Why Adeno-associated virus (AAV):
1, AAV infect both nondividing and dividing cells
2, Integrate into a single chromosomal site in the human genome.
3, Low pathogenic

Why use baculovirus-insect cell system to make AAV:
1, Easy to scale up. Some clinical studies require 10^15 rAAV particles, 5000 T175 flasks of 293 cells(10^11 cells) are needed. Only 10~20 liter of insect culture is required to produce 10^15 rAAV particles  if you use baculovirus-AAV insect cell system.
2, Low cost.
3, Efficient.

Baculovirus (BacMam)

• Requires BL1 containment
• Carries a large payload
• Can accommodate two transcription cassettes
• Efficiently infects both neurons and glia in vivo; neuronal or glial
  specificity of expression is achieved by placing the genes to be
  expressed under the control of neuronal or glial promoters
• Expression in vivo is transient, with a time-course that approximately parallels that of HSV
• Virus stock production takes about 2 weeks

Lentivirus

• Requires BL2+ containment
• Carries a restricted payload, up to 5-7 kb depending on the backbone
• Some backbones accommodate two separate transcription cassettes, but
  lentiviral vectors with two transcription cassettes package relatively
  poorly
• Infects both neurons and glia in vivo; neuronal specificity of
  expression is achieved by placing the genes to be expressed under the
  control of neuronal promoters
• Expression in vivo is persistent, and appears earlier (3-5 days) than does expression from AAV vectors
• Virus stock production takes 4-5 days

Adeno-Associated Virus (AAV)

• Requires BL2 containment
• Carries a relatively small payload; maximum is 3.5-4 kb
• With few exceptions, accommodates only one transcription cassette, due to size limitations
• Can be packaged with 11 possible serotypes; serotypes 2, 5, 8, and 9 confer optimal expression in neurons in the brain
• Efficiently and selectively infects neurons in vivo
• Expression in vivo is robust and persistent, beginning at about two
  weeks post-injection and peaking from 4 weeks post-injection onward
• Virus stock production takes about 4 weeks

Herpesvirus (HSV)

• Requires BL2 containment
• Can carry a large payload, theoretically up to 100 kb
• Accommodates two separate transcription cassettes, enabling the expression of more than one transgene
• Efficiently and selectively infects neurons in vivo
• Expression in vivo is robust , beginning at about 12 hours
  post-injection, peaking at days 3-5, and persisting thereafter with no
  diminution of expression
• Virus stock production takes about 3 weeks

Select region

Avoid 5′ and 3′UTRs, but it was reported both 5′ and 3′UTRs could be used to design shRNA against target genes.

siRNA size
Should be <30 nt. 19, 21, or 23 nucleotides are effective although 25~29 nt are considered better.

Special Requirements

The upper sense strand of the target sequence should start with a G (or an A), as RNA polymerase III prefers to initiate transcription with a purine. If a G or A is not present, then it must be inserted immediately upstream of the target sequence. A ‘G’ is added to the sequence at the 5’ end if it is not present. A terminator sequence consisting of 6 dTs is added immediately downstream of the target sequence.

The first nucleotide of the siRNA sequence should not be C or T.

Vector with 7SK promoter, G should be the starting nucleotide.
Vector with H1 promoter,  A should be the starting nucleotide.

GC Content
Ideally the GC content should be less than 50% (between 30% and 55%), although 50 to 60% may be OK.

Repeats
Avoids sequences with repeats of three or more G’s or C’s, as their presence initiates intra-molecular secondary structures preventing effective siRNA silencing hybridization.

Loop Sequence
TCAAGAG,  TTCG and GAAGCTTG (HindIII site).

BLAST against mRNA:
Canditate siRNA presenting more than 13 contiguous nucleotides of sequence identity with another mRNA should be discarded.

Day 1, Seeding 293T Cells

Seed HEK293T cells at 12-15×10^6 cells in a T175 flask with 25 ml complete medium or at a density of 3.5×10^6 cells per 100mm dish in 8 ml complete media. Cells should be seeded approximately 24 hours before transfection and incubated at 37°C, 10% CO2. Make sure cells is about  80-90% confluent at the time of transfection.

Day 2, Transfection
For T175 flask: In a 1.5ml eppendorf tube add:
30 μg pGag-Pol
10 μg pVSV-G
40 μg pGIPz DNA

For 100mm dish:

12 μg pGag-Pol
4 μg pVSV-G
16 μg pGIPz DNA

Add DNA to 5ml Optimem
Add 1μl of 10mM PEI to 5 ml  Optimem
(you may want to filter these solution using a 0.22μm filter).
Mix two solutions and let it sit at room temperature for 20 minutes.
Wash the cells with pre-warmed optimem
Add 10ml Optimem/DNA/PEI mix to the cells.
Culture cells  at 37 oC, 10% CO2 for 4 hours.
Replace the media with 25ml pre-warmed complete media and incubate overnight at 37°C, 10% CO2.

Day 3, Change medium

24 hours after the transfection, replace old medium with 8ml of fresh complete medium.

Day 4, Virus Collection

Harvest lentivirus containing supernatant 24 hours after media changing.
Collect medium into a 10ml syringe and filter through a 0.45μm filter into a 50ml Falcon tube
Replace fresh complete media on the cells if you wish to collect the following day (same volume as on Day 3)
Keep the virus at 4° overnight.

Day 5 – Virus Collection, Concentration and Storage

Collect virus as day 4.

Virus Concentration

Spin the medium containing virus at 100,000 xg at 4°C for 2 hours.

Decant the supernatant and allow the centrifuge tubes to sit upside down on towel to remove as much liquid as possible. Tissue may also be used to wipe around the tube and remove as much supernatant as possible.

Turn the tubes back up the right way and place into 50 ml falcons. Put storage media  (avoid FBS) into the tube without pipetting.

NOTE: Use protein containing medium as the proteins this will stabilize the viral particles and the
surfactant effect of the proteins will help to redissolve the pellet. Filter sterilized PBS or plain medium with 1% BSA is recommended.

Put tubes on ice for 30minutes.

After 30minutes, pipette up and down several times gently and carefully. The virus must be treated very gently, so as not to damage the virus membrane.

Place virus in a microcentrifuge tube and spin for 5 minutes at 4,000 rpm to remove any remaining debris.

Transfer the viral supernatant into a cryovial and store at -80 °C

Reagents
p8.91 (gag-pol)
pMDG (VSV-G)
pGIPZ clones from openbiosystems (grow in low salt LB with 100μg/ml Carbenicillin), make maxiprep using Qiagen Plus kit.

DMEM
Penicillin Streptomycin
FBS Heat inactivated
HEK293T cells (ATCC 293T/17 cell line)
PEI (Polyethylenimine), Polyethylenimine, branched – average Mw ~25,000 by LS, average Mn ~10,000 by GPC, 408727-100ML, Sigma
Phosphate Buffered Saline (PBS)
Trypsin EDTA, T4299, Sigma
TE Tris pH 8.0 10 mM, EDTA 1mM
Optimem with glutamax1, 51985-026, Invitrogen
10 ml syringe syringe disposable 20ml, SZR-150-052C, Fisher
0.22 μM Filter Nalgene Syringe Filters 0.22 μM 513-1901, VWR
0.45 μM Filter Nalgene Syringe Filters 0.45 μm, 513-1902, VWR
Ultracentrifuge tubes Polyallomer Konical 30 ml 358126, Beckman Coulter

10 mM PEI: Add 10ml water to 10 ml PEI and vortex, Add 12N HCl, 1ml at a time and vortex (approximately 10ml HCl in total) then add to 41.2 ml with water and vortex and then store in small aliquots at -80.

Maintain HEK293T cells in complete DMEM-10 (DMEM, 10% FBS, 1% pen/strep). Cells
should be split 3 times a week at a ratio of 1:4 – 1:6 using trypsin EDTA (e.g. Monday, Wednesday and Friday).