Mouse skin 4 days after inoculation

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Genetic Immunization Workshop
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Traditional Vaccines
Inactivate
Pathogen
Recombinant protein
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Protein
Recombinant protein is difficult and expensive to
produce
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Safety
Cases from natural infections
Number of cases of disease
Cases from vaccines
Years
Vaccine introduced
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Speed
Traditional vaccines can take decades to develop
Protein-based technologies are limited in their
throughput
Genomics
Throughput
Proteomics
Time
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The Two Arms of the Immune System
Protein-based vaccine

• Not effective against HIV, M. tuberculosis,
  Malaria

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Genetic Immunization
Johnston et al. (1992) Nature 356 p152-154
Stimulates BOTH humoral and CTL responses
Safe
DNA is stable and easy to produce
Fast
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!
Warning!
Genetic immunization is a completely different
technology from protein-based methods. DNA cannot
simply be substituted for protein in protocols.
Special considerations
-Different delivery methods
-Special care in the design of constructs to
ensure efficient expression
-Different kinds of adjuvants are required
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Genetic Immunization Plasmid
Antigen gene
Promoter
Terminator
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Codon Optimization
ATGTGGGCCGCCGACTCGGACAGGGAGGACCTGACCATGATGATCGACGA
CCTGGACCGCAGAGGCTTCAGATCCATCTACCGGCTGCTGGCCAGCCGCT
GCAAGCTGGCCGACGCGTTCCAGAATTGGGCGCGCTCCCGGCTCGCCCGC
TACATCATCCTGTCGGCCGCGATCGTCATCTACTACGTGCTCTCGTCGAT
CCTGTCCCTGTCGATCTCGATCTACCGGGCCTCCTCGATGTG
ATGTGCGCTGCTGATTCTGATAGGGAGGATCTTACTATGATGATAGATGA
TCTAGATCGTAGAGGATTTAGATCTATATACCGGCTATTGGCTAGTCGAT
GCAAGCTAGCTGATGCTTTTCAAAATTGGGCGCGCTCTCGGCTCGCTCGC
TATATTATTTTATCGGCTGCTATCGTCATCTACTATGTTCTCTCGTCGAT
ATTATCTCTATCTATTTCTATCTATCGGGCCTCTTCGATGTG
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Gene Synthesis
Pool of Oligos
Assembly (PCR)
etc
Amplify (PCR)
PCR assembly method (Gene v164 p49 1995)
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Gene Synthesis Example
Assembly
PCR
Gene 3
Markers
Gene 1
Gene 2
Gene 3
Gene 1
Gene 2
No natural DNA template required
1500-
1000-
500-
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Linear Expression Elements
Antigen
Control
Antigen
Promoter
Terminator
Terminator
Antigen
Promoter
Sykes Johnston (1999) Nat. Biotech. 17 p355-359
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Assembly of LEEs by Overlap PCR
Antigen
P
T
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DNA Delivery Methods
Needle delivers DNA outside cells and is
subsequently taken up
Gene gun delivers DNA directly into cells
Requires 1mg/shot
Requires 100?g/shot
Cheaper set up
Faster
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Needle Delivery
Muscle sac
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Helios Gene Gun
12 shot cartridge
400 psi optimal for mice
Helium
Gold particles carrying DNA are propelled into
the mouse tissues
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Gene Gun Cross-Section
He
Tube with gold dust inside
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Gene Gun Delivery
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Gene Gun Delivery
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Gene Gun Delivery
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Gene Gun Delivery
Ears are convenient sites to immunize
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Target cells for Immunization
High Throughput Antibody Production
Epidermis
DC
DC
DC
Dendritic cells are potent initiators of the
immune response
Large numbers are found in the skin
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Immune Response
DC
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Gene Gun Bullets

1mm Gold Particle
Nucleic acids
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Bullet Preparation
Nitrogen gas regulator
Syringes to inject slurry into tubing
Ethanol to resuspend gold
Modified 8-channel bullet dryer
Vortex to create gold/ethanol slurry
2ft section of Tefzel tubing (enough for 50
bullets)
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Bullet Preparation
Guillotine slices tubing into 14mm pieces
Tefzel tubing is fed in here
Cut bullets collect here
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Bullet Storage
Parafilm
Stable for at least 6 months if kept in a dry
environment
Bullets
Tissue paper
Drierite
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Workshop -Wednesday
Bullet making
Gene building part 1
LEE construction part 1
create experiments
Gene gun immunizations
Needle immunizations