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1. There are several viral gene delivery systems, including adenovirus, retrovirus and lentivirus. Which one should I use for my experiments?
1) Retrovirus: requires active cell division. In addition, there is a significant risk of integration into the host genome, leading to mutation of genes or activation of onco-genes in the host system, which is a concern for scientists.
2) Lentivirus: can infect both dividing and non-dividing cells. Like retrovirus, there is a significant risk of integration into the host genome, leading to mutation of genes or activation of oncogenes in the host system.
3) Adenovirus: 100% gene delivery efficiency in most cell types including dividing and non-dividing or primary cells. There is no integration with the host system.
4) AAV: has the capacity to produce high titer virus with broad spectrum of tropism in dividing and non-dividing cells and potential for long-term gene transfer with minimum immnunogenicity.
2. What is meant by “replication defective”?
Replication defective means that the parts of the viral genome which are used to replicate have been removed. After removing these genes, there is room for insertion of genetic material. For production of viral vectors that can transduce target cells, the genes which are used to replicate are expressed in trans in packaging cells. So that the mature viruses which are generated from the packaging cells contain the recombinant replication defective genome.
3. What is the difference between ecotropic, amphotropic and pantropic virus?
They infect different kinds of cells. Ecotropic virus can only infect mouse or rat cells, amphotropic can infect mammalian cells and pantropic virus can infect almost all kinds of cells. It is safe to use ecotropic virus because ecotropic virus can not infect human cells.
4. What is a typical titer for retrovirus?
It is 1×107~1×109 TU/ml. But there are many factors can influnence this value, for example the packaging cells, transfection efficiency, insert size and incubation time.
5. Can retrovirus be frozen?
Amphotropic and ecotropic viruses will degrade easily under a freeze/thaw cycle, so they should be used fresh. VSVG-pseudotyped retrovirus can be frozen, but titer will decrease.
6. Should antibiotics be used in the media during the transfection?
It is should be avoided to use any antibiotics to acquire stable cell lines when packaging the virus. Because the antibiotics may be toxic to the host cells during transfection.
7. Do retroviruses target particular locations in the DNA?
It would be wonderful if retroviruses can target precisely in DNA. If it is true, retroviruses can be an excellent tool for gene therapy, including cancer treatment, genetic engineering, anti-viral therapy and research. However, integrase, the enzyme that actually lead the integration of a new DNA sequence into the DNA of the host organism, does not target specific loci.