shRNA Lentivirus Production using HEK293T cells and
FuGENE
Biohazard Concerns
Lentivirus is a modified HIV virus and although unable to replicate in a host, it must be handled with caution. When working with these viruses, work only in BL2+ designated hoods or viral vector rooms. All handling, storage and disposal of biohazard waste must be in accordance with Institute rules and regulations, OSHA, EPA and MWA.
Notes
All rules of the virus room must be followed. Be sure you receive the proper training on policies and procedures of the virus room before use.
Materials
1.)293T packaging cells (ATCC # CRL-11268)
2.)Plasmid DNA for:
a.)shRNA-pLKO.1 plasmid
b.)pCMV-dR8.91 (Delta 8.9) plasmid containing gag, pol and rev genes
c.)VSV-G expressing envelope plasmid
d.)pLKO.ps control plasmid
3.)FuGENE 6 (Fisher # NC9666789 )
4.)100mm Corning CellBIND tissue culture plates (Fisher # 07-202-516)
5.)DMEM media (ATCC #30-2002)
6.)OptiMem Reduced Serum Media (Invitrogen # 31985-070)
7.)FBS (ATCC #30-2020)
8.)Pen/Strep (ATCC #30-2300)
9.)15mL Centrifuge Tubes (Fisher # 05-538-53F)
10.) 50mL Centrifuge Tubes (Fisher # 07-203-510)
Procedure
1 day before transfection:
Before Starting: Make and warm DMEM media supplemented with 10% FBS. No
antibiotic!
1.) Split 293T cells into twelve 100mm culture dishes at a density of 5x10^6
cells per plate using 10ml of DMEM media supplemented with 10% FBS
per plate.
2.) Be sure that each plate gets a consistent number of cells, and they are
evenly distributed throughout the plate.
3.) Incubate for the next 24 hours undisturbed at 37ºC and 5% C02.
Day of transfection:
Before Starting: Warm OptiMem media in a 37ºC water bath.
1.)To a 15mL centrifuge tube, add:
media
a.) 3096µl
OptiMem
b.) 504µl FuGENE 6
gently by swirling the pipet tip around or tapping with a finger
c.) Mix
(do not pipet or vortex to mix)
d.) Incubate at room temperature for 5 minutes.
2.)Meanwhile, to another 15mL centrifuge tube, add:
a.)7.2µg VsVg (1 tube, frozen at -20C)
b.)36µg of delta 8.9 (1 tube, frozen at -20C)
c.)72µg of DNA expression vector (1 tube, frozen at -20C)
d.)Bring volume up to 3600µl with OptiMem media
e.)Mix gently by swirling with the pipet tip or tapping with a finger
(do not pipet or vortex to mix)
Note: The ratio of Fugene to DNA is 7:1
The ratio of delta 8.9 to VsVg is 5:1
3.)Combine the tubes from steps 1 and 2 and mix gently by flicking to mix
the contents. Total volume will be 7200µl.
splitwise
4.)Incubate the combined mixture at room temperature for 30-45 minutes.
5.)Remove the 12 plates of 293T cells from incubator and bring them into the
hood. Add 600ul of the DNA mix to the dish drop wise and evenly to
each plate.
Note: 293T cells detach very easily from the plate. Take care in adding the
complex mix.
6.)Swirl the plate very gently to mix. Bring the plates into the virus room.
7.)Incubate the cells overnight in the virus room incubator at 37ºC and 5%
C02.
Day 1 after transfection:
Before Starting: Warm DMEM media supplemented with 10% FBS and 1% Pen/Strep in the 37ºC water bath.
1.) Change the media to fresh 10ml DMEM media supplemented with
10%FBS and 1% Pen/Strep.
Note: 293T cells can detach easily from the plate and you do not want to
disturb them, particularly at this point. Add the fresh media carefully. Day 2 after transfection:
Before Starting: Warm DMEM media supplemented with 10% FBS and 1% Pen/Strep in
a 37C water bath.
Note: Place SW-28 centrifuge tubes, SW-28 rotor buckets and caps into the hood to be sterilized by UV light overnight.
1.)Collect viral harvest at ~40 hours after transfection into six 50mL
centrifuge tubes (2 plates of virus into one 50mL tube). Place the 50mL
tubes into the cold room.
2.)Add 10ml of fresh DMEM media supplemented with 10% FBS and 1%
Pen/Strep.
Note: 293T cells can detach easily from the plate and you do not want to
disturb them. Add the fresh media carefully.
Day 3 after transfection:
Note: Look at any GFP control plates under the fluorescent microscope to be sure the cells are expressing GFP. If they are, you can assume the cells have taken in the DNA and are producing virus.
1.) Collect viral harvest at ~60 hours post transfection into the 50mL tubes
used the day before. Each 50mL tube will have approximately 40mL of
virus.
2.) Discard the 293T cells.
3.) Proceed with virus purification and concentration (see protocol
“Lentivirus Concentration and Titer”).
版权声明:本站内容均来自互联网,仅供演示用,请勿用于商业和其他非法用途。如果侵犯了您的权益请与我们联系QQ:729038198,我们将在24小时内删除。
发表评论