Lentivirus Vector System (Lentivurs packaging and expression system)

1. Customized cloning for any other gene ORF expression, shRNA/miRNA and CRISPR/Cas9.
No known immunogenic proteins generated.

2. High titer. 10⁸TU/ml or 10⁹TU/ml lentiviral titer for cell line transfection in medium or large scale.

3. With wide range of host. Mediate efficient transfection in both dividing and non-dividing cells.

4. Integration into host cell genome, mediating long-term and stable expression of exogenous genes.

5. Deliver complex genetic elements, such as intron-containing sequences

6. Easy system for vector manipulation and production

1. For detailed protocol about how to package and purify lentivirus, please see the Lentivirus User Manual.

1. What is the difference between 2nd generation and 3rd generation lentiviral systems?

Answer

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 plasmid such as psPAX2. 3rd generation packaging systems: express gag and pol from one packaging plasmid and rev from another, such aspMDLg/pRRE and pRSV-Rev. 3rd generation packaging systems do not express tat. Third generation lentiviral systems are considered safer than second generation systems, but may be more difficult to use, because they require transfection with four separate plasmids in order to create functional lentiviral particles.

2. What is the difference between lentivirus and retrovirus?

Answer

Lentiviruses are a subtype of retrovirus. From an experimental standpoint, the main difference between lentiviruses and standard retroviruses (γ-retroviruses) is that 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, they are different viruses and thus use slightly different isoforms of these packaging components. Therefore, lentivirus may not be efficiently packaged by retroviral packaging systems, and vice versa.

3. Which bacterial strain should be used for cloning and producing my lentiviral plasmids?

Answer

Due to the long terminal repeats found in lentiviral plasmids, we recommend using a strain that reduces the frequency of homologous recombination of unstable regions, such as Invitrogen Stbl3™ or NEB Stable cells. This will ensure that the repeats will be maintained and often results in a greater yield of DNA. However, if the plasmid contains a Gateway cassette containing the ccdB gene, a ccdB survival strain is necessary.

4. Which cell line should be used to produce lentivirus?

Answer

293T cells are usually used to produce lentivirus.

5. What dictates lentiviral host cell range (tropism)?

Answer

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, see the Cronin, et al. article on different envelopes and their tropism.

6. How can lentivirus be used to make stable cell lines?

Answer

Lentiviruses can be used to make stable cell lines in the same manner as standard retroviruses. That is, many lentiviral genomes have selectable markers, such as the puromycin resistance gene, conferring antibiotic resistance to infected host cells. When these antibiotics are added to the growth medium of the host cells, they kill off any cells that have not incorporated the lentiviral genome and those cells that survive can be expanded to create stable cell lines, which have incorporated the lentiviral genome and harbor the genetic information encoded by that genome. Many lentiviral transfer plasmids do not have selectable markers conferring resistance to an antibiotic, but do encode another marker, such as GFP. A researcher can use FACS to sort cells expressing GFP and later expand these cells into a cell line.

7. Where does lentivirus integrate?

Answer

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. Studies comparing the lentivirus HIV and the retrovirus MMLV indicate that the viral integrase plays a role in shaping integration site preferences. A major cellular determinant is LEDGF/p75, a lentiviral tethering protein that recruits the pre-integration complex to transcriptional units and facilitates integration. LEDGF/p75 binding sites are enriched in gene bodies and mostly absent in promoters and intergenic regions, mirroring patterns of lentiviral integration.

8. Can lentiviral plasmids be used in direct transfections as opposed to making virus?

Answer

Some (but not all) lentiviral transfer plasmids can be used in transient transfections to achieve expression of the transgene, and those that can are primarily third generation constructs. Lentiviral transfer plasmids are not designed specifically for transient transfections. Therefore, there may be limited transgene expression due to the lentiviral LTRs. While possible, it is not explicitly recommended that you use lentiviral transfer plasmids for simple transfections.

9. Is it feasible to express cDNA from a lentiviral plasmid normally used for shRNA expression?

Answer

Yes, it is feasible, but first the promoter within the transfer plasmid must be modified. Most shRNA‐expressing lentiviral plasmids, such as pLKO.1, use a U6 or H1 promoter in order to drive RNA pol III-directed transcription of shRNAs. cDNA expression requires RNA pol II, and thus requires an RNA pol II promoter, such as CMV or RSV.

10. What techniques can be used to clone an insert into a lentiviral plasmid containing only one restriction site?

Answer

If a lentiviral transfer plasmid contains a single 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 and ligating 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 wants 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. 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.

11. What safety concerns surround the use of lentiviral vectors?

Answer

As noted by the NIH, the two main safety concerns surrounding the use of lentiviral are:
1. The potential for generation of replication-competent lentivirus
2. The potential for oncogenesis
The potential for generation of replication-competent lentivirus is addressed by the design of the vectors and by safe laboratory practice. In terms of vector design, 2nd and 3rd 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, 3rd generation systems are considered safer than 2nd generation systems because the packaging vector has been divided into two separate plasmids (resulting in a four plasmid system in total). In addition, 3rd 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.
Many of the lentiviral transfer vectors that have been deposited with Hanbio 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.
Biosafety should always be considered with respect to the precise nature of experiments being performed, and your biosafety office can provide more information on your institution’s best practices with regard to lentiviral research.

12. How can I increase the titer of my lentivirus?

Answer

Here are some suggestions for higher-titer lentivirus production:
1. Because lentivirus contains LTR repeat sequences, recombination can happen after transformation, resulting in a plasmid of reduced size.  We recommend using Invitrogen Stbl3 competent cells for plasmid amplification, which will minimize recombination.  If you did not use these cell lines to amplify your plasmids, you should confirm that the plasmids have maintained the correct size by running a restriction digest.
2. The insert should not exceed the cloning capacity of the expression vector.  Viral packaging becomes less efficient with large inserts, so you can expect a low titer if you clone an insert size that approaches the maximum.
3. Transfection conditions should be optimized to achieve at least 80% transfection efficiency. Less than 80% may result in low titer.
4. Unlike VSVG pseudotyped virus, ecotropic viruses are not very stable, and can’t handle ultracentrifugation or freeze/thaw cycles. If you are making ecotropic virus, it is best to infect target cells immediately upon harvesting the supernatant.
5.We recommend using 293T cells that are healthy and at a low passage number for packaging virus.