Lentiviral Plasmids- Frequently Asked Questions
1- What is the difference between 2nd generation and 3rd generation lentiviral systems?
2- What is the difference between a lentivirus and a retrovirus?
3- What cell line should be used in order to produce lentivirus?
4- What dictates lentiviral host cell range (tropism)?
5- How can lentiviral vectors be used to make stable cell lines?
6- Can lentiviral vectors be used in direct transfections as opposed to making virus?
10- What safety concerns surround the use of lentiviral vectors?
Answers
1- What is the difference between 2nd generation and 3rd generation lentiviral systems?
For a full description of 2nd and 3rd generation lentiviruses please see the page on packaging plasmids. Briefly, 2nd generation lentiviral systems use more HIV proteins (on fewer plasmids) in order to produce functional lentiviral particles than 3rd generation systems.
• 2nd generation packaging systems – express the HIV gag, pol, rev, and tat genes all from a single packaging vector such as psPAX2
• 3rd generation packaging systems – express gag and pol from one packaging vector and rev from another, such as pMDLg/pRRE and pRSV-Rev. 3rd generation packaging systems DO NOT express tat. 3rd generation lentiviral systems are considered safer than second generation systems, but are more difficult to use because they require transfection with four separate vectors in order to create functional lentiviral particles.
IMPORTANT: A 3rd generation transfer vector can be used with a 2nd generation packaging system, but a 2nd generation transfer vector cannot be used with a 3rd generation packaging system.
2- What is the difference between a lentivirus and a retrovirus?
Lentiviruses are a subtype of retrovirus. The main difference between lentiviruses and standard retroviruses from an experimental standpoint is lentiviruses are capable of infecting non-dividing and actively dividing cell types whereas standard retroviruses can only infect mitotically active cell types. This means that lentiviruses can infect a greater variety of cell types than retroviruses.
Both lentiviruses and standard retroviruses use the gag, pol, and env genes for packaging. However, the isoforms of these proteins used by different retroviruses and lentiviruses are different and lentiviral vectors may not be efficiently packaged by retroviral packaging systems and vice versa.
3- What cell line should be used in order to produce lentivirus?
293T cells are usually used to produce lentivirus. The 293T cell line for producing lentiviral particles can be obtained from GenHunter.
4-What dictates lentiviral host cell range (tropism)?
Lentiviral tropism is determined by the ability of the viral envelope protein to interact with receptors at the host cell surface. The VSV-G envelope protein is commonly used in lentiviral particle production because it confers broad tropism over a range of species and cell types. For more information on different envelopes and their tropism see the following article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1368960/
5-How can lentiviral vectors be used to make stable cell lines?
Lentiviral vectors can be used to make stable cell lines in the same manner as standard retroviral vectors. That is, many lentiviral vectors have selectable markers, such as the puromycin resistance gene, conferring resistance to antibiotics. When these antibiotics are added to the growth medium, they kill off any cells that have not incorporated the vector and those cells that survive can be expanded to create stable cell lines which have incorporated the vector and express the insert.
Many lentiviral transfer vectors do not have selectable markers conferring resistance to an antibiotic, but do express some other maker such as GFP. A researcher can use FACS to separate cells expressing fluorescent and other types of markers and later expand these cells into a cell line.
6-Can lentiviral vectors be used in direct transfections as opposed to making virus?
Lentiviral transfer vectors can be used in transient transfections to achieve expression of the transgene. Indeed, transgene expression from the lentiviral vector after transfection during the packaging process sometimes has detrimental effects on viral titre. Lentiviral transfer vectors are not designed specifically for transient transfections, therefore there may be consequences on transgene expression due to the lentiviral LTRs. It is not explicitly recommended that you can use lentiviral transfer vectors for simple transfections, but it is possible.
7- Is it feasible to express cDNA from a lentiviral transfer vector normally used for shRNA expression?
Yes, it is feasible, but first the promoter within the transfer vector must be changed. Most shRNA expressing lentiviral vectors such as pLKO.1 use U6 promoters in order to drive RNA pol III transcription of shRNAs or sometimes the H1 promoter. cDNA expression requires the use of a RNA pol II promoter such as CMV or RSV.
8- What techniques can be used to clone an insert into a lentiviral vector containing only one restriction site?
If a lentiviral vector contains only one restriction site, one can use standard cloning techniques to ligate the insert into this site. If it is not immediately feasible to digest and clone the insert from a parent vector, some possible approaches to using this site include subcloning or appending compatible restriction sites onto the insert of interest using PCR. The process of subcloning consists of digesting the insert of interest from its parent vector into a second vector in such as way that the insert may later be digested from this new vector and cloned into the lentiviral vector (this is basically shuffling restriction sites between vectors until the gene of interest is flanked by sites compatible with those in the vector into which one ultimately want to ligate the insert). Often times it is less time consuming and easier to simply add restriction sites onto the insert of interest using PCR. This is accomplished by PCR amplifying the insert sequence using primers that contain the restriction sites needed. Functional restriction sites must be a certain number of bases from the ends of the primers used. For help designing primers see Lab Life’s plasmid design page:
For more information on designing primers containing restriction sites, see New England Bioblabs’ website:
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp
Alternatively, you could ligate a multiple cloning site (MCS) from a separate vector into the single site in the lentiviral vector and generate more useful restriction sites.
9- How do I clone my insert into a transfer vector with no viable restriction sites but compatible with the Gateway ® cloning system?
Gateway® compatible vectors use recombination in order to generate clones containing the insert of interest. In brief, the insert is first cloned into an entry vector at a region flanked by sequences (called attP1 and attP2) that allow the insert to recombine with the destination vector (in this case the destination vector would be the lentiviral transfer vector). The destination vector contains attB sequences with which the attP sequences recombine. More information on the Gateway® cloning system can be found at invitrogen’s website.
10- What safety concerns surround the use of lentiviral vectors?
The two main safety concerns surrounding the use of lentiviral vectors noted by the NIH include:
1) The potential for generation of replication-competent lentivirus
2) The potential for oncogenesis
These concerns are primarily addressed by the design of the vectors used and by safe laboratory practice. In terms of vector design, second and third generation lentiviral systems provided by Addgene separate transfer, envelope, and packaging components of the virus onto different vectors. The transfer vector encodes the gene of interest and contains the sequences that will incorporate into the host cell genome, but cannot produce functional viral particles without the genes encoded in the envelope and packaging vectors. Unless recombination occurs between the packaging, envelope, and transfer vectors, and the resulting construct is packaged into a viral particle, it is not possible for viruses normally produced from these systems to replicate and produce more virus after the initial infection. In this regard, third generation systems are considered safer than second generation systems because the packaging vector has been divided into two separate plasmids (resulting in a four plasmid system in total). In addition, third generation systems do not use the HIV protein tat in order to produce full length virus from the transfer vector during the viral production stage (See Lentiviral main page[LINK]).
Many of the lentiviral transfer vectors that have been deposited with Addgene are self-inactivating (SIN) vectors. These vectors have a deletion in the 3’LTR of the viral genome that is transferred into the 5’LTR after one round of reverse transcription. This deletion abolishes transcription of the full-length virus after it has incorporated into a host cell.
The potential for oncogenesis is largely based on the specific insert contained within the lentiviral transfer vector (dependent upon whether or not it is an oncogene) and should be considered on a case by case basis.
BL2 is appropriate for most uses of lentiviral vectors, but bio-safety should always be considered with respect to the precise nature of experiments being performed.
The NIH provides more information on lentiviral safety considerations here.