L10: How we study cells

Question 1

What is the main goal of studying cells in cancer research?

Answer 1

To understand how cellular processes are deregulated in cancer and to find drug targets that can regulate these processes for treatment

Question 2

What role do protein arginine methyltransferases (PRMTs) play in cells?

Answer 2

They add methyl groups to arginine residues, affecting cellular processes like signaling, DNA repair, and gene expression, which are often deregulated in cancer

Question 3

What are some key questions scientists ask about proteins in cell studies?

Answer 3

Scientists ask about protein substrates, enzyme location, interaction with other proteins, and changes in behavior in disease processes

Question 4

What is RNA interference (RNAi) used for in cell biology?

Answer 4

RNAi is used to knock down or reduce protein expression by degrading messenger RNA

Question 5

How does CRISPR-Cas9 function in genetic manipulation?

Answer 5

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

Question 6

What is reverse genetics?

Answer 6

A method where scientists start with a known gene/protein and manipulate it to study its function, often using CRISPR or RNAi

Question 7

What are monoclonal antibodies and what is a benefit of using them?

Answer 7

Monoclonal antibodies target one specific antigen, providing high specificity for detecting proteins

Question 8

Why are polyclonal antibodies advantageous in experiments?

Answer 8

They can detect multiple epitopes, allowing for tolerance to small changes in protein structure and providing more versatile detection in experiments

Question 9

What are the limitations of polyclonal antibodies in experiments?

Answer 9

They may vary in specificity between batches, recognise multiple epitopes, and lack the consistency required for reproducible experiments

Question 10

What is Western blotting used for in cell studies?

Answer 10

• 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

Question 11

What are some applications of fluorescently tagged proteins in cell biology?

Answer 11

Visualising protein localisation, observing cell movement, and monitoring cellular processes in live cells

Question 12

How can CRISPR be used to create transgenic animal models?

Answer 12

By editing genes in fertilised eggs, researchers can generate animals with specific genetic modifications for studying diseases

Question 13

What is the difference between forward and reverse genetics?

Answer 13

Forward genetics starts with a phenotype to identify the gene responsible, while reverse genetics begins with a known gene to study its function

Question 14

What is the purpose of using antibodies in cell studies?

Answer 14

Antibodies are used to detect specific proteins, assess protein-protein interactions, visualise protein location within cells, and study cellular processes like signaling

Question 15

What is the difference between knockout and knockdown in genetic manipulation?

Answer 15

Knockdown reduces protein levels by degrading mRNA, while knockout completely removes the gene using CRISPR-Cas9, preventing protein production entirely

Question 16

Why is CRISPR-Cas9 referred to as ‘genetic scissors’?

Answer 16

It can precisely cut DNA at targeted locations, allowing for gene editing, knockout, or the insertion of mutations

Question 17

How does the RISC complex function in RNA interference?

Answer 17

The RISC complex binds to double-stranded RNA, unwinds it, and then pairs with messenger RNA, leading to its degradation and reducing protein expression

Question 18

What are two main types of antibodies used in research?

Answer 18

Monoclonal antibodies, which recognise a single antigen site, and polyclonal antibodies, which recognise multiple epitopes on an antigen

Question 19

What are fluorophores, and why are they used with antibodies?

Answer 19

Fluorophores are fluorescent molecules attached to proteins or antibodies that allow scientists to visualise the location of proteins within cells under a fluorescence microscope

Question 20

What is GFP, and why is it important in cell biology?

Answer 20

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

Question 21

How does CRISPR-Cas9 repair DNA after it cuts it?

Answer 21

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

Question 22

What is the purpose of SDS-PAGE in Western blotting?

Answer 22

SDS-PAGE separates proteins based on size, allowing specific proteins to be identified and analysed on a gel

Question 23

How are fluorescently tagged proteins used to study cell movement?

Answer 23

Fluorescent tags allow scientists to observe real-time protein dynamics and cell movement, providing insight into processes like cell migration and division

Question 24

What is mass spectrometry used for in cell biology?

Answer 24

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

Question 25

What is a wound scratch assay, and what does it test?

Answer 25

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

Question 26

Why is studying protein post-translational modifications important?

Answer 26

Post-translational modifications like phosphorylation and methylation can alter protein function, localisation, and interactions, affecting cellular processes

Question 27

What is multiplexing in fluorescence microscopy?

Answer 27

Using different fluorophores to label multiple proteins in a single cell, enabling visualisation of several cellular components simultaneously in a single sample

Question 28

What challenges arise from using polyclonal antibodies?

Answer 28

Polyclonal antibodies vary between batches and may recognize multiple epitopes, which can cause inconsistencies in experimental results

Question 29

What is the role of Cas9 in the CRISPR system?

Answer 29

Cas9 is an enzyme that cuts DNA at specific sites, guided by an RNA sequence, enabling precise gene editing

Question 30

How does using viral vectors in gene expression studies work?

Answer 30

Viral vectors introduce genes into cells with high efficiency, allowing stable gene expression over time, unlike transient transfection which is temporary

Question 31

What are some applications of CRISPR technology beyond gene knockout?

Answer 31

CRISPR is used to insert specific mutations, create transgenic animals, and is being tested in clinical trials for disease treatments like HIV

Question 32

What is the Hallmarks of Cancer concept?

Answer 32

It’s a framework identifying essential traits that cancer cells acquire, such as sustained proliferation, evading growth suppression, and resisting cell death

Question 33

What is forward genetics, and how does it differ from reverse genetics?

Answer 33

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

Question 34

Why is CRISPR considered a breakthrough for creating genetically modified animal models?

Answer 34

It allows precise gene editing in animal genomes (e.g., mice or zebrafish), accelerating the creation of models for studying genetic diseases

Question 35

What role do endosomes play in cell biology?

Answer 35

Endosomes are membrane-bound compartments involved in transporting and sorting cellular material, which can be tagged with specific antibodies for visualisation

Question 36

Why are mitochondrial proteins often tagged in cellular imaging?

Answer 36

Mitochondrial tags help visualise energy production centers of cells, revealing insights into cellular health, energy metabolism, and disease states

Question 37

What are transfection methods, and what are their main types?

Answer 37

Transfection introduces foreign DNA into cells, using either non-viral (e.g., liposomes) or viral methods (e.g., retroviruses) for gene delivery

Question 38

What is a stable cell line, and how is it created?

Answer 38

A stable cell line expresses a gene of interest permanently, created by integrating foreign DNA into the genome, often using viral vectors

Question 39

What is the primary purpose of using fluorescence microscopy in cell biology?

Answer 39

It enables visualization of cellular structures, protein localisation, and interactions within live or fixed cells using fluorescent tags

Question 40

How do retroviruses function in gene delivery systems?

Answer 40

Retroviruses integrate their genetic material into host cell DNA, allowing for stable gene expression in transfected cells

Question 41

What is the RISC complex, and how does it aid in gene silencing?

Answer 41

The RISC complex binds to and degrades mRNA, reducing protein production as part of RNA interference (RNAi) in gene silencing

Question 42

How does the CRISPR-Cas9 system target specific DNA sequences?

Answer 42

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

Question 43

What is the importance of disulfide bonds in antibody structure?

Answer 43

Disulfide bonds stabilise the antibody’s structure, linking heavy and light chains for proper antigen binding and immune function

Question 44

Why is CRISPR considered advantageous for studying complex diseases?

Answer 44

CRISPR can edit multiple genes simultaneously, enabling research into diseases caused by multiple genetic factors