNAs?
What is a potential disadvantage of creating an 'exon-intron'-fusion?
What is a potential disadvantage of creating an 'exon-intron'-fusion?
Which step in the CRISPR stable cell line workflow typically takes the longest?
Which step in the CRISPR stable cell line workflow typically takes the longest?
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Which of the following techniques is easier to analyze according to the strategies for gene knock out?
Which of the following techniques is easier to analyze according to the strategies for gene knock out?
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What is typically the first step in the Knock-Out Workflow time line?
What is typically the first step in the Knock-Out Workflow time line?
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Which of the following statements about UTRs is correct?
Which of the following statements about UTRs is correct?
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What is the role of introns in gene structure?
What is the role of introns in gene structure?
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What is the purpose of RNA interference (RNAi) in cell culture techniques?
What is the purpose of RNA interference (RNAi) in cell culture techniques?
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Which of the following techniques is used for genomic editing?
Which of the following techniques is used for genomic editing?
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Which method would be least effective for detecting apopotic cell death?
Which method would be least effective for detecting apopotic cell death?
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What role does CRISPR/Cas9 play in genetic modification?
What role does CRISPR/Cas9 play in genetic modification?
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Which option is a tool used for genetic modification that involves introducing foreign DNA?
Which option is a tool used for genetic modification that involves introducing foreign DNA?
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What is the primary focus of cell-based assays in the context of cancer research?
What is the primary focus of cell-based assays in the context of cancer research?
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Which of the following techniques would be considered a form of overexpression?
Which of the following techniques would be considered a form of overexpression?
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What is the significance of using antibodies in microscopy?
What is the significance of using antibodies in microscopy?
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What is the total time required for gene modification using ES cells (mouse)?
What is the total time required for gene modification using ES cells (mouse)?
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Which of the following methods is NOT commonly used for analyzing gene modification?
Which of the following methods is NOT commonly used for analyzing gene modification?
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When targeting a gene for C-terminal knock-in, what site is focused on?
When targeting a gene for C-terminal knock-in, what site is focused on?
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What is the function of a donor oligo in gene knock-in strategies?
What is the function of a donor oligo in gene knock-in strategies?
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What is a key characteristic of a bicistronic expression system?
What is a key characteristic of a bicistronic expression system?
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How much time is estimated for designing or selecting gRNAs for knockout?
How much time is estimated for designing or selecting gRNAs for knockout?
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What is the primary function of RNA interference (RNAi) in the context of gene regulation?
What is the primary function of RNA interference (RNAi) in the context of gene regulation?
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Which of the following describes a potential drawback when utilizing fluorescent protein knock-in?
Which of the following describes a potential drawback when utilizing fluorescent protein knock-in?
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Which mechanism is mainly utilized by RNA interference to carry out its effects?
Which mechanism is mainly utilized by RNA interference to carry out its effects?
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What is a common misconception regarding the relationship between mRNA and protein levels?
What is a common misconception regarding the relationship between mRNA and protein levels?
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What is the first step in the knock-in workflow timeline for gene modification?
What is the first step in the knock-in workflow timeline for gene modification?
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Which of the following statements regarding the outcomes of gene knockdown through RNAi is true?
Which of the following statements regarding the outcomes of gene knockdown through RNAi is true?
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Which statement is true about the expectations during the knockdown process of a gene of interest?
Which statement is true about the expectations during the knockdown process of a gene of interest?
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What does dCas9 primarily enable in the field of genetics?
What does dCas9 primarily enable in the field of genetics?
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Which system is utilized for direct repression of gene expression using dCas9?
Which system is utilized for direct repression of gene expression using dCas9?
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In the context of dCas9 applications, what is the function of DNA labeling?
In the context of dCas9 applications, what is the function of DNA labeling?
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What does the term 'epigenetic landscape' refer to in CRISPR applications?
What does the term 'epigenetic landscape' refer to in CRISPR applications?
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What is a primary characteristic of dCas9 when it is described as a 'nuclease-deficient mutant'?
What is a primary characteristic of dCas9 when it is described as a 'nuclease-deficient mutant'?
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Which of the following is NOT an application of dCas9?
Which of the following is NOT an application of dCas9?
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What is a distinguishing feature of CRISPR pooled screening?
What is a distinguishing feature of CRISPR pooled screening?
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What is the primary purpose of using a whole genome library in screening with CRISPR?
What is the primary purpose of using a whole genome library in screening with CRISPR?
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What does CRISPRa utilize for direct gene activation?
What does CRISPRa utilize for direct gene activation?
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Why is the fusion of dCas9 with an epigenetic modifier significant?
Why is the fusion of dCas9 with an epigenetic modifier significant?
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What is the primary function of the Cas9 protein in CRISPR technology?
What is the primary function of the Cas9 protein in CRISPR technology?
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What type of DNA repair mechanism does non-homologous end joining (NHEJ) primarily utilize?
What type of DNA repair mechanism does non-homologous end joining (NHEJ) primarily utilize?
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Which component is critical to guide the Cas9 protein to the target DNA sequence?
Which component is critical to guide the Cas9 protein to the target DNA sequence?
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What are the potential outcomes of using homology-directed repair (HDR) in genome editing?
What are the potential outcomes of using homology-directed repair (HDR) in genome editing?
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Which of the following statements is true regarding CRISPR/Cas subtypes?
Which of the following statements is true regarding CRISPR/Cas subtypes?
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What is required to effectively utilize Cas9 in eukaryotic cells?
What is required to effectively utilize Cas9 in eukaryotic cells?
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Which of the following is a key benefit of utilizing CRISPR/Cas9 for genome editing?
Which of the following is a key benefit of utilizing CRISPR/Cas9 for genome editing?
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What was significant about the advancements in CRISPR technology that became apparent in 2014?
What was significant about the advancements in CRISPR technology that became apparent in 2014?
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Study Notes
Cell Culture Techniques Lecture 7+8
- Lecture covered genetic modification, RNAi, and CRISPR/Cas applications.
- The lecture covered various aspects of cell biology, including cell cycle, senescence, proliferation, differentiation, death (apoptosis, necrosis), and diseases.
- Microscopy techniques, and immunological tools like FACS, light, dyes, and antibodies, were also mentioned.
- Metabolic analysis, toxicity testing, and cell-based assays were included in the discussion of cell diseases.
- Methods to evaluate gene/protein function including genomic editing and overexpression were presented along with specific tools, such as reporter assays and RNAI (siRNA/shRNA).
RNA Interference (RNAi) History
- RNAi was discovered by Fire and Mello, and they won the Nobel Prize in Physiology or Medicine for it.
- The first clinical trial of siRNA (siRNA-027) for treatment of WAMD, and for cancer treatment (CALAA-01) were mentioned.
- Key events in the development of siRNA such as the discovery of dicer and RISC, siRNA triggers RNAi in human cells and development of siRNA-based therapy in mice, and the first published study using RNAi-based treatment for cardiovascular disease, and approval the first RNAi-effector (Patisiran) as a treatment for HATTR were discussed.
RNA Interference (RNAi) Mechanism
- RNA interference (RNAi) is an endogenous cellular process utilizing an endogenous cellular machinery.
- It involves the production and use of small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) to silence the expression of specific genes.
- The process includes producing dsRNA, creating siRNA duplex, formation of RISC complex, and mRNA degradation to achieve gene silencing.
- The diagrams illustrated pathways for RNAi-mediated gene silencing.
CRISPR/Cas9 Overview
- CRISPR/Cas9 is used for genome editing.
- The process involves genomic editing/interference, RNA editing/interference, and reporter assays in cells.
- It was explained that this technology is based on the bacterial adaptive immunity system.
- This technology allows the modification of genomes in eukaryotic cells.
CRISPR/Cas9 History
- Timeline of CRISPR-Cas9 and Genome editing research fields were presented.
- Key players and technologies were identified.
- The discovery that Cas9 is an RNA-programmable DNA endonuclease, leading to advancements in genome engineering was presented.
- Cas9-RNA-mediated site-specific genome editing in human cells, and other eukaryotic cells was also mentioned.
Mechanisms of DNA repair and genome editing
- Different Mechanisms of DSB or Double-Strand Break repair were outlined.
- These mechanisms include Non-homologous end joining (NHEJ) and Homology-directed repair (HDR) pathways used for genome editing were documented.
CRISPR/Cas Workflow
- Tools available for CRISPR/Cas9 experiments in cell culture including: plasmid-based, viral, mixed versions, and plasmid-free methods.
- Strategies for generating knockouts (KO) of genes were explained.
- These strategies include creating random indels, deleting exons, and creating exon-intron fusions, methods for successful knock out, cell types, and timeframe for research were detailed.
- Workflow for generating knockout cell lines, including sgRNA design and cloning, plasmid transfer, cell selection and analysis.
- Overview of different CRISPR-Cas systems (SpCas9, SaCas9, Cas12, Cas13) and their subtype variations.
- Detailed timelines of CRISPR/Cas9 experiment were also provided.
CRISPR Screening
- CRISPR pooled screening workflow (4-8 weeks) and arrayed screen were presented.
dCas9
- dCas9 is a nuclease-deficient Cas9 variant.
- It can be fused with different proteins or domains to perform various functions beyond genome editing.
- Methods including activation, repression, DNA labeling of genes or epigenetic modification of genes were also shown.
Degron Systems - dTag system
- Degron tagging for rapid protein degradation in cells and mice.
- Degron tagging enables the controlled regulation of protein degradation via chemical stimuli were described.
- Different types of Degron systems and their characteristics were presented.
- Experimental design considerations for in vivo studies, time-resolved consequences of target perturbation were detailed.
Strategies for gene Knock In (KI)
- Introduction of genetic material into a specific location within a DNA molecule in human cells and mice.
- Different types of knock-in strategies were detailed.
Fluorescent Protein Knock In - Fusion vs Bicistronic
- Description of bicistronic and fusion strategies for tagging proteins with fluorescent proteins.
- Fusion and bicistronic systems to insert genes of interest into certain locations in cells were outlined.
Additional Information
- Methods for analyzing results and determining the success of gene modifications.
- Information about generating and testing edited cells and potential cell types were included.
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Description
This lecture delves into advanced cell culture techniques, focusing on genetic modification methods like RNAi and CRISPR/Cas. It covers essential aspects of cell biology, including the cell cycle and metabolic analysis, alongside various microscopy and immunological tools. A historical overview of RNA interference, including key discoveries and clinical applications, is also provided.
