Methods in molecular cell biology Flashcards
What is in a tissue culture media?
blend of nutrients:
- glucose
- vitamins
- essential amino acids
- salts
What is the life span of primary cell cultures?
Passage cycle can be repeated 50-100 times (diluting etc)
- after that, cells will lose the capacity to divide and become senescent
What are immortal cell lines?
- cells that can grow forever
- cycle can be repeated indefinitely
- often developed form tumour cells
What are the limitations of cell culture techniques?
- 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
How can you find the location of a protein within a cell?
- antibodies with fluorophores an be used to detect proteins using immunocytochemistry
- antibodies with fluorophores can be used to detect surface expression levels by flow cytometry
- protein of interest can be engineered t be tagged with green fluorescent protein (GFP)
What are the disadvantages of immunocytochemistry?
- antibodies might not bind well
- antibodies might not be specific
- cells must be fixed to preserve them
- cells are often permeabilised with detergent
What are the disadvantages of flow cytometry?
- sub cellular localisation is not determined
What is the green fluorescent protein (GFP)?
- 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
How can you engineer a GFP fusion protein?
- generate an expression plasmid
2. introduce the plasmid into cells in which it will be transcribed and translated
How can you introduce a plasmid into mammalian cells?
- Viral transduction
- Engineer a virus to express the gene of interest
- Virus can infect the cell
- Introduce into the nucleus - Microinjection
- microinject plasmid directly into a nucleus - Electroporation
- Apply electric current to the cell
- Mix dna with cell
- Damages plasma membrane
- Some plasma dna will be taken in - Liposomes
- Buy liposomes
- Mix plasmid DNA
- DNA incorporated into the liposome
- The cell will take up the dna by endocytosis
What are the disadvantages of GFP fusion proteins?
- must have cDA of protein of interest
- must be able to transfect cells
- adding GFP might affect protein function
What are the advantages of GFP fusion proteins?
- allows for live cell imaging
- can investigate protein dynamics
How can you identify with proteins another protein is interacting with?
- immunoprecipitation of the endogenous protein
- immunoprecipitation of the protein following introduction of an epitope tag
- fusion protein pulldowns
- yeast two-hybrid system
How does the co-immunoprecipitation technique work?
1) Lyse cells in a detergent to solubilise the lipid bilayer
2) Capture protein X with a specific antibody
3) Wash away unbound proteins
How does the co-immunoprecipitation technique work when there is no antibody to your protein of interest?
- generate an expression plasmid, introducing a tag
- introduce the plasmid into cells
- able to capture the protein with the anti-tag antibody
What is the GST fusion protein pull down technique?
- make fusion between protein X and glutathione S-transferase (GST)
- fusion protein bound to glutathione-coated beads
- when cell extract is added, interacting proteins bind to protein X
- glutathione solution removes fusion protein together with proteins that interact with protein X
How do you identify the interacting proteins from immunoprecipitation or protein pulldown experiments?
- run on a protein gel
- mass spec
- can guess via their Mr
What is the yeast two-hybrid system for detecting protein-protein interactions?
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
What are the two methods of knockdown of protein expression in cells?
- RNA interference (RNAi)
- CRISPR-Cas9 genome editing
How does RNAi mRNA degradation work?
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
What are the advantages of RNAi knockdown?
- easy to reduce expression of target gene by 80-100%
- stable knockdown can be achieved by introducing a plasmid that expresses the siRNA
What are the disadvantages of RNAi knockdown?
- off target effects are possible
- knockdown with siRNA doesn’t last a long time (effective between 2 - 5 days after transfection)
How can you overcome the RNAi knockdown being off-target?
- 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
How does CRISPR-Cas9 work?
- guide RNA binds to DNA and ‘guides’ Cas9 to the right part of the genome
- Cas9 acts as a pair of ‘molecular’ scissors that cuts both strands of DNA creating a double stranded break.
- Can either undergo error-prone non homolous end-joining
- deletions
- insertions with indels - Or can undergo homology directed repair if a donor template is present
- precise insertion or modification into the break
- homologous recombination
