Commonly Known Facts About Viral Packaging -That Might Not Be Correct…

Packaging lentiviruses or retroviruses is not a routine procedure that every biology lab performs even if there is need to use it. A viral packaging protocol normally begins with preparation of purified transfer plasmid DNA, a miniprep should be enough for a few transfections. The virus backbone plasmid is either co-transfected into commonly used cells with helper plasmids that provide the essential proteins required for particle packaging, or transfected into established helper cell lines that express the required proteins from integrated transgenes. After incubation of packaging cells for a couple of days, viruses are collected and tittered. Titer determination is somewhat tricky for the inexperienced. Using a control virus expressing a fluorescent protein can make this step convenient.

Commonly known facts:

1) Lentiviruses are packaged at a titer of 10^6 IU/ml without concentrating steps.

This needs update since with more advanced technologies lentiviruses can be packaged routinely at 10^8 IU/ml. With further concentrating, the titer can be easily above 10^11 IU/ml. Retroviruses can be packaged to similar titers as well.

2) Using packaging cell lines gives the highest possible titer

While packaging cell lines (such as Allele’s popular Phoenix Eco and Ampho cells for retrovirus packaging) provides maybe the most convenient method for packaging, the yield will not reach the highest potential. Packaging cell lines may also lose their capability for packaging after continued culturing, requiring periodic selection with antibiotics and functional tests, as we do here at Allele.

3) Retroviruses are always collected in one shot after transfection into packaging cells

If the transfer vector has oriP/EBNA1 episomal maintenance system, such as some of the Phoenix vectors Allele offers, the plasmids may continue to express for up to 30 days. With puromycin selection, the titer of retrovirus produced from Eco or Ampho cells can reach 10^7 IU/ml.

This week’s promotion (102509-103109): 10% off across the board of Allele Biotech’s custom services, for an example, check out our world-leading baculovirus protein expression.

New Product/Service of the Week: Introduction of Custom Viral Packaging Service. Routine titer of 10^8 IU/ml, as high as 10^10 IU/ml, option to include cloning. Signature service ABP-CS-MERV002 provides more than 200 million particles at $7/million particles. These are game-changing prices for the viral packaging service market based on superior technologies!

Retroviral Vectors with Integrated oriP/EBNA1 for IPSC

The new product of the week of Sep 21-27 is the retrovirus plasmid sets that contain a built-in episomal expression system. As we have discussed previously, OriP/EBNA1 system originated from Epstein-Bar virus, which allows the establishment of stable episomes at 5-20 copies per cell, and duplication once per cell division.

By using the oriP/EBNA1 episomal system, reprogramming cDNAs can be expressed at prolonged time period in reference to plasmid transfection, without integration into chromosomal DNA. A paper published in PLoS One on Sep 18, 2009 by Marchetto et al. showed that by using such a system (on different plasmids) the authors were able to create induced pluripotent stems cells (iPS cells,) effectively from human embryo neural precursor cells.

The Allele pCHAC-EBNA system has dual functions: it can be ready-to-use plasmids for episomal expression of Oct4, Sox2, c-Myc, Klf4, or Nanog and Lin28 by a simple transfection into target cells; it can also be packaged into retroviruses by transfecting into the Allele Phoenix Retrovirus packaging Eco or Ampho cells. This product group is officially launched today. It should become a highly convenient and unique tool for iPSC-related studies.

Difference between hESCs and NSC-Derived Integration-Free iPSCs

By introducing Oct4 and Nanog into human fetal neural progenitor cells, the Muotri lab at UCSD, in collaboration with colleagues from the Yeo and Gage labs of UCSD and Salk, was able to create induced pluripotent stem cells (iPS cells) using the oriP/EBNA1 episomal system to introduce the aforementioned Oct4 and Nanog. While it has been shown by others that with less differentiated cells, such as progenitor cells, it is relatively easy to reprogram using two, or sometimes even one, of the commonly used iPSC generating factors (Oct4, Sox2, Klf4, c-Myc, Nanog, or Lin28) as reported in the AlleleNews article “iPSC generated by using a single reprogramming factor” on August 3rd, 2009, the new publication in PLoS ONE analyzed in detail the differences between embryonic stem cells (ES cells) and iPS cells.



There were three groups of genes that changed significantly between hESC versus iPSC and iPSC versus NSC; genes that are important in early embryonic fate including iPSC-expressed factors that are not sufficiently repressed and genes that are upregulated in iPSCs but are silenced in both NSCs and hESCs which may be downstream to the reprogramming genes during dedifferentiation. Their conclusion is that iPSCs may retain the gene expression signature of donor cells in human reprogrammed cells, even with integration-free gene transfer vehicles such as oriP/EBNA1 element containing plasmids. It should be pointed out that this episomal vector system has been previously reported for iPS cell generation by the Thomson group, see:

(http://allelebiotech.com/blogs/2009/03/episomal-expression-of-ips-inducing-genes-no-trace-of-transgenes-afterwards/). In this paper it was also shown, not unexpectedly, that myc-immortalized cell lines can be efficiently reprogrammed.



Marchetto et al. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007076



Note from Allele: Congratulations to Maria, Gene, Alysson and the Gage lab.