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BIOL483- Comprehensive Exam Notes .docx

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University of Waterloo
BIOL 483
Vivian Dayeh

What are two direct ways to determine the number of cells in your culture? - Hemocytometer o Manual way of counting cells o Neubauer is the most commonly used - Electronic counter or particle counter o Electronic way o Coulter Counter  Rapid counting  Known volume of cell suspension buffered into saline solution  Solution drawn up through a suction hole  As particles travel into hole it passes 2 electrodes, one inside, one outside  This creates pulse that is detected by counter, can set upper and lower size limits of particles and eliminate particles that are not the size of the cells you are counting. What do cells undergo before passing through the coulter counter? Why do they undergo this process? - Undergo FIXATION o Must be fixed because that way you get less clumping of the cells o Fixation is where a cell is no longer capable to divide. It is a dead cell. What are the indirect methods to find out the density of cells? - Protein determination o Measures total amount of protein in cell culture o Uses BRADFORD ASSAY  Lyse cells, add NaOH + Comassie blue which causes BLUE COLONIES TO FORM  Compare it to standard results on specto - Glucose determination o Measure changes in glucose levels in medium with respect to cell density o Technique is good for immobilized cells found in bioreactors – used when isolation of a cell sample is difficult due to immobilization What is cell viability? How many viability tests are there? - Measure of metabolic state of a cell population – is the potential for cell growth in the culture - 5 viability tests. 1. Colony formation a. Direct measure of ability of cells to grow b. Place known number of cells at LOW DENSITY in petri dish c. Each viable cell in initial count will produce a distinct colony of cells d. Count number of colonies to determine plating efficiency 2. Cell membrane integrity a. TWO TYPES OF ASSAYS – dye exclusion & retention Dye exclusion - Ability of cell membrane to exclude bulky charged molecules o TRYPAN BLUE MOST COMMON DYE o Dye penetrates through cell membrane of dead cells and accumulates o Dead cells stained BLUE. Retention of a marker molecule - Measures ability of cells to RETAIN MARKER MOLECULE - LDH (lactate dehydrogenase) - If cell membrane damaged it allows for LDH to leak out into surrounding medium - Measures LDH activity in culture medium - DEAD CELLS WILL RELEASE LDH into medium T/F – If LDH is found outside a cell, it means it is a viable cell –FALSE means its dead cell Retention of esterase substrates such as CFDA - Is NON POLAR, NON FLOURESCENT - Diffuses into cells rapidly - Converted by esterases of living cells to CF which IS polar and fluorescent - CF diffuses out slowly - DEAD CELLS WILL NOT BE FLOURESCENT as CFDA will move in but WONT BE CONVERTED. - LIVE CELLS will have CFDA MOVE INTO CELL, CONVERTED INTO CF and show FLOURESCENCE. LIVE= CFDA  CF  FLUORESCENCE How many ways can you measure metabolic activity of cells? What are they? - Two ways o Tetrazolium Assay  Measure of MITOCHONDRIAL ACTIVITY  MTT (dye) reduced by succinate dehydrogenase  Measured spectrophotometrically for FORMATION OF BLUE COLORED PRODUCT from YELLOW MTT reagent. This indicates METABOLLICALY ACTIVE CELLS o Resazurin reduction assay  NONFLOURSECANT DYE that permeates cell membrane  Reduced in living cells by MITOCHONDRIAL ENZYMES AND DIAPHORASES  Is a GENERAL measure of MA.  FORMS PINK PRODUCT from BLUE DYE. Gene Modification What is the most widely used cell for introducing recombinant DNA into a mamammlian cell? - CHO cells – Chinese Hamster Ovary cells. What must you have to genetically modify cells in culture? - Gene of interest – (seq you want to introduce in cells) - Cells – - Transfection mechanism – (how the gene of interest is introduced in the cells (artificial, natural)) - Selection mechanism – (Dominant marker for WT genes, Selectable marker for Mutant genes, which of your cells have taken up the gene of interest) How do we get cells to fuse? - By using agents that promote cell fusion What are the agents that promote cell fusion known as? - Known as FUSOGENS How many types of fusogens are there? - 3 types o Biological – Sendai Virus o Chemical – Poleytheylene Glycol (PEG) o Electrofusion – electric pulses fuse cell How do Fusogens act on the cell? - They disrupt the cell membrane of the cell that is being exposed, this will produce an unstable region of that cell membrane. If two neighbouring cells have both their cell membranes disrupted, they can come together and fuse. Describe the process of AB Hybrid Synkaryon - Parent cell A and Parent Cell B are exposed to fusogen - They fuse together by cell membrane - Then they become one cell with two nuclei to form a heterokaryon (two different nuclei) - Undergo mitosis - Form AB hybrid synkaryon with half genes of parent A and half of Parent B Define: Homokaryon – Multinucleate cell = all nuclei are the same Heterokaryon – Multinucleate cell = two or more different nuclei Synkaryon – mononucleate cell that has formed from a hetero or homokaryon Cell hybrid – proliferating synkaryons Chromosome transfer How many ways can you transfer chromosomes? - Two ways o Isolated chromosomes  Arrest chromosome at metaphase with COLCHICINE  Transfer directly – not very successful o Microcells  Arrest cells in MITOSIS – unstable and forms many small nuclei  Treat with CYTOCHALASIN B – which interrupts microfilaments and disrupts cytoskeleton  Centrifuge (forming microcells)  Use fusogens to introduce microcell into another cell What is transfection? - Transfer of nucleic acids into an animal cell aka DNA MEDIATED TRANSFORMATION What are the steps? - Extract DNA (or isolate RNA and prepare cDNA and proceed to step 3) - Cut with restriction endonuclease - Incorporate into plasmid with selectable markers - Clone in bacteria - Transfect into cell line - Use selectable media to grow transfected cells How is the DNA introduced into the cells? - By ARTIFICIAL or NATURAL methods of transfection What are the artificial methods of transfection? How many types of are there? - Two types of artificial methods o Direct o Indirect Direct methods - Microinjection o Inject a known amount of DNA via a microneedle into nucleus of individual cell o Disadvantage = o few cells can be injected at one time o Is nonspecific Indirect methods - Calcium Phosphate method (simple method) o Take dna that you have in terms of seq that u isolated and bring it together with CaPO4. o CaCl2 + PO4 + DNA forms fine precipitate which cells take up for endocytosis and therefore you get transfection. o Disad = only a few types of cells will take up the precipitate - DEAE dextran o Treat cells with DMSO as it will DESTABILIZE CELL MEMBRANE (same compound used to cryopreserve cell) o Cells then take up DNA and few cells will incorporate the new DNA and express gene - Liposomes o Formed from a positively charged lipid and DNA o Readily taken up by cells o High level of gene expression - Protoplast fusion o Gene of interest is inserted into plasmid which is grown in bacteria o Bacteria treated with lysozyme to remove cell wall o Fuse protoplast with animal cell using PEG o Disad – potential to introduce unwanted bacterial components into animal cells - Electroporation o Expose cells to high levels of electrical impulses for short time o Leads to reversible increase in cell membrane permeabilitiy to promote DNA uptake - Microprojectiles o Mostly used for transfection of plant cells o Coat projectiles with DNA and shoot into cell T/f – microprojectiles can be used for animal cells – TRUE t/f - Electroporation leads to irreversible increase in cell membrane permeability = FALSE, reversible. What is the natural method? - Uses viruses to genetically manipulate cells in culture - Efficient way to introduce genes via viral infection What is transduction? - Is the process of using a viral vector to introduce DNA into a cell Why is there an increase in efficiency of transduction? - Capsid proteins on viruses provide uptake mechanism into cell - Contain promoter elements for gene expression - Produce several copies of gene within each cell - Efficient integration of gene into certain cell types What are the two most common used viral groups for transduction? - SV40 – SIMIAN VIRUS o Causes neoplastic transformation IN VITRO o Has two phases of transcription in normal SV40 infection  Early and Late phase  Early phase = T antigen proteins synthesized that unwinds the viral DNA, for neoplastic transformation and immortalization  Late phase = transcription and production of capsid proteins  EVENTUALLY LEADS TO CELL LYSIS and RELEASE OF SV40 cells What is the recombinant viral vector used for? - To transfect animal cells Gene of interest replaces part of the viral genome, the virus then infects cell and introduces foreign DNA efficiently. ONLY DISADV is the need for a host cell that is derived from a monkey. NEEDS A PERMISSIVE CELL LINE! What is a retrovirus? - Is an RNA virus - Incorporates genes into nuclear DNA - Forms complementary DNA seq from RNA genome by reverse transcriptase - Normal infection incorporates DNA without cell lysis - Also used for cell immortalization - DON’T GET LYSIS AT END OF NORMAL INFECTION ROUTE. CAN BE USED TO CREATE CONTINUOUS CELL LINE However, you still need a way to select whether or not cells have been transfected with that gene of interest What is a non-selectable gene? - Is gene of interest - The gene you want to introduce into your cell T/F = regardless of the transfection technique a large proportion of cells take up and express the DNA in a stable manner = FALSE, only SMALL PROPORTION do that What is a selectable gene? Why is it important? - Selectable gene used depends on whether a mutant or wild type cell is used - It is important because you need to have a way of selecting those cells that have been stably transfected, which requires use of a selectable gene as it is brought in with expression NT biosynthesis What are they commonly used for? - Used for markers for transfection of genes of interest into both WT and Mutant cells. - Allow the state of transfection to be determined – whether or not gene of interest has succesfuly been transfected into cells in culture. Non selectable markers = are DNA seq that you are introducing into the cell but are not able to select for Selectable gene = are able to select whether or not cell takes up the Seq. What 4 NTs are incorporated into DNA? - dATP, dGTP = purines - dTTP, dCTP = pyramidines What are the two pathways that are used to create NT? - De Novo pathway – from new synthesis of NT from simple precursors - Salvage pathway – NTs that are synthesized from other NAcid intermediates. How do you create a genetic variant? - Deliberate or accidental modification of the normal genome that it survives but is mutated in a way that you could use to introduce your genes of interest. o Eg take cells that come from someone who already has condition that has a mutation within tissue metabolism What is the Lesch-Nyhan syndrome? - Cells that lack Hypoxanthine-guanine phosporibosyl transferase = HGPRT NEGATIVE - HGPRT is involved in salvage pathway BUT in these cells HGPRT does not function, therefore cells take de novo pathway of NT synthesis to survive. What are drug resistant cell lines? - When u start with normal cell and expose them to chemicals that are toxic – cells that survive the exposure are resistant. - Incorporated as the base guanine with important enzyme = HGPRT - Incorporation of these guanine analogues are eventually toxic to cells Why is it toxic? - Due to disturbed transcription and translation - Reduction in level of NT triphosphates (eg ATP) - Makes HGPRT mutant = deficient in HGPRT BromoDeOxyUridine (BUdR) resistence = analoge of thymidine which is incorporated into DNA. - Incorporated for base THYMADINE with important enzyme TK = Thymadine Kinase - BUdR is toxic to cells at HIGH concentrations o Due to blocking synthesis of deoxycytidine NTs - Few cells may grow with BudR either with or without prior mutagen treatment o Causes TK- mutants. What is transfection selection in mutant cells? What does it use? - Uses HAT medium to determine if transfection has occurred in mutant cells o H = Hypoxanthine o A = Aminopterin o T = Thymadine BLOCKS De Novo PATHWAY! Cells must use SALVAGE PATHWAY to survive, therefore cells must be HGPRT+!! It is blocked because of the AMINOPTERIN block on De NOVO pathway. If a mutant cell is exposed to HAT medium = WILL NOT SURVIVE Why cant HGPRT- cells grow in HAT medium? - Cant grow cus they cant utilize Hypoxanthine in HAT medium Why cant TK- cells grow in HAT medium? - Cant grow cus they cant utilize Thymadine in Hat medium Wild type cells can overcome the block by aminopterin by utilizing H and T in medium Use mutant cells (either TK- or HGPRT-), transfect selectable gene along with gene of interest (non selectable gene) using calcium phosphate copreciptation method - Transfer TK gene to TK- cells and select on HAT medium - Transfer HGPRT gene to HGPRT- cells and select on HAT medium Successful transfection of mutant cell with gene of interest and marker gene – mutant cells will express WT phenotype and will grow in HAT medium (expressing gene of interest) What will Hat medium allow for in terms of detection? - Detects drug resistant cells that have reverted back to normal wild type cells Transfection selection in WILD TYPE CELLS - When using a WILD TYPE CELL to transfect genes into, a DOMINANT SELECTABLE MARKER GENE must be used - Can be introduced into any WT cell - Only transfected cells with introduced cell will grow What are the 3 examples of Dominant Selectable genes? - DiHydroFolate Reductase = DHFR o Mutant DHFR gene isolated and cloned from mouse line, exhibits increased resistance to methotrexate which blocks de novo pathway o Introduce DHFR into WT cells using calcium phosphate coprecipition method o Select for transfected cells by growing in medium with high methotrexate concentration and no hypoxanthine o Cells must rely on de novo pathway - those without DHGFR mutant gene don’t survive o Only cells transfected with mutant DHFR gene grow = means these cells are methotrexate resistant and able to grow under selectable pressure. o Is a MUTANT form of DHFR gene  Mutant form is RESISTANT to compound known as methotrexate  Normal form is IRRESISTANT TO IT  Methotrextate BLOCKS de novo pathway - neoR gene o codes for AGPT (amino glycoside phosphotransferase) o resistant to antibiotic G418 o normal mammalian cell wont grow when exposed to this o if you add neoR and take dominant selectable gene and put into WT cell and it takes it up and is expressing it they can grow o only cells that have taken up neoR can grow in G418 media. XGPRT – Xanthine Guanine PhosphoRibosylTransferase - enzyme found in bacteria - not found in mammalian cells - converts Xanthine to xanthine monophosphate - introduce it using calcium phosphate copreciptiation method What does XGPRT contain, what does it select with? - Hypoxanthine - Xanthine - Aminopterin – BLOCKS DE NOVO pathway - Mycophenolic Acid – BLOCKS inosine monophosphate dehydrogenase (IMP) Aminopterin and Macid are blockers of NT synthesis - If WT takes up XGPRT it can bypass the blocks that are produced by aminopterin and Macid Two types of expression - what are they? - Transient expression o Expression of gene for short period of time after transfection o After this period the gene ceases - Permanent expression o Stable expression of transfected gene – capable of indefinite synthesis of the protein What are the two approaches to transfect non-selectable genes permanently? - Covalently ligate non selectable to a selectable gene o Eg growth hormone gene to TK gene  Transfect into TK- cells – SELECT ON HAT MEDIUM o Eg growth hormone gene (non selectable gene) to bacterial neoR gene (dominant selectable gene)  Transfect into mammalian cell = select in medium containing antibiotic G418 - Cotransfection o Use calcium phosphate coprecipitation method with a mixture of selectable and non selectable genes o Don’t know whether or not cells have taken up both or if they will express it. You want cell culture to produce Protein X – how do u do this? Start = either with mutant cell (HGPRT- or TK-) or WT cell (use dominant selectable marker, HGPRT+ or TK+) For a mutant = it can be a natural mutant OR take a normal cell and expose it to chemical treatment How do you make HGPRT- cells? - Take WT cell and expose it to Azaguinine or Thioguanine - If cell is exposed and dies – it is WT cell, meaning HGPRT+ - If cell is exposed and survives = HGPRT- What are the 2 pathways for NT synthesis? - De novo - Salvage Start with TK mutant cell you can follow same pathway as HGPRT mutant cell - Come up with construct - Protein that you want ur cells that are easy to work and grow with but not expressing = NON SELECTABLE GENE o You have no way of finding out has ur cell taken up gene protein X o Ligate or attach gene for selectable marker and in this case you dealing with TK- mutant cell. o Easiest way is to ligate them to one another Gene for protein X and TK, they transfect TK- cells = transfection techniques can be artificial or natural (calcium phosphate coprecipitation) Take gene with protein X = (gene for TK+) - Make construct - Transfect cells - Use selectable medium (HAT MEDIUM) Hat medium – two possible results 1. - TK- mutant cells that did NOT take up construct - CELLS WILL NOT GROW - Wont grow because they don’t have TK gene, which means they cant rely on THYMADINE because DE NOVO pathway is BLOCKED 2. - Mutant cell that HAS taken up construct = WILL GROW IN HAT MEDIUM - WILL REVERT TO WT CELL - Check to see if produces protein X What are the three general goals for introducing a gene into an animal cell? - Immortalization o Advantageous to use a continuous cell line over a finite cell line - Expression o Can introduce gene of interest into cells that do not express it or for over expression - Secretion o Want product to be secreted into culture medium  Easier to have product secreted than cell lysis to obtain product  Concerned with downstream processing and purification of product Immortalization IN VITRO SENESCENCE - Mammalian cells in culture = finite lifespan - Human fibroblast = most well studied (50-60 doubling) What is the difference between Senescence versus quiescence? - Quiescence is PHYSIOLOGICAL WITHDRAWAL from cell cycle = could be due to nutrient deprivation o IS REVERSABLE as stimulation with appropriate growth factors will result in proliferation - Senescent cells are maintained but DON’T PROLIFERATE o IRREVERSABLE o Distinct from cell death that leads to loss of cell viability What is the correlation between IN VITRO lifespand witih age of donor and LE of species? - Longer SP = more cell doublings - Decreased IN VITRO LS in cells with premature aging syndrome (werners) What is the cause of senescence due to? - Telomere Experimental Immortalization of Cells - How is immortalization usually done? o Done with finite cells to convert them into continuous cell lines but also done with primary cultures Why is immortalization an advantage over the use of primary cell cultures? - Easier to obtain continuous cell line expressing differentiated functions T/F = immortalizing rodent cells is harder to do than human cells = FALSE – easier to do than humans What are the 5 classes of methods for immortalization that could be used? - Oncogenic Virus (SV40) - Oncogenes – inappropriate expression in cells - Chemical carcinogens – causes mutation in DNA directing senescence mechanism - Irradiation – same as for carcinogens - Telomerase MEMORIZE PAGE 56 Telomeres What are telomeres? Why are they necessary? What would happen if we did not have any telomeres? - Special DNA seq found at ENDS OF EACH CHROMOSOME - Necessary because our chromosomes are linear and they allow for precise replication of DNA - DNA would be prone to degredation if we did not have any teloemres and end-to-end fusion T/F = each time cell divides the telomeres shorten = TRUE What happens if one or more telomere lengths is at minimum length? - Cell division stops, preventing damage to codin region of genome What is the role of Telomerase? - Is an enzyme that replaces missing repeated sequence of each telomere that would be lost at DNA replication o Activity is low in most cells o Immortal and neoplastic cells exhibit high telomerase activity o Transfecting gene for telomerase into fibroblasts EXTENDS LS. Why is end protection important? - Important because it prevents damage to the coding region of DNA otherwise dna can stick together, causing unreadable strands and molecules cant code for a particular product. Once telomeres get short enough it wont take it to senescence Why are malignant cells having telomerase activity turned back on? - Its because when you introduce the TELOMERASE GENE it can be turned on and can extend LS. - You need to introduce the GENE that encodes for telomerase and if expressed, it’s the telomerase that extends LS of a culture. Protein itself WONT create an extended LS, it’s the integration of the GENE for telomerase into GENOME OF CELL that will turn on expression (if integrated correctly) SV40 What is SV40? - It is a small DNA tumourigenic virus that causes tumours in rodents under lab conditions - Is a circular DNA molecule - Divided into an early and late phase What does the early region code for? - Large T antigen (transformation) = expression important for neoplastic transformation and immortalization - Small T antigen = promotes cell transformation What does the late region code for? - Codes for structural viral proteins = VP1, VP2, VP3 How many types of interactions can a susceptible SV40 cell undergo? - 2 reactions Lytic cycle - Occurs in PERMISSIVE CELLS ONLY (monkey cells) - Leads to production of new infectious virus particles and CELL DEATH - Requires large T antigen as viral DNA is replicated Neoplastic transformation of cells - Happens in NON PERMISSIVE CELLS – rodent and some primate cells - Viral DNA integrated into cellular DNA (nonspecific) - Large T antigen is oncogene product of SV40 T/F = large T antigen is capable of immortalizing primary cell cultures & cell lines from rodents = TRUE What are the two tumour suppressor genes? - Rb & p53 T/F = immortalizing often results in a continuous cell line that don’t retain differntiatied cell functions = FALSE, they DO RETAIN differentiated cell functions What are the ways to introduce large T antigens into primary cell culture? - As part of the intact virus o Rely on natural infectivity = specific species are infected by SV40 o Must remove origin of viral replication to prevent killing of cells via lytic cycle that will allow for SV40 rep o Want cell to continuously express large T antigen but don’t want viral rep which would lead to host cell lysis - Transfection of large T antigen gene o Link large T antigen gene to neoR gene using calcium phosphate coprecipitation o Select with medium containing G418 – antibiotic to select for cells expressing dominant selective gene Experimental immortalization of human cells - Human cells that have been manipulated in culture to bypass senescence only extend IN VITRO lifespan =do not make cell line immortal t/F = using SV40 large T antigen extends lifespan and immortalization = FALSE, only extends lifespan, NO immortalization. (In human cells, in vitro) - Need to bypass senescence AND keep telomeres intact What is crisis caused by? - Caused by bypass of senescence – leads to critical telomere shortening on many chromosomes - Causes breakdown of chromosome stability What happens if some cells escape crisis? - All cells that escape exhibit telomerase maintenance mechanism - Somehow telomerase activity is activated in some cells during crisis LEADING TO IMMORTALIZATION OF CELLS. Expression What are the main reasons for expression? - Want to obtain high levels of gene expression of a gene of interest (higher than the cell line may normally express) - May want to express a gene the cell does not normally express What is a viral expression vector? How many types are we looking at? - Viral expression vector is a viral DNA fragment that carries necessary regions to express an adjacent gene – these regions are enhancers and promoters SV40 - If you’re looking for way to have cells to take up and express gene of interest, this can be method to use - Acts as a transient expression vector (expression of gene of interest lasts short time) - Remove SV40 genome and early region and replace with gene of interest o This puts gene of interest under promoter control for early region - Transfect construct into COS cell line = SV40 early region is already integrated into dna of COS cells – stably transformed with partial SV40 genome HAVE TO HAVE CELLS that are not only permissive but also ones that contain early region already integrated! What does the stable change in cell genome allow? - Allows for SV40 construct to come in and express gene of interest that you’ve introduced in cell. Where does the COS name come from? - CV-1 (African green monkey cell line giving rise to COS) - Origin of replication - SV40 It also supplies essential large T antigen needed for rep of viral construct WITH gene of interest - Viral construct with gene of interest will be transcribed What are the disadvantages? - Only MONKEY cells will allow for virus rep (eg cos cell) - Size of gene insert into vector is limited - Cells support rep of large number of viral particles – this is what kills the cell o Get massive production within days of transfection but cells do die – not useful. Retroviruses - Acts as a stable expression vector = stable expression in continuous cell line - Usually contain two expression units under LTR control o LTR integrates the viral DNA Retrovirus is designed to integrate genome into host cell - LTRs STABILIZE vectors - Are on ends of Viral genome Retroviruses allow stable expression of gene of interest Can propagate retrovirus infected cells that continuously produce and release viral progeny into medium. Making transmission vectors - What do transmission vectors allow for? o Allow for introduction of genes into mammalian cells by viral infection What are the 4 steps in creating transmission vectors 1. Create expression vector a. Replace structural genes in retrovirus with: i. Selectable gene ii. Biotech gene of interest b. What are LTRs and what do the left and right LTRs do? i. Provides stability ii. Left LTR at 5’ end contains packaging signal 1. Necessary for RNA genome to be packaged into mature virons 2. Select packaging cell line a. Cell line that already contains an integrated provirus What does a provirus direct? - Directs synthesis of viron BUT cannot package their RNA genomes into virons = only produce EMPTY VIRAL PACKAGES 3. Transfection of packaging cell line with expression vector a. CaK copreicitpation method used to introduce expression vector into chosen packaging cell line b. Select for transfectants on selective medium (HAT, or G418) c. Grow up on colonies of transfected packaging cell line which becomes the producer cell line (product is transmission vector) LOOK AT PAGE 61 4. Release of transmission vectors via producer cell line a. What does the transmission vector consist of? i. Consists of RNA GENOME from expression vector packaged into virons from packaging cell line (these are complete virons complete with reverse transcriptase) T/F = using transmission vectors results in high efficiency of gene transfer to other cells using this natural method of transfection = TRUE SECRETION - Protein needs to have the correct following 3 aspects o Modified post translationally  Removal of AAcids  Chemical modification of R groups of individual AAcids o Folded – to get a functional protein o Secreted = allows for cheaper production than having protein retained in cell If a protein is secreted, there are no additional steps to break cells open and purify protein product. Protein Synthesis (PS) T/F = PS occurs on the Smooth endoplasmic reticulum = FALSE, occurs on RER T/F = proteins fold in the ER = TRUE, proteins not folded correctly are eliminated After folding, they move to transition vesicles and go to the GOLGI APPARATUS to be modified. After Golgi, the protein is released from secretion vesicle via exocytosis. Protein folding What are the two mechanisms of protein folding? - Spontaneous folding – does NOT rely on external forces - Protein catalyzed protein folding o Use CHAPERONES (holds N terminal region of protein in exposed conformation)  Permits region to be available for association with later synthesized C terminal regions Why is it critical to have a proper folding? - To have functional protein Glycosylation - What is the general function of glycosylation? o To allow for correct folding and stability in the conformation of glycoprotein What is a glycan? - Addition of carbohydrate side chain Glycoproteins =attached to protein Where does initial glycosylation occur? Terminal glycosylation? - ER - Golgi What are glycosyltransferases? - Group of enzymes that produce GLYCANS. What are the two types of glycans secreted in proteins? - N linked and O Linked o N linked  Carbohydrate linked to ASPARAGINE residues of polypeptides o O linked  Carbohydrate linked to SERINE OR THREONINE residues What are mammalian cells commonly used for? - Production of human glycoproteins Animal Cell Culture Scale Up Why scale up animal cell cultures? - To produce significant quantities of an animal cell culture product Anchorage independent cells - Growing mammalian cells in suspension culture to produce high quantities of culture product What are potential problems to scale up of mammalian cells? - Ability to keep cells in suspension - Absolute sterility of culture - Foaming of serum - O2 supply Mode of Culture What waste product is produced? - Lactic acid = from glycolysis o pH shifts are noticed from too much Lacid (red to yellow) o SOLVED by not using glucose in culture medium (substitute for galactose) - Ammonia = inhibits growth of culture o Solved by keeping low concentration of glutamine o Can also inhibit glycosylation of mammalian protein products  Can reduce reactions in O linked glycosylation pathway Mode of culture can limit metabolic end product, mode depends on culture product being produced - Batch culture ALL ON PAGE 65 o Whole culture harvested at a certain point after inoculation  Large vessel, holds media that suspends cells  Mixing impeller blade, entire media processed after certain period of time  ENTIRE SYSTEM REMOVED - Semi Continuous o PORTION of the culture removed at INTERVALS o Fresh culture medium introduced to replenish the loss o Gently mixed with a propeller blade  Take small subsamples of cultures out + introduce small amount of media back in = semi continuous o Whatever sample you take out you add that back in with NUTRIENTS. - Continuous system o Culture medium is continuously harvested with respect to cells or product o New culture continuously added o OVERFLOW RATE = FEEDING RATE. o As cells grow you CONTINUOUSLY r
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