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Flashcards in Chris Deck (8)
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1
Q

Why are CHO cells used? What are the advantages and disadvantages of using recombinant cell systems such as these?

A

CHO cells can stably express large quantities of recombinant proteins
Adv = stable transfection, authentic post-translational modifications
Disadv = Expensive, difficult to scale up due to complex growth requirement, lengthy expression time

2
Q

What is a point mutation?

A

Changing a single nucleotide

3
Q

What is sucrose doing, and how?

A

Sucrose prevents sequestration through inhibition of clathrin-coated vesicle formation. Hypertonic sucrose (0.45 M) causes cells to swell which prevents membrane invagination

4
Q

How does the sequestration data relate to the functional data, and why?

A

Blocking of sequestration led to no reversal in desensitisation (i.e. no resensitisation)
Wt beta2AR showed resensitisation after agonist-free incubation
Suggesting sequestration of beta2AR is involved in the recycling and resensitisation of desensitised beta2AR

5
Q

What is a splice variant?

A

Different mRNAs are produced from RNA splicing

The MOR gene has many introns/exons - exons can be ‘spliced’ together in various ways

6
Q

What is monensin doing, and how?

A

Monensin prevents recycling of internalised receptors back to the plasma membrane
It inhibits acidification of endosomes which prevents dissociation of the ligand from the receptor, meaning the receptor cannot be trafficked back to the plasma membrane so remains intracellular

7
Q

Radioligand binding assays

A

Quantify how many/proportion of receptors with DAMGO bound

8
Q

Confocal microscopy

A

Gives information about the localisation of the receptors in the cell