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Week 1 BN - Coggle Diagram
Week 1 BN
Monday
Viral vectors
General importance
Natural vehicle to introduce geentic information
Small and simple
Capsid protects the virus allowing for stable transport
Use of hostcell protein machinery
Generally safe and non-toxic
Types
Herpes simplex virus
Characteristics
Naturally neurotrophic
can't target non-neurons
Short incubation period --> days until peak of expression
However declines quickly
Very large cloning capacity (~150kb)
High immune response
Infects axons --> retrograde transport
Episomal -> not integrated into host DNA
Double stranded DNA
Use
One of the first recombinant vectors
Retroviruses (eg lentivirus)
Characteristics
Single stranded RNA
carry reverse transcriptase to convert ssRNA into DNA and integrate in host genome
Large cloning capacity (~8kb)
Lasting expression
3 months
Doesn't naturally infect CNS
limited diffusion
Endogenous integration
Can disrupt other genes
Non-integrating versions can be used
Moderate immune response
Use
Mainly used in vitro
due to differing transduction efficiency vs in vivo
Adeno-associated virus (AAV)
Characteristics
Single stranded DNA
Smaller capacity
<4.7kb
Long lasting / permanent
At least >5 months?
Diffuses well
Low immune response
Longer incubation time
self complimentary AAV (scAAV) as alternative
faster incubation
Double stranded -> halved cloning capacity
Quite safe to work with
level 1 in NL
Use
Most used for in vivo
Not suitable for lineage tracing studies and transgene expression in mitotic cells
With each cell division, the episomal DNA is not copied. Remains in only one of the daughter cells.
Structure
Inverted terminal repeats (ITR)
145 nucleotide seq at either end of AAV
Needed for replication
natural viral genome between ITRs
Replaced with transgene
Capsid
AAV packaged into capsid via helper plasmids
Capsid proteins specific to target cell
Process
AAV altered
Packaged into capsid via helper plasmids
capsid proteins bind with target cell
endocytosed
1 more item...
Factors impacting AAV expression
Volume
Titer
Flow rate from syringe
Direction
ie if wanting to avoid a certain area
AAV serotype
primary factor
Route of delivery
Usually stereotaxic surgery (intracranial)
Intra-CSF
Systemic (eg IV)
needs to be able to cross BBB
gene regulatory elements (eg promotor)
Method 1: strong cell-type specific promotors
CamKII most used
Often used to target excitatory neurons but is not actually specific to excitatory neurons
Used in WT animals
Method 2: weak cell-type specific promoters
Used in transgenic models
Use of transgene 1 promotor to selectively express recombinase in target cells (Cre most used)
Transgene 2 in double inversed open reading frame (DIO)
Will only be expressed in recombinase-expressing target cells
Misc
Cloning capacity due to virus size