Term
| What large scale processes are automated in regards to adherent cell cultures? |
|
Definition
| roller bottles, (attached to microcarriers which allows them in stirred tank reactors) |
|
|
Term
| how are mammalian cells adapted to protein free media? |
|
Definition
| long process- repeatedly exchanging 80% of culture medium against protein free medium- cells learn to adapt |
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Term
| cell growth rates can be increased by insertion of specific genes such as___,___,___,___ |
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Definition
proto-oncogenes
cell cycle control genes (cyclins)
growth factor genes
anti-apoptotic genes |
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Term
| Why is serum-free, protein-free media defined in large scale productions? |
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Definition
| reduce risk of transmitting infections |
|
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Term
| Recombinases such as ____,___ allow homologous recombination with specific attachment sites on donor and recipient DNA providing targeted gene integration |
|
Definition
bacteriophage P1Cre
Yeast FlP |
|
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Term
| List three strategies to overcome the effects of random integration of genes into the mammalian cell genome? |
|
Definition
1. protective cis elements
2. block histone deacetylation
3. targeted gene integration |
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Term
| ___- influences expression levels, frequently leading to gene silencing and has to be taken into account when developing a new mammalian cell line |
|
Definition
|
|
Term
| What three things must be considered when developing mammalian cell cultures? |
|
Definition
1. mammalian expression vectors
2. effects of recombination and random integration
3. medium optimization and cell host engineering |
|
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Term
| what are 5 advantages of filamentous fungi expression systems? |
|
Definition
1. Gras
2. Excellent enzyme secrection
3. large scale cultivation techniques
4. High production yields
5. no excessive hyper mannosylation sometimes glycostructure compatible to mammalian structures |
|
|
Term
| what are 5 disadvantes of filamentous fungi expression systems |
|
Definition
1. difficult genetic manipulation
2. genetics cell biology not well characterized
3. abundant produciton of endegenous and extracellular proteases
4. potential toxin producers
5. heavily patented |
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|
Term
| What are 4 main components of a mammalian expression vector? |
|
Definition
1. eukaryotic/viral promoters
2. regulatory enhancer elements
3. selection markers
4. elements for bacterial cell propagation |
|
|
Term
| Briefly describe how a mammalian cell line is generated and how the development of cell culture processes for recombinant proteins of interest is achieved? |
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Definition
cell line generation starts with a protein and a selector-> then you linearize and transfect and clone the cell lines
after that there is a screening process until the right one is found |
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Term
| What are 5 important features of stable mammalian cell lines? |
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Definition
1. takes 6-12 months to develop
2. can be used for human use- clinical trials
3. heterologoius DNA is integrated into the host genome and is maintained throughout many generations
4. high cost
5. after ampflication and producer selection expression level is relatively high |
|
|
Term
| What are 6 important characteristics of transint mammalian cell lines? |
|
Definition
1. expression of transfected genes are usually within 16-96 hours but expression does not propagate through generations
2. trasnfection requires a large quantity of plasmid DNA
3. expression level usally relatively low
4. expressed product is good for pre-clinical assesment but not for human use
5. takes only a short period to express a product for "proof of principal" demonstration
6. low cost |
|
|
Term
| What are 4 applications of BEVs in terms of protein production? |
|
Definition
1. insect larvae
2. insect cells
3. mammalian cells
4. baculodisplay |
|
|
Term
| By 2006 production of therapeutic proteins by mammalian systems reached 20 billion dollars mainly due to use of ____ for the production of flu vaccine and ___ for the industrial production of recombinant protein therapeutics |
|
Definition
|
|
Term
| Why are CHO preferred for protein production |
|
Definition
|
|
Term
| list three examples of top selling biopharmaceuticals in 2007 derived from mamlian cells |
|
Definition
|
|
Term
| What are 6 advantages of mammalian cell expression systems |
|
Definition
1. post translational modifications similar to human cells
2. proper protein folding
3. flexibility of protein size
4. efficient cleavage of signal peptides
5. multiple genes expressed simultaneously
6. high value market products |
|
|
Term
| what are 6 disadvantages of mammalian cells |
|
Definition
1. low levels of expression
2. poor secretion
3. expensive
4. time consuing process
5. potential for product contamination by viruses
6. long and costly FDA approval |
|
|
Term
|
Definition
chinese hamster ovary
dog cocker spaniel kidney
human cervix carcinoma
retina derived human cells |
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Term
| adherent or anchorage dependent insect cells require a compatible growht surface list 3 |
|
Definition
1. multi well plates
2. roller bottles
3. t flasks
4. microcarrier beads |
|
|
Term
| three examples of suspension adapted cell lines growht |
|
Definition
1. spinner flasks
2. shake flask
3. stirred bioreactor |
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|
Term
| What are 5 steps in recombinant protein expression using engineered baculovirus systems? |
|
Definition
1. transfection with recombinant baculoviral DNA
2. amplification of viral stock in adherent cultures
3. protein expression in suspension cultures
4. protein purification
5. scaling up protein expression for industrial production |
|
|
Term
| give three examples of insect cell lines that can be infected with recombinant baculovirus for protein expression |
|
Definition
|
|
Term
primary host infection is caused by an ____
while secondary host infection is caused by a ____ with a late infection phase of ____ hours |
|
Definition
occulsion derived virus
budded virus
6-24 hours |
|
|
Term
| characteristics of direct in vitro cloning |
|
Definition
k.transfect into insect cells
E. Restriction endonucleus site
J.digestion w ecori |
|
|
Term
| characteristics of homologous recombination |
|
Definition
c. transfer plasmid
d. polyhedrin gene locus
k. transfect into insect cells |
|
|
Term
| site specific transposition |
|
Definition
k. transfect into insect cells
b. bacertial host
I. recombinant bacmid DNA
G. shuttle plasmid |
|
|
Term
| site specific recombination |
|
Definition
k. transfect into insect cells
F. LR clonase
Gateway entry clone
polyhedrin promoter |
|
|
Term
| list the four strategies for engineering recombinant DNA for the purpose of transfecting into insect cells to obtain a stock of recombinant viruses |
|
Definition
1. homologous recombination
2. site specific transporisiton
3. direct in vitro cloning
4. site specific recombination |
|
|
Term
| what is the basis for engineered baculoviruses? |
|
Definition
| based on the introduction of a foreign gene into nonessential for baculovirus replication genome region via homologous recombination or trasnposition with a transfer vector containing target gene of interest |
|
|
Term
| What are the main features of bevs |
|
Definition
production of high levels of properly post trasnlationally modified biologically active and functional recombinant proteins in insect cells
resulting recombinant baculovirus lacks one of the nonessential genes replaced with a foreign gene encoding heterologous protein which can be extressed in cultures of insect cells or larvae |
|
|
Term
| provide three examples of insect cell expression system products with applications in the pharmaceutical industry? |
|
Definition
| cytokines, hormones, vaccines |
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