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Cell and Developmental Biology > Methods in molecular cell biology > Flashcards

Methods in molecular cell biology Flashcards

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1
Q

What is in a tissue culture media?

A

blend of nutrients:

  • glucose
  • vitamins
  • essential amino acids
  • salts
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2
Q

What is the life span of primary cell cultures?

A

Passage cycle can be repeated 50-100 times (diluting etc)

- after that, cells will lose the capacity to divide and become senescent

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3
Q

What are immortal cell lines?

A
  • cells that can grow forever
  • cycle can be repeated indefinitely
  • often developed form tumour cells
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4
Q

What are the limitations of cell culture techniques?

A
  • cell lines retain some characteristics of the original cell type but are often less differentiated
  • cells often have more than the normal no. of chromosomes/ fused chromosomes
  • properties of cells in this artificial environment may not reflect natural behaviour
  • some difficult to culture
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5
Q

How can you find the location of a protein within a cell?

A
  1. antibodies with fluorophores an be used to detect proteins using immunocytochemistry
  2. antibodies with fluorophores can be used to detect surface expression levels by flow cytometry
  3. protein of interest can be engineered t be tagged with green fluorescent protein (GFP)
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6
Q

What are the disadvantages of immunocytochemistry?

A
  • antibodies might not bind well
  • antibodies might not be specific
  • cells must be fixed to preserve them
  • cells are often permeabilised with detergent
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7
Q

What are the disadvantages of flow cytometry?

A
  • sub cellular localisation is not determined
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8
Q

What is the green fluorescent protein (GFP)?

A
  • discovered in fluorescence jelly fish
  • protein that has a beta barrel type structure
  • chromophore that emits green light in the core
  • UV & blue light excite the chromophore
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9
Q

How can you engineer a GFP fusion protein?

A
  1. generate an expression plasmid

2. introduce the plasmid into cells in which it will be transcribed and translated

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10
Q

How can you introduce a plasmid into mammalian cells?

A
  1. Viral transduction
    - Engineer a virus to express the gene of interest
    - Virus can infect the cell
    - Introduce into the nucleus
  2. Microinjection
    - microinject plasmid directly into a nucleus
  3. Electroporation
    - Apply electric current to the cell
    - Mix dna with cell
    - Damages plasma membrane
    - Some plasma dna will be taken in
  4. Liposomes
    - Buy liposomes
    - Mix plasmid DNA
    - DNA incorporated into the liposome
    - The cell will take up the dna by endocytosis
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11
Q

What are the disadvantages of GFP fusion proteins?

A
  • must have cDA of protein of interest
  • must be able to transfect cells
  • adding GFP might affect protein function
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12
Q

What are the advantages of GFP fusion proteins?

A
  • allows for live cell imaging

- can investigate protein dynamics

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13
Q

How can you identify with proteins another protein is interacting with?

A
  • immunoprecipitation of the endogenous protein
  • immunoprecipitation of the protein following introduction of an epitope tag
  • fusion protein pulldowns
  • yeast two-hybrid system
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14
Q

How does the co-immunoprecipitation technique work?

A

1) Lyse cells in a detergent to solubilise the lipid bilayer
2) Capture protein X with a specific antibody
3) Wash away unbound proteins

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15
Q

How does the co-immunoprecipitation technique work when there is no antibody to your protein of interest?

A
  1. generate an expression plasmid, introducing a tag
  2. introduce the plasmid into cells
  3. able to capture the protein with the anti-tag antibody
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16
Q

What is the GST fusion protein pull down technique?

A
  1. make fusion between protein X and glutathione S-transferase (GST)
  2. fusion protein bound to glutathione-coated beads
  3. when cell extract is added, interacting proteins bind to protein X
  4. glutathione solution removes fusion protein together with proteins that interact with protein X
17
Q

How do you identify the interacting proteins from immunoprecipitation or protein pulldown experiments?

A
  • run on a protein gel
  • mass spec
  • can guess via their Mr
18
Q

What is the yeast two-hybrid system for detecting protein-protein interactions?

A

Engineer protein of interest with a BAIT protein

  • Binding protein that is bound to a transcriptional activation domain will be complementary to your target protein and will bind in the yeast
  • Will bring together the transcriptional activation domain and DNA - binding domain
19
Q

What are the two methods of knockdown of protein expression in cells?

A
  • RNA interference (RNAi)

- CRISPR-Cas9 genome editing

20
Q

How does RNAi mRNA degradation work?

A

Start with a double stranded RNA (small interfering RNA = siRNA)

  • Introduce into cells with lipid nanoparticles
  • Released in the cell
  • Picked up by the risk complex
  • Carries around the antisense strand and finds out complementary strand
  • Will knock down and replace the strand
21
Q

What are the advantages of RNAi knockdown?

A
  • easy to reduce expression of target gene by 80-100%

- stable knockdown can be achieved by introducing a plasmid that expresses the siRNA

22
Q

What are the disadvantages of RNAi knockdown?

A
  • off target effects are possible

- knockdown with siRNA doesn’t last a long time (effective between 2 - 5 days after transfection)

23
Q

How can you overcome the RNAi knockdown being off-target?

A
  • use at least two different siRNAs and check to see if the result is the same for each
  • reconstitute the cell with what has been knocked down, to ‘rescue’ the phenotype
24
Q

How does CRISPR-Cas9 work?

A
  1. guide RNA binds to DNA and ‘guides’ Cas9 to the right part of the genome
  2. Cas9 acts as a pair of ‘molecular’ scissors that cuts both strands of DNA creating a double stranded break.
  3. Can either undergo error-prone non homolous end-joining
    - deletions
    - insertions with indels
  4. Or can undergo homology directed repair if a donor template is present
    - precise insertion or modification into the break
    - homologous recombination

Cell and Developmental Biology (16 decks)

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