L10: How we study cells Flashcards
What is the main goal of studying cells in cancer research?
To understand how cellular processes are deregulated in cancer and to find drug targets that can regulate these processes for treatment
What role do protein arginine methyltransferases (PRMTs) play in cells?
They add methyl groups to arginine residues, affecting cellular processes like signaling, DNA repair, and gene expression, which are often deregulated in cancer
What are some key questions scientists ask about proteins in cell studies?
Scientists ask about protein substrates, enzyme location, interaction with other proteins, and changes in behavior in disease processes
What is RNA interference (RNAi) used for in cell biology?
RNAi is used to knock down or reduce protein expression by degrading messenger RNA
How does CRISPR-Cas9 function in genetic manipulation?
It creates targeted DNA cuts using guide RNA, leading to gene knockout or specific mutations by inducing DNA repair pathways that alter the genetic sequence
What is reverse genetics?
A method where scientists start with a known gene/protein and manipulate it to study its function, often using CRISPR or RNAi
What are monoclonal antibodies and what is a benefit of using them?
Monoclonal antibodies target one specific antigen, providing high specificity for detecting proteins
Why are polyclonal antibodies advantageous in experiments?
They can detect multiple epitopes, allowing for tolerance to small changes in protein structure and providing more versatile detection in experiments
What are the limitations of polyclonal antibodies in experiments?
They may vary in specificity between batches, recognise multiple epitopes, and lack the consistency required for reproducible experiments
What is Western blotting used for in cell studies?
- Separate and identify specific proteins from complex samples, in cell lysates based on size, using antibodies for detection
- Help analyse expression, modifications and disease markers
What are some applications of fluorescently tagged proteins in cell biology?
Visualising protein localisation, observing cell movement, and monitoring cellular processes in live cells
How can CRISPR be used to create transgenic animal models?
By editing genes in fertilised eggs, researchers can generate animals with specific genetic modifications for studying diseases
What is the difference between forward and reverse genetics?
Forward genetics starts with a phenotype to identify the gene responsible, while reverse genetics begins with a known gene to study its function
What is the purpose of using antibodies in cell studies?
Antibodies are used to detect specific proteins, assess protein-protein interactions, visualise protein location within cells, and study cellular processes like signaling
What is the difference between knockout and knockdown in genetic manipulation?
Knockdown reduces protein levels by degrading mRNA, while knockout completely removes the gene using CRISPR-Cas9, preventing protein production entirely
Why is CRISPR-Cas9 referred to as ‘genetic scissors’?
It can precisely cut DNA at targeted locations, allowing for gene editing, knockout, or the insertion of mutations
How does the RISC complex function in RNA interference?
The RISC complex binds to double-stranded RNA, unwinds it, and then pairs with messenger RNA, leading to its degradation and reducing protein expression
What are two main types of antibodies used in research?
Monoclonal antibodies, which recognise a single antigen site, and polyclonal antibodies, which recognise multiple epitopes on an antigen
What are fluorophores, and why are they used with antibodies?
Fluorophores are fluorescent molecules attached to proteins or antibodies that allow scientists to visualise the location of proteins within cells under a fluorescence microscope
What is GFP, and why is it important in cell biology?
GFP (Green Fluorescent Protein) and its colour variants (e.g. YFP, RFP) is a protein that fluoresces green and is used to tag proteins, allowing scientists to track protein location and movement in live cells in real-time analysis
How does CRISPR-Cas9 repair DNA after it cuts it?
The cell tries to repair the double-strand break, often using non-homologous end joining (NHEJ), which is error-prone and can result in mutations
What is the purpose of SDS-PAGE in Western blotting?
SDS-PAGE separates proteins based on size, allowing specific proteins to be identified and analysed on a gel
How are fluorescently tagged proteins used to study cell movement?
Fluorescent tags allow scientists to observe real-time protein dynamics and cell movement, providing insight into processes like cell migration and division
What is mass spectrometry used for in cell biology?
To identify proteins in a complex sample by analysing protein fragments and the composition of cellular structures, allowing scientists to study protein complexes and interactions
What is a wound scratch assay, and what does it test?
A wound scratch assay creates a “wound” in a cell layer to study cell migration and wound-healing properties, useful for observing how cells move and respond
Why is studying protein post-translational modifications important?
Post-translational modifications like phosphorylation and methylation can alter protein function, localisation, and interactions, affecting cellular processes
What is multiplexing in fluorescence microscopy?
Using different fluorophores to label multiple proteins in a single cell, enabling visualisation of several cellular components simultaneously in a single sample
What challenges arise from using polyclonal antibodies?
Polyclonal antibodies vary between batches and may recognize multiple epitopes, which can cause inconsistencies in experimental results
What is the role of Cas9 in the CRISPR system?
Cas9 is an enzyme that cuts DNA at specific sites, guided by an RNA sequence, enabling precise gene editing
How does using viral vectors in gene expression studies work?
Viral vectors introduce genes into cells with high efficiency, allowing stable gene expression over time, unlike transient transfection which is temporary
What are some applications of CRISPR technology beyond gene knockout?
CRISPR is used to insert specific mutations, create transgenic animals, and is being tested in clinical trials for disease treatments like HIV
What is the Hallmarks of Cancer concept?
It’s a framework identifying essential traits that cancer cells acquire, such as sustained proliferation, evading growth suppression, and resisting cell death
What is forward genetics, and how does it differ from reverse genetics?
Forward genetics identifies genes causing a phenotype (e.g., disease), often through random mutagenesis, while reverse genetics studies the effects of modifying specific known genes
Why is CRISPR considered a breakthrough for creating genetically modified animal models?
It allows precise gene editing in animal genomes (e.g., mice or zebrafish), accelerating the creation of models for studying genetic diseases
What role do endosomes play in cell biology?
Endosomes are membrane-bound compartments involved in transporting and sorting cellular material, which can be tagged with specific antibodies for visualisation
Why are mitochondrial proteins often tagged in cellular imaging?
Mitochondrial tags help visualise energy production centers of cells, revealing insights into cellular health, energy metabolism, and disease states
What are transfection methods, and what are their main types?
Transfection introduces foreign DNA into cells, using either non-viral (e.g., liposomes) or viral methods (e.g., retroviruses) for gene delivery
What is a stable cell line, and how is it created?
A stable cell line expresses a gene of interest permanently, created by integrating foreign DNA into the genome, often using viral vectors
What is the primary purpose of using fluorescence microscopy in cell biology?
It enables visualization of cellular structures, protein localisation, and interactions within live or fixed cells using fluorescent tags
How do retroviruses function in gene delivery systems?
Retroviruses integrate their genetic material into host cell DNA, allowing for stable gene expression in transfected cells
What is the RISC complex, and how does it aid in gene silencing?
The RISC complex binds to and degrades mRNA, reducing protein production as part of RNA interference (RNAi) in gene silencing
How does the CRISPR-Cas9 system target specific DNA sequences?
It uses a guide RNA to match and bind a DNA sequence, enabling the Cas9 enzyme to cut DNA at precise locations for gene editing
What is the importance of disulfide bonds in antibody structure?
Disulfide bonds stabilise the antibody’s structure, linking heavy and light chains for proper antigen binding and immune function
Why is CRISPR considered advantageous for studying complex diseases?
CRISPR can edit multiple genes simultaneously, enabling research into diseases caused by multiple genetic factors
