What is quiescence?
A physiological withdrawal from the cell cycle (could be due to nutrient deprivation). Reversable if appropriate growth factors are provided to the cells.
What are five classes of methods for immortalizing a cell line?
Oncogenic viruses (ie. SV40)
Oncogenes - inappropriate expresseion in cells
Chemical carcinogens - may cause mutation in DNA directing senescence mechanism
Irradiation - same as carcinogens
Telomerase - enzyme
How does telomerase affect the lifespan of a cell line?
Introducing telomerase into a cell will prolong its life beyond a finite lifespan. Neoplastic cells often have high telomerase activity.
How does SV40 virus immortalize a cell line?
SV40 can immortalize non-permissive cells (rodent cells and some primate cells) by integrating viral DNA into cellular DNA. This is a non-specific insertion.
The large T antigen is capable of immortalizing primate cell cultures & cell lines from rodents by knocking out tumour-suppressor genes Rb & P53 by complexing.
How is large T antigen introduced into primary cell cultures?
As part of intact virus - relying on natural infectivity of SV40, removing origin of replication to prevent virus from kill cells via lytic cycle
Transfection - link large T antigen with neoR gene using calcium phosphate coprecipitation. Grow on G418 medium to select for cells.
How can SV40 be used to express a gene of interest?
Start by removing the genome in the early region and replacing with the gene of interest (gene is under the control of promoter for early region). Transfect construct into COS cell line (CV-1, Origin of Replication, SV40).
The disadvantages of this is that it only works for monkey cells, insert size is limited and cells cannot support large number of replication
Explain how retroviruses are used as expression vectors
They act as a stable expression vector in continuous cell lines; only so because of their retroviral properties. They integrate the viral DNA (controlled by the LTRs). Retroviruses can also continuously produce and release viral progeny into the medium.
What is a transmission vector and how is it made?
Transmission vectors allow for the introduction of genes into mammalian cells via virual infection.
1. Create expression vector - replace structural genes in retrovirus with gene of interest and selectable marker. The LTR on the left (5') contains packaging signal.
2. Select a packaging cell line - a cell line that contains an integrated provirus that directs synthesis of viron (but cannot package their DNA).
3. Transfect packaging cell with expression vector using calcium-phosphate coprecip method to introduce expression vector into packaging cell. Select for the transfectants using selective media (HAT or G418).
4. Release of transmission vectors via producer cell line - the transmission vectors will consist of RNA genome from the expression vector packaged into virons from packaging cell line. These are complete virons complete with reverse transcriptase.
After expression, protein products must be correclty...
Modified post-transcriptionally - removal of amino acids, chemical modification of R groups of individual amino acids
Folded - to get functional proteins (assisted by chaperones)
Secreted - allows for cheaper production than having the protein retained in the cell
List the steps of protein synthesis
- Occurs on rough endoplasmic reticulum
- Some modifications occur in the ER
- Proteins fold in the ER (proteins not folded properly are eliminated)
- Proteins move to the transition vesicles then the Golgi apparatus (modifications done completely here)
- Proteins are excreted via exocytosis
What are the 2 mechanisms of protein folding?
1. Spontaneous folding - protien folding that does not rely on any external forces
2. Protein catalyzed protein folding - Done through the help of chaperones that have a duel 'holding' and 'folding' fuctions. They hold the N-terminal region of the nascent protein in an exposed conformation and allows it to be associated with the later synthesized C-terminal regions.
What is glycosylation?
Glycosylation is the addition of carbohydrate side chains (glycans) which serves to allow for the correct folding and stability of the final glycoprotein.
What are the two types of glycans in secreted proteins?
N-linked glycans: carbohydrate linked to asparagine (Asn) residues of polypeptides
O-linked glycans: carbohydrate linked to serine (Ser) or theronine (Thr) resudues of polypeptides