Is anyone aware of any papers citing the optimum temperature for lentiviral delivery into mice? ie, what temperature should the virus be at?
Dear Nathan,
The following review articles cover the answer to your question. I have copied the protocol from the review for quick view (see bold text).
Nucleic Acid Delivery: Lentiviral and Retroviral Vectors
Eric P O’Keefe (erinisok at gmail dot com)
Princeton University, United States
DOI
http://dx.doi.org/10.13070/mm.en.3.174
Date
last modified : 2015-06-15; original version : 2013-03-22
Cite as
MATER METHODS 2013;3:174
http://www.labome.com/method/Nucleic-Acid-Delivery-Lentiviral-and-Retroviral-Vectors.html
Abstract
A review and guide of lentiviral and retroviral vectors for nucleic acid delivery
In recent years, retroviral and lentiviral vectors have become increasingly vital tools for the delivery of nucleic acids to many cell types in a variety of experimental systems. In addition to being important in laboratory settings for use in both in tissue culture and animal models, they have also been applied in clinical trials to treat genetic diseases. Using lentiviral or retroviral systems allows for the stable, heritable integration of a specific nucleic acid sequence into the target cell’s genome. Utilizing this feature of lentiviral and retroviral vectors allows for the theoretically permanent expression of a gene construct, such as an siRNA or protein coding sequence, in a population of cells.
Protocol Summary
Transfection of packaging cells
The first step in lentiviral vector production is co-transfection of the packaging cells with the transfer, envelope, and gag-pol plasmids (or single transfection of the transfer plasmid in the case of retroviral vector production).
Note: Packaging cells should not be allowed to reach confluence as this reduces their transfection efficiency.
Plasmid DNA quality can also affect transfection efficiency. The use of commercial kits, such as the QIAGEN Endotoxin-free Plasmid Kit, can help if plasmid quality is a problem.
HEK 293T-derived cells are highly transfectable by either calcium phosphate mediated or lipid-based transfection (see Reagents section below). Depending on the transfection protocol used, the cells may need to be washed, followed by the addition of fresh media within 12 to18 hours. At about 24 hours post transfection, the media should be removed and replaced with the media to be applied to the target cells. The packaging cells are now allowed to produce virus for the next 48-72 hours. Since lentiviruses are more stable at 32 °C than 37 °C, the packaging cells can be incubated at this temperature until the virus is collected.
Virus Collection
The highest concentration of virus is typically produced between 48-72 hours. To collect the virus, remove the supernatant from the packaging cells and place it in a 15 mL tube. To remove cellular debris, the supernatant can either be centrifuged at 1500 rpm for 5 minutes or it can be filtered through a 0.45 um filter.
Note: The purified virus can be stored at -80 °C until needed. However, the titer drops by about 50% with each freeze-thaw cycle.
Media can be added back on to the packaging cells and the harvest can be repeated out to 96 hours post transfection if more virus is desired.
Note: If desired, the titer of the virus can be determined by various methods, as discussed later.
Transduction
The virus-containing medium can now be added to the subconfluent target cells. Actively dividing cells will take up the viruses more efficiently; in the case of retroviral vectors, the cells must be dividing or the construct will not reach the nucleus. Depending on your experiment, you may choose a specific multiplicity of infection (MOI) or you can test a range of volumes of the purified virus to determine which gives you the appropriate results.
Lentiviral and Retroviral transduction can be enhanced by the addition of polybrene (SantaCruz sc-134220; Millipore TR-1003-G; Sigma 107689). Also known as hexadimethrine bromide, this cationic polymer is used to increase the efficiency of retrovirus transduction. It is thought to act by neutralizing the virus-cell charged repulsion [32] and enhancing receptor independent adsorption of the viruses [33].
Change the media the following day
Note: The media can be changed in a little as 4 hours if toxicity of the lentiviral particles is a concern.
Note: Reverse transcription and integration of the construct takes place within 24-36 hours.
The transduction process can be repeated if needed. When the virus-containing medium is removed from the packaging cells, fresh media is replaced. This can be collected 24 hours later and used to repeat the transduction step.
Selection of transduced cells
Some transfer vectors also contain a marker such as a fluorescent reporter or drug selection marker.
FACS sorting can be used to enrich for cells which express a fluorescent reporter (such as GFP).
If it contains a drug selectable marker, follow the protocol for the particular drug. For example, puromycin selection is usually carried out at 1-10 ug/mL, depending on the target cells’ sensitivity.
Hoping this will be helpful,
Rafik
We normally bring the virus to room temperature before injecting.
Usually the volume of virus suspension is fairly small, from practical standpoint it may not make difference. All you have to do is to make sure that the virus is not overheated (in your exuberance to thaw it quickly).
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Thermodynamics