Trisha BasuCluster 1: Biotechnology2010
Vaccine: A biological concoctionof pathogenicsubstances that isinserted into the bodyin order to produce orenhance an immuneresponse to aparticular disease. Theimmune system is ableto actively fight off thecurrent “infection” andproduce memory cellsto fight against futuretransmission.
The Immune System: The body’s natural response to destroyand remove any detrimental foreignmaterial from the body. Two main types of immunity:• Innate: nonspecific, immediate response,universal protection, basic• Adaptive: specified to pathogens andantigens, delayed (relatively), producesimmunological memory,
Components of the ImmuneSystem:InnateSkinTearsMucus / SalivaLeukocytesMast CellsAdaptiveLymphocytes:- T-cells (killer / helper)- B-cells (memory)Phagocytes:- Macrophage- Neutrophils- Dendritic Cells- Basophils and eosinophils T-Cell- γδ T cells- Natural Killer Cells
DNA Vaccination: An Overview First developed during 1990’s, DNA vaccinescontinue to be under clinical trial and testingtoday. An alternative method to produce immunitywhen modified pathogen/ pathogen-likesubstances were hard to obtain or produce. A modified, double-stranded bacterial plasmidthat has been transformed to contain aeukaryotic promoter sequence, a reporter gene,and our gene of interestI t d i t li ll t d Inserted into mammalian cells to produce animmune response and prevent future success ofthe disease later on.
Gene forAntigenBacterialPlasmidPathogenModified PlasmidSyringeGenetic MaterialGene Gun
DNA Vaccines: ConstructionContains:• A selectable marker• Eukaryotic promoter• A gene insert with vaccine/cytokineadjuvant• Resistance gene• Polyadenylation l signals○ Polyadenylation signals: the addition ofa poly-A tail to an RNA moleculel
How it Works:Modified plasmidinserted into cell andinto nucleus• Encodes a protein(s) withinits modified sectorThe protein(s)• Translated from plasmid• Presented on the surface ofthe transformed cells(through MHC class I andclass II molecules)
Proteinpresentationtieffects:• Fighter and helper T-cellsrecognize the protein asa foreign body and attackthe transformed cells• Specific B-cells produceantibodies to match thepresented protein• Memory B-cells form toinitiate response againstfuture infection
Methods of Vaccination:DNA vaccines are insertedinto body throughhypodermic injections andgene gun with gold beads.• Gold beads require less DNAto be put into the bodycompared to hypodermicinjections as the gold beadsare coated with a fewplasmids.Administered at areas withlittle or no hairThe preferred method tovaccinate food animals is toapply the vaccine throughthe skin• keeps the meat clean andstimulates a natural infectionand proper response
Researchable Question Can we use pUC19 based plasmid,such as pVAX1, to determine if theplasmid in a DNA vaccine is actuallyincorporated into an animal hostgenome?• What are the effects of an integrated DNAvaccine?
The Experiment: Objectives 1. Determine if DNA vaccine genome isincorporated into host genome. 2. If 1 proves true, observe the effect ofan integrated DNA vaccine plasmid in amammalian cell in vitro.
Materials: Transformed pVAX1 2 cultures of body cells (muscle and/orskin) E.Coli GFP
Materials cont’d PCR machine/ kit Gel electrophoresis equipment Gene gun + gold pellets Agarose w/ Kanamycin FACS machine/kit
pVAX1 Modified pUC19plasmid Contains a CMVpromoter with ihintron A, BGHterminator, and akanamycinresistance gene CMV promoter =CytoMegaloviruspromoter
FACS methodology: FACS- Fluorescence-Activated Cell Sorter Used to sort out cells according to theprotein that is produced. After plasmid has been added to cell, thecell produces a protein from the insertedsequence A laser tags the cell and activatesfluorescence and charge in the celldepending d on the protein produced Cells are sorted with electrodes intoseparate containers
Gene Gun: Tiny pellets of gold are coated with theDNA vaccine The pellets are ejected onto the skin andenter the cells and transport the DNAvaccine Replicates a natural infection as theforeign body enters through the skin asin many other infections.
GFP A reporter gene which can easily provideinformation about the transcription andtranslation of plasmid by the host cell. Easy to obtain and manipulate withouthaving to worry about disrupting the hostcell.
Procedure: Overview1.Transform plasmid2. Put plasmid into mammalian cell througheither gene gun or hypodermic needle3. Observe cells on day 1, 5, 10, 20, 30 andcheck for protein production using FACS4. Determine integration and observe5. Observe over longer period of time toexamine trends or oncogenesis
Procedure: Step 1 Insert the desired GFP protein sequenceinto the pVAX1 after the CMV promotersequence. Since the CMV promoter region is aeukaryotic promoter, the cell should beable to transcribe and translate t thetarget sequence. Put transformed plasmids into E.colicells for replication though heat shockand cultivate t for 2 days.
Procedure: Step 2 Remove vector from bacterial cell Prepare gene gun with half of modifiedplasmids Shoot gene gun at a culture of cleancells and infect cells Take rest of modified plasmid intohypodermic needle Inject half other culture of cells withhypodermic needle
Procedure: Step 3 Observe cells on after day 1, 5, 10 , 15,20, 30 Screen cells through FACS method tocheck for plasmid production and recordthe amount and rate of proteinproduction. Observe amount of GFP fluorescenceproduced by cell under UV light. Record observations
Procedure: Step 4 Analyze observations of fluorescence todetermine integration of plasmid into cellgenome. Check if GFP production follows bellcurve with steady production as timeprogresses.
Procedure: Step 5 Observe cells periodically Record cell growth and division todetermine if insertion of plasmid hascaused oncogenesis in cells.• If oncogenesis starts and persists try toIf oncogenesis starts and persists, try todetermine why
DNA Vaccines:Potential Problems DNA Vaccines differ with differentspecies and diseases• Unique promoter regions determine how DNA istranscripted and translated to form proteins• Proteins must be properly modified to bedisplayed by the cell and produce the desiredeffect.• DNA vaccines cannot be used for bacteria or RNAviruses○ Genes code for wrong sequences or lack importantsequences to translate the correct proteins
Animal / Human DNA vaccines:• Highly restricted by price, versatility, rateof dispersion throughout the organism, durability,etc.• Can hopefully be used in young organisms for avaried set of diseases and to last throughout life• Must be controlled depending on the speciesbeing used and the future of that organism.• Must also be tested for their specific proteins andtheir response to "challenges from the infectiousagent".• Must be controlled to reduce mutationti
Bibliography: Brandsma, Janet L, et al. DNA Vaccines: Methods and Protocols. Ed. W. Mark Saltzman, HongShen, and Janet L. Brandsma. 2nd ed. Vol. 127. Totowa: Humana Press, 2006. SpringerProtocols. Web. 21 July 2010.1597451681>. Cengage Learning. N.p., n.d. Web. 27 July 2010.. “DNA Vaccines, HIV/ AIDS.” National Institute of Allergy and Infectious Disease. Department ofHealth and Human Services, 14 Jan. 2010. Web. 15 July 2010.. Lerner, K Lee, and Brenda Wilmoth. “DNA Vaccine.” Gale Encyclopedia of Science. 4th ed.2008. Gale Virtual Reference Library. Web. 14 July 2010.. Mor, Gil, and Mariel Eliza. “DNA Vaccination: Tolerance and Autoimmunity.” Methods inMolecular Medicine 29: 489-497. SpringerProtocols. Web. 14 July 2010. Mumper, Russell J., et al. “Controlled Plasmid Delivery and Gene Expression: Applications forNucleic Acid-Based Vaccines .” Methods in Molecular Medicine 29: 267-286. PDF file.Rice, Jason, Christian H. Ottensmeier, and Freda K. Stevenson. “DNA vaccines: precision toolsfor activating effective immunity against cancer.” Nature Reviews 8 (Feb. 2008): 108-120. PDFfile. Van Drunen, Sylvia, Ralph P. Braun, and Lorne A. Babiuk. “Veterinary DNA Vaccines.” Methodsin Molecular Medicine 29: 79-94. PDF file.
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