日产无码久久久久久精品 -久久久91人妻无码精品蜜桃HD-久久久久精品成人网站-国产97视频人人做人人爱免费

Many gene delivery experiments utilize viral vectors for efficient delivery of genetic information to target cells. To make your recombinant virus, plasmids containing your GOI and the instructions for producing the virus are propagated in bacterial cells, then transfected into packaging cells. These cells produce the live recombinant virus, which is isolated, purified, and concentrated. Like most molecular biology adventures, these many steps and many days can lead to heartache or victory. Viral vector heartache can be due to problems including design, transfection, or gene toxicity. If your particular adventure ends in low quantities of virus, here are some tips that we at VectorBuilder have collected to ensure victory.

Titration station

After isolating your virus from the packaging cells, it is important to determine how much was collected, called the viral titer. This step is imperative for ensuring efficient transduction of your target cells. However, there are many different ways to measure viral titer, and deciding which method to use can be complex and context dependent. A major differentiating factor is whether you need physical titer (the physical amount), functional titer (the level of biological activity), or both. Which method varies depending on the virus used. The table below compares titration methods for popular viruses.

Each titration approach has advantages and disadvantages, especially depending on the space and resources used for titration. Differences in measured titer is a common problem because various methods, models, reagents, and equipment can yield different results. Additionally, measurement after viral degradation can result in low titer.

The titer checklist

When generating viral vectors in the lab, low titer can be attributed to one of the many upstream steps in production. Ensuring correct design of your vectors is imperative: cloning inserts into vectors that are beyond carrying capacity will decrease viral titer, as will using plasmids that contain >70% GC content across a hundred or more bases. AAV’s inverted terminal repeats (ITRs) are GC rich and can have errors introduced during replication, which decreases viral titer. Ensuring ITR integrity is important for maintaining high titers.

Additionally, researchers must ensure that they are using appropriate helper plasmids and packaging cells. For instance, second generation lentiviral transfer plasmids cannot be used with third generation lentiviral packaging plasmids. Next, packaging cells must be transfected with the transfer plasmids and any helper plasmids. If transfection efficiency is low, for instance because the packaging cells exhibit senescence, then viral titer will be low. Finally, harvesting the resulting virus must be optimized: the harvesting time point and conditions (e.g. whether collecting from the supernatant, cell pellet, or both).

Genes that kill

If all steps through transfection have been optimized and viral titer is still low, the problem could lie in what the transfer plasmid is carrying. There are a number of genes which are toxic to packaging cells, e.g. pro-apoptotic genes like Bax and GSDME, cell cycle regulators like BABAM2 and NEK1, proliferation modulators like F2RL1 and Foxn1. When these genes are cloned into transfer plasmids, packaging cells are not able to produce high levels of virus, often due to cytotoxicity (e.g. in McClean et al., 2009 and Janus et al., 2020). Notably, we and others have consistently found low viral titer when using vectors carrying CRISPRa trans activators p65/HSF1.

To achieve a high viral titer when using toxic genes, the most straight-forward approach is to change the promoter to a weaker promoter. In the table below, an AAV vector carrying Foxn1 produced low viral titers when using strong promoters to drive Foxn1 expression, but changing to a weaker promoter resulted in titers comparable to control virus containing non-toxic EGFP.

Additionally, inducible or tissue specific promoters can be utilized to keep transgene expression levels low in packaging cells. If a strong promoter is needed in the transfer plasmid, then packaging cells can be modified to prevent activity of the toxic gene. For instance, if RNAi is being utilized, DICER can be inhibited in packaging cells to prevent shRNA activity, which is a common strategy for packaging vectors containing multiple shRNAs. There are also methods which repress transgene expression specifically in the packaging cells to prevent damage. Finally, a toxic transgene may be broken up across multiple transfer plasmids, as can be done to accommodate larger transgenes when using AAV. This approach requires careful design and would be best utilized if other methods are not possible.

Viral vectors allow for gene delivery to a variety of cell types. However, depending on your application and your target cells, achieving a concentration of virus that is high enough can be difficult. After ensuring your vectors are properly designed and your transfection rate is optimized, there are multiple approaches for increasing viral titer. Adjusting promoters on the transfer plasmid or downstream targets in the packaging cells are common strategies for bypassing the effects of toxic genes. We can help you optimize your experiment from design to packaging using VectorBuilder’s extensive experience at every step, ensuring toxic genes don’t stand in your way.

The table below lays out the genes that we have found to consistently produce low viral titer, which benefit from alternative or inducible promoters.

Sources

Janus P, Toma-Jonik A, Vydra N, Mrowiec K, Korfanty J, Chadalski M, Wid?ak P, Dudek K, Paszek A, Rusin M, Polańska J, Wid?ak W. Pro-death signaling of cytoprotective heat shock factor 1: upregulation of NOXA leading to apoptosis in heat-sensitive cells. Cell Death Differ. 2020 Jul;27(7):2280-2292. doi: 10.1038/s41418-020-0501-8. Epub 2020 Jan 29. PMID: 31996779; PMCID: PMC7308270.

McLean JE, Datan E, Matassov D, Zakeri ZF. Lack of Bax prevents influenza A virus-induced apoptosis and causes diminished viral replication. J Virol. 2009 Aug;83(16):8233-46. doi: 10.1128/JVI.02672-08. Epub 2009 Jun 3. PMID: 19494020; PMCID: PMC2715773.

Sena-Esteves M, Gao G. Titration of Lentivirus Vectors. Cold Spring Harb Protoc. 2018 Apr 2;2018(4). doi: 10.1101/pdb.prot095695. PMID: 29610363.

Tiscornia G, Singer O, Verma IM. Production and purification of lentiviral vectors. Nat Protoc. 2006;1(1):241-5. doi: 10.1038/nprot.2006.37. PMID: 17406239.

Wang F, Cui X, Wang M, Xiao W, Xu R. A reliable and feasible qPCR strategy for titrating AAV vectors. Med Sci Monit Basic Res. 2013 Jul 5;19:187-93. doi: 10.12659/MSMBR.883968. PMID: 23828206; PMCID: PMC3706409.

Weaver LS, Kadan MJ. Evaluation of adenoviral vectors by flow cytometry. Methods. 2000 Jul;21(3):297-312. doi: 10.1006/meth.2000.1010. PMID: 10873484.