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FAQs for lentivirus

1. What is lentivirus plasmids package system?
2. What is the difference between second generation and third generation lentivirus plasmids package system?
3. Is handling the lentivirus package plasmids system safe? What are the safety concerns in case of the use of lentiviral plasmids?
4. What are the differences between recombinant adenovirus and recombinant lentivirus?
5. Is there any unfeasible host to be infected for lentiviral gene transduction? What determine the lentiviral host cell range (lentiviral tropism)?
6. What are the advantages to use lentivirus over adenovirus?
7. We want to examine effects of transient expression of protein X in human primary cells. But plasmid transfection does not work well. Should we use adeno or lentivirus?
8. We want to create cell-line by lentivirus. How should we do?

9. What points do I need care about viral plasmid amplification?


Answers:

1. What is lentivirus plasmids package system?

A lentivirus plasmids package system is designed to reduce the chance of recombination between helper and gene transfer vector sequences by using the constitutive transport element (CTE) derived from Mason-Pfizer monkey virus for expression of the viral proteins and the Rev-Rev response element (RRE) combination for expression of the gene transfer vector. Basically, current system contains transfer plasmid, packaging plasmid (helper plasmids: gag, pol, rev and tat) and envelope plasmid (VSV-G) for assembling lentivirus particles for gene delivery purpose.

2. What is the difference between second generation and third generation lentivirus plasmids package system?

A number of years, researchers investigated HIV gene products to determine minimal requirements (Miyoshi et. al. J. Virology 72:8150, 1998) which became the first generation. The second generation was made to split these essential gene to separate expression vector in addition to replacement of envelop from original to vesicular stomatitis  virus (Burns et. al. Proc. Natl. Acad. Sci. USA 90:8033, 1993). In the third generation vector, original U3 region of LTR was modified to make virus self-inactive (SIN vector: infected cells do not express viral structural proteins). Third generation of lentivirus vector expresses only internal promoter driven gene of interest. On the other hand, the second generation vector has an original LTR promoter activity which expresses some viral structural proteins along with inserted gene of interest. To package virus, the second-generation vector requires tat protein expression vector (Dull et. al. J Virology 72:8463, 1998). Clontech claims their vector is “fourth generation” in the advertisement. Although it employs a sophisticated packaging system to reduce the recombination risk, the vector itself has an original LTR, thus it is categorized into the 2nd. generation.

3. Is handling the lentivirus package plasmids system safe? What are the safety concerns in case of the use of lentiviral plasmids?

There are two main safety concerns for handling lentiviral plasmids noted by NIH.  

A. The potential replication-competent lentivirus 

B. The potential oncogenesis

Lentiviral gene delivery is due to the ability of lentivirus to efficiently enter non-dividing or quiescent cells. Attempts are being made in many laboratories to optimize the packaging and plasmid constructs to ensure safety without compromising vector titer, so that lentiviral plasmids can eventually be used in a clinical setting.

The third generation lentiviral systems provided by ATCGbio Life Technology separates the viral transfer, envelope, and packaging components onto different plasmid. The transfer (lentivirus) plasmid 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 plasmids. Unless recombination occurs between the packaging, envelope, and transfer plasmids, 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 viruses after the initial infection. In term of this, third generation system is considered safer than second generation system because the packaging vector has been divided into two separate plasmids resulting in a four plasmid system in total. In addition, third generation system does not use the HIV protein tat in order to produce full length virus from the transfer vector during the viral production stage.

The lentivirus plasmids provided by ATCGbio Life technology 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 in the website at http://oba.od.nih.gov/rdna_rac/rac_guidance_lentivirus.html

4. What are the differences between recombinant adenovirus and recombinant lentivirus?

Adenovirus

 

Lentivirus

DNA virus      

 

RNA virus

36 kb in size (parental type 5)

 

9 kb in size (parental HIV I)

Non-integration

 

Integrate in host genome

No envelop

 

VSVG envelop

Receptor mediated infection

 

Non-receptor mediated infection

Fast expression (within 12 hr)          

 

Slow expression (2-3 days)

Can be amplified in HEK293 cells

 

No amplification. Package in HEK293T cells

Stable in the culture medium

 

Unstable in the culture medium

Multiple infection (high expression)

 

Single or a few virus infection per cell

High titer solution can be prepared

 

Relatively low titer

about 2 weeks to make

 

2 days to make

No selection possible

 

Antibiotics selection possible

Biosafety level-2 regulation

 

Biosafety level-2 (or 2+) regulation

 

5. Is there any unfeasible host to be infected for lentiviral gene transduction? What determine the lentiviral host cell range (lentiviral 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 a 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/

6. What are the advantages to use lentivirus over adenovirus?

1). It can be made in short time. Using our lentiviral vector plasmid (pLBB-SLIC), plasmid can be prepared in 2-3 days and virus can be made another 2 days. So, target gene expression experiment can be performed within 1 week. 

  
2). Expression level is more controllable. Adenovirus infection typically results in bare or too much expression of target proteins. Sometimes, multiple viruses (e.g. 10-20 viruses) can infect just one single cell that causes too much protein expression overwhelms the cellular system. This effect may be unphysiological and causes different results depending on protein expression level. Contrary to adenovirus, lentivirus infection is much modest, typically one or a few virus per cell. Thus, interpretation of the results is much easier. 


3). No virus protein is expressed. In the third generation of lentivirus vector, original virus promoter located in LTR is inactivated (SIN vector). Therefore, only internal promoter driven protein is expressed. On the other hand, recombinant adenovirus vector expresses a number of virus proteins, thus, control virus set-up is essential to interpret the result. 


4). Virus is Integrated into the host genome. With antibiotics selection, it allows to make cell-line easy. To establish stable cell line, cloning is still required. 

 

5). Host range is wide. With VSVG envelop, the virus can be infected in many types of the cells. In our hand, only the cell type failed to work is adult cardiomyocytes.       

 

7. We want to examine effects of transient expression of protein X in human primary cells. But plasmid transfection does not work well. Should we use adeno or lentivirus? 

Both viruses can do this job. However, the protocol needs to adjust based on the nature of each virus. If recombinant adenovirus to express protein X is available, we provide purification kit suitable for such experiment including determining titer. With proper titer determination, the target cells should be infected at high confluence, and the effects can be observed within 1-2 days. 

Lentivirus vector is another choice which can be created quickly. With our lentiviral vector plasmid pLBB-SLIC and its kit, users can make lentivirus construct by one-step PCR in a single day. Although the vector does not have antibiotic selection, for transient expression it is not problem. Our lentivirus production kit offers high titer virus suitable for human cells. Target primary  cells should be infected at low density (20-30%) and experiments is performed in 2-4 days later. Since virus is integrated in host genome, even after cell division, cells still can express protein X. 

8. We want to create cell-line by lentivirus. How should we do? 

Since lentivirus integrates host-genome, it is easy to make cell-line by antibiotic selection. Caution must be taken that to establish stable cell-line, cloning (typically done by limited dilution) still is necessary.  Otherwise, phenotype may change by passaging. This is due the fact that virus integrates randomly to host genome, so there is cell to cell variation. 

Keep above caution in mind, antibiotics selection can be done by the following way. Infect target cells at 20-30% confluence in 12 or 6 wells, wait for 3 days till antibiotics gene is expressed. Incubate with antibiotics (1/2 dose: 5 µg/ml for blasticidin or hygromycin) overnight and re-plate the cells to 2 or 3 wells. Increase antibiotics to 1 dose (10 µg/ml for blasticidin or hygromycin). Once the cells become confluent, re-plate it to 100 mm dish. Split the cells and do the experiment. At this point, all the cells are already antibiotics resistant. 

9. What points do I need care about viral plasmid amplification?

Virus plasmids are very prone to mutation in E.Coli resulting a short size products, typically, about 3-4 Kb plasmids. But, it is very easy to avoid such problem if you practice the following suggestions.

1). Decrease the incubation temperature (both on agar plate and in LB medium) to 32-34 °C. This is the most effective way to avoid mutation. Plasmids still can grow almost normally under this condition, so there is no trade-off.

2). Use virus-stable E.Coli such as Stbl3®. Avoid DH5alpha. Most common mistake is using DH5alpha E.Coli to amplify lentivirus plasmid under 37 °C. If users do not have such E.Coli, we recommend to use our VP-Easy kit (VP1001) which provide reagents making  competent E.coli. suitable for viral plasmid amplification.

3). Amplify in higher volume of LB medium than purification kit usually recommended. Typically, we suggested that using 250 ml LB for midi-scale column purification kit instead of the recommended culture volume 40-100 ml; and using 10 ml but not 4 ml of suspension, lysis and neutralization solution. The washing and elution volume are kept the same as original described in the protocol of midi-scale plasmid purification kit. By doing this, plasmid concentration in isopropanol step is high enough to be effectively precipitated.

4). Keep plasmid in TE buffer at 4 °C. With airtight tube, the plasmid is stable for years.

5). Do not vortex the plasmid solution vigorously in order to avoid mechanical damage. 


 



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