太鼎生物科技/太鼎食安科技

Lentivirus 純化產品. 其能有效增強病毒力價。

 Both recombinant retrovirus and lentivirus are widely used in the transduction of a varity of target cells and can be produced by transient transfection, from which viral supernatant can be collectedand used for target cell transduction. The titer of viral supernatant by transient transfection is approximately 10⁶ cfu (colony forming unit)/ml, which is sufficient Purification Experimental Flow Chart for the generation of most stable cell lines in vitro. However, there are applications that require higher purity and titers. These applications include experiments demanding higher gene transduction efficiency and in vivo gene delivery. In addition, crude viral supernatants are not suitable for in vivo administration due to various contaminants contained in cell culture supernatant. Thus, the viral supernatant needs to be further concentrated and purified before use.
  Traditionally, both recombinant retrovirus and lentivirus have been concentrated via ultracentrifugation. Although the method works well, it requires specific ultracentrifugation equipment and it is technically demanding. In addition, the total viral supernatant volume is limited to the size of ultracentrifugation tubes. Reports have also stated that the ultracentrifugation process has some detrimental physical effects on the recombinant virus.
  Due to limitations on the existing technique, there is great necessity for a quick and efficient method to purify and concentrate recombinant retrovirus and lentivirus. Scientists at ABM Inc. have had years of experience with Ion-Exchange-based filter membranes and have successfully developed PuRetro™, the most effective retroviral and lentiviral purification method based on this technology.

Advantages of PuRetro™ Lentivirus Purification Kits

·        easy to use

·        very reliable

·        very affordable

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Citations

1.    Buchschacher, G. L., Jr., and Wong-Staal, F. (2000) Development of Lentiviral Vectors for Gene Therapy for Human Diseases. Blood 95, 2499-2504.

2.    Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J.-K. (1993) Vesicular Stomatitis Virus G Pseudotyped Retroviral Vectors: Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells. Proc. Natl. Acad. Sci. USA 90, 8033-8037.

3.    Demmer, W. and Nussbaumer, D. 1999. Large-scale Membrane Adsorbers. J. Chromatogr. A., 852:73-81.

4.    Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., and Naldini, L. (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72, 8463-8471.

5.    Graham, R.L., Smiley, J., Russel, W.C., and Narin, R. 1977. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol., 36:59-72.

6.    Hoganson, D.K., Ma, J.C., Asato, L., Ong, M., Printz, M.A., Huyghe, B.G., Sosnowshi, B.A. and D'Andrea, M.J. 2002. Development of a stable adenoviral vector formulation. Bioprocessing J. 1(1):43-48.

7.    Hutchins, B. 2002. Development of a reference material for characterizing adenovirus vectors. Bioprocessing J., 1(1):25-28.

8.    Lochmuller, H., Jani, A., Huard, J., Prescott, S., Simoneau, M., Massie, B., Karpati, G., and Acsadi, G. (1994). Emergence of Early Region 1-Containing Replication-Competent Adenovirus in Stocks of Replication-Defective Adenovirus Recombinants (Delta E1 + Delta E3) During Multiple Passages in 293 Cells. Hum. Gene Ther. 5, 1485-1491.

9.    Wang, I. I. , and Huang, I. I. (2000). Adenovirus Technology for Gene Manipulation and Functional Studies. Drug Discovery Today 5, 10-16.

10. Wivel, N. A. (1999) Adenoviral Vectors. In The Development of Human Gene Therapy, T. Friedmann, ed.( Cold Spring Harbor , NY : Cold Spring Harbor Laboratory Press), pp. 87-110.

11. Yamada, K., McCarty, D.M., Madden, V.J., and Walsh, C.E. (2003). Lentivirus Vector Purification Using Anion Exchange HPLC Leads to Improved Gene Transfer. Biotechniques 34:1074-1080.

What is the difference between Retro-, Lenti-, and Adeno- viruses?

Retrovirus: Classic, can integrate into the genome but with low transduction efficiency. They are useful for gene transfer and protein expression in cells that have low transfection efficiency with other transfection reagents.

Lentivirus: Can integrate into the genome with relatively high transduction efficiency and they are very useful for cells that have low transfection efficiency with other transfection reagents. No special competent cells required, as they are stable plasmids. Lentiviruses are a powerful tool for stable gene transfer to both dividing and non-dividing cells in vitro and in vivo.

Adenovirus: Only work transiently (about 7 days) but have almost 100% transduction efficiency. Adenoviruses can infect a broad range of cell types with the highest efficiency and infection is not dependent on active host cell division. A second key feature is that high virus titers and high-level gene expression can be obtained in most mammalian cells.

What are the correct concentration units for each recombinant viral particle?

For lentiviruses and retroviruses, they are measured in CFU/ml (colony-forming units per millilitre). Transduction with lentiviruses and retroviruses can cause the formation of colonies, which can be quantified for concentration. Adenoviruses are measured as PFU/ml (plaque-forming units per millilitre). Transduction with adenoviruses will kill packaging cells, forming plaques in the process for quantification. The concentration for all three types of viruses can also be classified as IU/ml (Infectious Units/ml). Ultimately, the units refers to the viral particles and different units reflect the different assays involved.

Both recombinant retrovirus and lentivirus are widely used in the transduction of a varity of target cells and can be produced by transient transfection, from which viral supernatant can be collectedand used for target cell transduction. The titer of viral supernatant by transient transfection is approximately 10⁶ cfu (colony forming unit)/ml, which is sufficient Purification Experimental Flow Chart for the generation of most stable cell lines in vitro. However, there are applications that require higher purity and titers. These applications include experiments demanding higher gene transduction efficiency and in vivo gene delivery. In addition, crude viral supernatants are not suitable for in vivo administration due to various contaminants contained in cell culture supernatant. Thus, the viral supernatant needs to be further concentrated and purified before use.
  Traditionally, both recombinant retrovirus and lentivirus have been concentrated via ultracentrifugation. Although the method works well, it requires specific ultracentrifugation equipment and it is technically demanding. In addition, the total viral supernatant volume is limited to the size of ultracentrifugation tubes. Reports have also stated that the ultracentrifugation process has some detrimental physical effects on the recombinant virus.
  Due to limitations on the existing technique, there is great necessity for a quick and efficient method to purify and concentrate recombinant retrovirus and lentivirus. Scientists at ABM Inc. have had years of experience with Ion-Exchange-based filter membranes and have successfully developed PuRetro™, the most effective retroviral and lentiviral purification method based on this technology.

Advantages of PuRetro™ Lentivirus Purification Kits

·        easy to use

·        very reliable

·        very affordable