freeze at -80 in small aliquots and use each tube just once. For short term storage, keep the vector at 4degrees (fridge) for
3 or 4 days
, rather than freezing and thawing, but for longer storage definitely store at -80.
How long does lentivirus survive in culture?
The half-life in culture medium ranged from
8 to 40 h
for the different envelope-pseudotyped vectors, with 35 h for VSV-G-envelope-pseudotyped vector particles.
How long does it take to make lentivirus?
All lentiviral vectors present in the transduction mix need
at least 5 hours
to penetrate the cells of interest. Based on the experiment, the transduction can be left from 5 hours to an overnight incubation.
Principle: Lentiviruses—a subclass of retroviruses—
have the ability to permanently integrate into the genome of the host cell
. After the virus has entered the cell, the viral RNA is transcribed by the reverse transcriptase to produce double-stranded DNA that enters the nucleus.
How long is lentivirus stable at room temperature?
Lentivirus Storage Conditions: Upon collection, the viral media was distributed into 2 mL aliquots and these were stored at the following conditions: (1) room temperature for
less than one hour
, (2) -80oC for 24 hours, (3) 4oC for 3 days and (4) 4oC for 7 days.
If storing for less than a day, lentivirus can be kept at 4°C
. For long-term storage, viral preps should be divided into single-use aliquots, and stored at -80°C. Some reports suggest rapidly freezing virus in a dry ice/ethanol bath or liquid nitrogen prior to storing.
How do you do lentivirus transduction?
When do you change media after transduction?
24 hours after transduction
, remove media containing virus (hazardous waste – transfer to container with bleach) and replace with 2ml fresh media.
Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of
stable cell lines
.
How does lentivirus infect?
Lentiviruses (a genus of retrovirus)
express reverse transcriptase, which converts the viral RNA to double stranded DNA, and integrase, which inserts this viral DNA into the host DNA
. Once the viral DNA is integrated into the host DNA, it divides along with host cell and none are the wiser.
Lentiviral vectors are a versatile tool for gene transfer, because they can efficiently transduce dividing and non-dividing cells and stably integrate their viral DNA into the host genome. So far, the most common way to concentrate lentiviral preparations is to
pellet the viral particles using ultracentrifugation
.
How do you filter lentivirus?
Harvest lentiviral supernatant: Use Lenti packaging kit (cat# TR30037) to make lentiviral particles. Collect the lentiviral supernatant, centrifuge at 500g for 10 min, then
filter through 0.45 μm filter to remove any cell debris
. Note: a. Peak lentivirus production is 48 hours post transfection.
What does a lentivirus vector do?
Lentiviral vectors are a type of retrovirus that can
infect both dividing and nondividing cells
because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell.
Does lentivirus integrate?
Genome-wide studies of viral integration have shown that
lentiviruses most often integrate into actively transcribed genes
, and that this preference is conserved across target species. Although chromatin availability facilitates integration, it does not explain the lentiviral preference for transcribed genes.
What is the difference between stable and transient transfection?
In stable transfection, the plasmid DNA successfully integrates into the cellular genome and will be passed on to future generations of the cell. However, in transient transfection, the transfected material enters the cell but does not get integrated into the cellular genome.
How do you freeze a stock virus?
Keep the virus stock on ice.
Sonicate twice for 30 sec in ice water, with a 30 sec rest on ice between, and divide it into 0.5- to 2-ml aliquots
. Store the aliquots indefinitely at
−
80°C.
Is lentivirus enveloped?
Lentivirus is an enveloped retrovirus
with a single-stranded RNA genome. Current recombinant lentivectors are derived from human immunodeficiency virus (HIV) and other nonhuman lentivirus, such as feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV).
Why are lentiviruses used?
Gene therapy vectors derived from lentiviruses offer many potentially unique advantages over more conventional retroviral gene delivery systems. Principal amongst these is their ability to
provide long-term and stable gene expression and to infect non-dividing cells, such as neurons
.
What temperature are viruses stored at?
We recommend that viruses in wastewater be stored in the dark at
4 degrees C
unless storage for >40 days is necessary.
How do you thaw lentivirus?
How should lentiviral vectors be thawed? Just before use,
remove the lentiviral vectors from the -80°C freezer and thaw the particles according to the volume of your vial
as described below. Once thawed, the vectors should be used for transduction as soon as possible to avoid degradation.
How do you thaw lentivirus supernatant?
Thaw the recombinant retrovirus or lentivirus supernatant
in a 37°C waterbath
and remove it from the bath immediately when thawed.
Why are lentivirus used for transduction?
Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and
unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines
.
How do you find the MOI of lentivirus?
- (total number of cells per vessel or well) x (desired MOI) = total TU needed.
- (total TU needed) / (TU/mL functional titer) = total mL of lentiviral particles to add to each well.
What does a Spinoculation do?
In general, during spinoculation, cells are centrifuged at a low speed (approximately 1,000 to 2,000 × g) for several hours. This leads to
a dramatic enhancement of viral replication of up to several-hundred-fold
.
How long does Stable transfection last?
| Transient Transfection Stable Transfection | Cells are typically harvested within 24–96 hours of transfection. Requires 2–3 weeks of selection for the isolation of stably transfected colonies. |
|---|
How do you make a cell stable?
- Generate a kill curve to determine the optimal selection antibiotic concentration.
- Transfect cells with desired plasmid construct(s)
- Select and expand stable polyclonal colonies.
- Identify single clones by limited dilution and expansion.
