Virus transduction troubleshooting

It is important to have an estimation of the concentration of infection-forming units IFUs in the supernatant, as this measurement will determine how much supernatant needs to be used to achieve a desired multiplicity of infection MOI , the number of virions per cell in a transduction.

Non-functional methods, however, tend to overestimate infectious titer because of free p24 protein, viral RNA, and immature or empty particles present in all packaging supernatants.

Therefore, non-functional titers should be used based on previous knowledge of how p24 or genome measurement relates to infectivity. Functional methods include assessment of the number of colony forming units following antibiotic selection, or, if the vector contains a fluorescent protein, flow cytometry analysis of transduced cells.

If the vector does not express a fluorescent protein, determining the number of integrated proviral DNA copies per cell by qPCR is the fastest, easiest, and the best method for assessing functional titer. The exact correlation will vary for each construct, packaging system, and packaging conditions used.

Again, the exact correlation will vary for each construct, packaging system, and packaging conditions used. Lenti-X GoStix also measures p24 protein in the packaging cell supernatants. This rapid test 10 minutes can be used to quickly determine if virus production is within a usable range or for selecting the best time to harvest your virus.

For Lenti-X vectors containing a fluorescent marker , cells can be transduced and the number of fluorescent cells can be determined 24—48 hours after transduction using flow cytometry. For Lenti-X vectors that contain a selectable marker , cells can be infected with serial dilutions of the virus stock, and stable transductants can be selected using the appropriate antibiotic. Titer is calculated from the number of drug-resistant colonies that develop after selection. In general, titers determined using this method are lower than those determined by flow cytometry due to the extra stress placed on cells during drug selection and to loss of transductants during colony expansion.

Measuring provirus, the viral genome after integration into the DNA of target cells, provides the most accurate functional titer. The transduction efficiency of different cell types can vary widely, so measuring virus titer from packaging supernatant often will not provide an accurate estimate of how much virus is needed to transduce one cell type versus another. For example, HT cells can be transduced with lentivirus more easily than Jurkat cells; thus, using the same amount of virus supernatant will result in far fewer integrated proviruses and lower multiplicity of infection M.

In 13 independent packaging reactions performed with 10 different fluorescent protein constructs, the average titer obtained for pLVX vectors packaged using the Lenti-X system was 1. This method is scalable, easier and faster than ultracentrifugation, and requires no specialized equipment.

If the titer is low, additional troubleshooting should be performed to increase titer and increase the likelihood of successful transduction. To start, evaluate upstream factors that may have led to lower packaging efficiency. The presence or absence of different vector sequence elements e. Our pLVX vectors are optimized for both titer and expression, and will produce higher titers than other vectors.

Thus, a relative titre is obtained. If the retrovirus contains a reporter gene, such as lacZ, then infection, integration, and expression of the recombinant virus is measured by histological staining for lacZ expression or by flow cytometry 8, The general thought has been that it is not possible to concentrate retrovirus by centrifugation or over sucrose gradients due to the lability of the retroviral envelope.

Concentration has also thought to have been hindered by the production of "defective" virion particles, containing incomplete genomes or otherwise impaired core structures or envelope proteins that co-purify and compete with active retroviral particles.

Recently, two findings have emerged which challenge the view that retroviral titres are limited by concentration techniques or defective particle formation.

The first involves the biophysical consideration that a particle as large as a retrovirus will not move very far by brownian motion in liquid. Using pure physicodynamic considerations of movement in fluids it was postulated that in fact, the half-life for the time of an average retrovirus to move 3 mm in a theoretical standing solution would be Thus, although more viral supernatant can be added to cells, fluid dynamics would predict that much of that virus never comes in contact with cells to initiate the infection process.

Therefore the effective "infective" volume around cells is quite small, and is rapidly depleted in an environment free of fluidic current flow. Of course, there is no such thing as a theoretical standing solution. One might try rocking the cells in virus supernatent, but experiments in our lab have shown this actually LOWERS the infectivity. However, if cells are grown or placed on a porous filter and retrovirus is allowed to move past cells by gradual gravitometric flow, one can effectively maintain a high concentration of virus around cells at all times.

Experiments in our laboratory confirm this finding, as we observe up to ten-fold higher infectivity by infecting cells on a porous membrane and allowing retrovirus supernatent to slowly flow past them over a period of hours.

This can alleviate the need to buy expensive concentrating devices or devise other more complicated means to generate high-titre virus. Studies show this is in fact the case, and we have confirmed this in our labora tory. Interestingly the experiments by Morling and Russell 63 show that not only is it possible to precipitate the virus in this manner but that the host range of the retrovirus, whether ecotropic or amphotropic, is not altered.

Also, it is possible to chelate away the calcium with EDTA, without affecting retrovirus infectivity, and obtain calcium-free retrovirus concentration by a simple series of centrifu gation-chelation steps Morling et al. By concentrating virus in this manner we observe a remarkable fold increase in effective titres on 3T3 cells. Palsson, B. Methods of increasing rates of infection by directing motion of vectors. April 20, Morling, F. Enhanced transduction efficiency of retroviral vectors coprecipitated with calcium phosphate.

Gene Therapy. We and many others have found that "spinning" the virus onto cells can result in up to a ten-fold increase in effective titer of the virus. Plates are sealed and placed in a microtiter rotor and spun at rpm for up to 45 minutes at room temperature.

Fresh virus can be applied and the cells spun again for 45 minutes. For adherant cells the same protocol is followed, usually in 6-well plates. Obviously, spinning cells at rpm for 45 minutes is not enought to sediment free virus. It is thought that virus on membrane fragments is spun onto cells in a manner which effects greater infection. All we know is that it works and I strongly recommend you try it to increase infection. Additionally, NEB is focused on strengthening alliances that enable new technologies to reach key market sectors, including molecular diagnostics development.

New England Biolabs is a privately held company, headquartered in Ipswich, MA, and has extensive worldwide distribution through a network of exclusive distributors, agents and seven subsidiaries located in Canada, China, France, Germany, Japan, Singapore and the UK. For more information about New England Biolabs visit neb.

The use of viral delivery systems to transduce cells for gene and protein investigations has become prominent over the last 20 years. In particular, the use of lentiviral vectors permits stable expression of your gene of interest. This is all possible with a little bit of nucleic acid magic. The main difference between retrovirus and lentivirus is that retrovirus infects only dividing cells whereas lentivirus infects both dividing and non-dividing cells 1 , thus making it possible to infect post mitotic cells like neurons 2.

So one of the most important aspects of lentiviral vector delivery system experiments is the actual production of lentiviral vectors, which often takes place in HEK cells or some variety.

Before starting any experiment, it is always a good idea to plan ahead and order all the required components. Core facilities in universities may also offer to design and synthesize shRNAs according to your preferences. Always make sure to include a fluorescent marker with the shRNA for easy detection of infected cells. Once the shRNA is obtained, it is a good idea to use midi or maxi-prep, to produce enough for upcoming experiments. It is essential to maintain cells in good condition.

HEK cells are fast growing and trypsinize quickly, so do not allow for HEK cells to incubate for long in trypsin s should do it , or you could witness large cell death. Cells thawed from frozen stock should be passaged at least twice before seeding for the transduction experiment. Stay tuned to the Addgene blog and be sure to check out our viral vector guide pages to get up-to-date tips for your virus experiments.

Anthony, Simon J. Rohwer, Forest. Chen, Sifeng, et al. Krajden, Mel, et al. Ugai, Hideyo, et al. Davis, Howard E. Morgan, and Martin L. Springer Berlin Heidelberg, Murnane, John P. Yezzi, and Barbara R. Add Comment. Addgene is a nonprofit plasmid repository. We archive and distribute high quality plasmids from your colleagues.