A summary of the experimental experience of virus packaging

In doing virus packaging experiments, some researchers often encounter: with the same virus carrier system for virus packaging experiments, why do some people do very well, the second success, some people are often can not do out, stability is very poor, is not low titration or not poison at all,
From our many years of experience in virus packaging of different vector systems, the following aspects are mainly learned:First, several key nodes of virus packaging are cellular factors, carrier systems (using mature commercial carrier systems as far as possible), building recombined granules correctly or not, The purification of protons, packaging transflection control (24, 48 hours of cell and fluorescence status judgment), purpose
gene
on viral packaging (gene size, sequence condition, protein functional toxicity, etc.) will affect the success of packaging.
Second, if there are cells
culture
and the general experience of cell transfeding experiments, cell factors should be no problem (
cell culture
personnel must pay attention to, packaging or transfed infection must pay attention to the cell clean and subtle pollution or not, full stereoscopic sense is good, the spread of uniform cell density moderate no), cell production during the toxic process of vitality is normal packaging and out An important link of virus titration (normal packaging out of the virus situation, ropovirus, retrovirus packaging a cell out of a virus particle, adenovirus is 1000, adeno-related viruses are 10000), so the practice shows that the virus packaging transposing cell density to be controlled, so that the cell density at 90%-95% when the virus packaging transposes.This third, it is recommended to use commercialized mature poison carrier system (not used for road confusion or basically eliminated from the system that few people use), from the literature and our practice for many years can conclude that such a system is stable, so the analysis of the reasons as little effort as possible on the verification of the carrier system, which will be in vain.Fourth, as for the details of the operation control when packaging trans-dyeing and its trans-dyeing
reaction
factors, as long as the operation according to the relevant instructions and operating procedures, there should be no major problems, so do not spend too much energy on this.Fifth, another factor that needs to be focused on and checked is the expression of the destination gene
carrier construction
and the purification of pumping, does it lead to the recombination of the prosurge vector or the loss of a component, or the contamination of other particles or debris? Is the purification of the mid-pumping contaminated? Are other protons mentioned? To develop the habit of doing negative control at the same time (whether the purpose gene carrier and negative control GFP vector is the same batch, recommended the same batch, recommended that each packaging is so operated), if this link is not a problem, in the construction of recombination and loss, or the possibility of purification failure is not great.sixth, implementing those factors above, and that's the last reason. The size of the gene of the purpose, sequence condition, the functional toxicity of the protein of the purpose can not be successfully packaged (in practice, this situation is there, we occasionally encounter, possibly because some gene expression translated to the packaging cell toxicity, resulting in abnormal cell state), then what we can do is to change the virus packaging carrier system, try other carrier systems can be packaged out. But the chances of this happening are very low, and if you make 100 genes, you may not be able to touch one.So in general, we should pay attention to packaging materials - cells and expression pros granules (to take the cells and vector system to verify, often appear packaging cells, empty expression carrier pros granules have problems, we have to regularly purify the production of packaging cells, empty expression vectors), cell culture let it "white fat, energetic", the expression vector of the aim gene to build a good purification, the proportion between the protons can have better results.