Cell Factories - Mammalian Cell Systems Flashcards
Key considerations for mammalian cell systems..
1) Means of transgene delivery
2) selection of transformant
3) Yield
Advantages of mammalian cell systems..
System of choice
- Most therapeutic proteins require PTMs (normally glycosylation) to be fully active -> only mam systems can do this
- Well characterised lines:
Chinese Hamster Ovary cells (CHO)
Baby Hamster Kidney Cells (BHK)
Mouse Myeloma NS0 cells (favoured for mAb production)
Disadvantages of mammalian cell systems…
- ^ complexity of cell culture
- ^ expense to ensure conditions remain sterile (microbial infection is common)
Transgene delivery in mammalian cell systems
Easy to transfect & deliver DNA naked (linear or circular plasmid)
Integrated by…
-> Action of endogenous pathways for DNA double strand break repair
Integration of liner DNA = more efficient
-> circular DNA gets nicked w/in cell and subsequently integrated
Cells integrate at random locations, different for each cell in the population:
-> typically transforming DNA will integrate at a single locus but as multiple tandemly repeated copies
-> No. of copies varies in DNA
Methods of transgene delivery?
- Electroporation
- Liposome-mediated uptake
- Polymer-mediated uptake
- Calcium phosphate co-precipitation
Electroporation
1) Cells mixed w/ DNA & placed in a cuvette between a pair of electrodes.
2) ^ voltage passed through causing transient pored to be generated in cell membranes
3) DNA can enter
Liposome-mediated uptake
1) DNA is mixed with a mixture of phospholipids and water, and the mixture is emulsified.
2) The phospholipids become organised into circular micelles, and DNA gets trapped in these.
3) By designing the lipid content to be similar to that of the mammalian cell membrane, mixing the liposomes with cells in culture results in fusion between them and the cell membrane.
4) releasing the DNA into the cell.
Polymer-mediated uptake
1) DNA is mixed with Polyethylene Glycol and Calcium Chloride
2) the PEG polymer will transiently solubilise the cell membrane and permit DNA uptake.
Calcium Phosphate co-precipitation.
1) DNA is mixed with Calcium Chloride + Sodium Phosphate = Calcium Phosphate (highly insoluble, and will precipitate out as nanoparticles)
2) DNA becomes wrapped around these nanoparticles.
3) Cells are incubated with these nanoparticles and “ingested” by the cell via endocytosis
4) DNA is released and can be integrated into the genome.
Selectable markers in mammalian cell systems
These can be resistances to aminoglycoside antibiotics - but more commonly method used =
the complementation of a mammalian mutant gene.
DFHR as a selectable marker for mammalian systems
DFHR = most efficient
- enzyme critical for synthesis of nucleotides that make up DNA
- Following mutagenesis, deficient DFHR lines have been isolated
Typical vector for transformation when using DFHR as a selectable marker for mammalian systems
Contains:
- gene for desired recomb proteins adjacent to DFHR gene
- driven by weak promoter
After transfection cells transferred to a medium that lacks DFHR activity products = only cells w/ integrated wt DFHR gene will survive
Why is transgene weakly expressed when using mammalian systems?
Ensures transgene that are low copy or in unfavourable genomic domains are selected out -> only multicopy transgenes that can produce sufficient DFHR will survive.
=> Cell density is limited t/ best way of maximising protein production = identify lines containing many copies of transgene.
- can be enhanced by treatments that induce selective amplification of transgene copy number w/in host genome: chemostat approach used.
Gene amplification when using mammalian cell lines
Initial selection based on DFHR activity generates a population of transformed cells with different levels of DFHR activity
- mixed population can be treated w/ anticancer drug and inhibitor - MTX
Methotrexate (MTX)
Anticancer drug and inhibitor of DFHR
- at low conc it kills expressing DFHR marker at a low level.
Gradually ^ing MTX conc = applying a selection pressure for high copy (6 expression) transformants & for cell lines in which selective amplification of DFHR takes place t/ this will also be amplified
Individual cells then clonally propagated in absence of MTX and screened for level of production of recombinant product (western blot e.c.t) and best lines are maintained as production strains.
T/ ^ copy ^ expression cells are selected.
