Hi Dina,

Both viral vectors should work fine in a cell line; even regular transfection should give you good results using SH-SY5Y. You could based your decision depending on your ability to buy, produce or use the different virus (biosafety level). People normally use adenoasociated virus (AAV) and/or lentivirus because there are relatively safe (I am not sure about adenovirus). Regarding the production, due to the normal viral cycle, lentivirus can be collected from the cell culture supernantat, which can be directly used for infecting cells in culture or further purified; on the other hand, for AAV the lysis of the host cell is required thus having a more complicated purification protocol.

Of course there are many more differences between these different virus, including the ability to infect replicating and non replicating cells, amount of DNA load, etc. but for your particular case I would say to use what ever virus is easier to obtain or produce in your lab as both should work just fine.

Hope this helps,

Ezequiel

Dear Ezequiel,

Your comments really help us. We can work with lentiviral vector ,however I worried about if it is good system.

Thank you, upon your advice, we will start ASAP.

Dina

Greetings,

In gene therapy, for transfer the gene of interest to the target cells, different viral vectors are used including Flavor Viruses، Adeno-Associated Viruses، Adeno Viruses، Retro Viruses، Herpes Simplex Viruses. Each of these vectors has their own limitation. For example, Herpes Simplex Viruses and Adeno Viruses by inspiring the immune system impede the constant transfer of gene to the host genome. In addition, Adeno-Associated Viruses is dependent to the helper vectors.

Lentiviruses: On the other hands, Lentiviruses which are belonging to the Retoviridea family are of those viruses that are used for the constant transfer of gene to cells. These vectors has the particular capability for gene transfer including the ability of gene transfer with high efficiency to different mammalian cells, the capability of gene transfer to both dividing and non-dividing cells, lack of an unwanted immune response, long-term expression of recombinant genes, integration of gene of interest in the target cells genome and the ability to express high levels of gene in a variety of mammalian cells such as stem cells. This feature makes such vectors to be greatly used in vitro and in vivo research. HEK293T is employed to produce the lentivirus including gene of interest. By transfection the lentiviral vectors containing the gene of interest to HEK293T cells, lentivirus can be produced in vitro and would be collected from the culture media.

A good characteristic of lentiviruses is that unlike retroviruses they can integrate in all cells including dividing and non-dividing cells. This is important especially for the primary neuron cells. When the virus enters the cell, the viral genome which is in the form of RNA is converted to DNA using reverse-transcriptase enzyme and then integrates to the genome by the integrase enzyme haphazardly. This vector which is now called provirus would be existed in the genome and after the cell proliferation and division would be transferred into both daughter cells. The integration site is unpredictable that could be causing the problem. Provirus could disturb the function of cellular genes and cause oncogene to be activated leading ultimately being the origin of cancer which raises concerns in gene therapy by the use of lentiviruses. Nevertheless, as you are not intending to work on gene therapy, you do not deal with such problem. In addition, results of studies showed that the lentiviral vectors have less tendency to integrated to the position that have high potential for carcinogenesis. For the safety, the lentivital vectors have never the genes that are necessary for their transcription ever. In this system, one or two vectors are used for the packaging that encodes the virion proteins such as capsid, integrase and reverse transcriptase. The other plasmid contained the gene of interest which must be transfer by the vector. This plasmid has the ψ (psi) sequence which is necessary for the packaging of the gene in the virion.

Adenovirus: unlike the lentivirus, the adenovirus would not integrate in the genome of the cells and during the cell division would not be transcripted. So, they are limited in the basic sciences research. The primary use of them is in gene therapy and vaccination. Given that human is commonly exposure to adenoviruses which cause the respiratory, gastrointestinal and eye infections, these viruses cause an immune response to human rapidly with potentially dangerous consequences. To overcome this problem, scientists are investigating the human adenovirus that does not have immunogenicity.

Adeno-associated viruses (AAV): AAV is still not known as a cause of any disease in human, and thus the virus causes a mild immune response. This virus can infect both dividing and non-dividing cells and may integrate its genome to the host genome. This feature makes it a good candidate to production of viral vectors for the purpose of gene therapy. Due to high potential of this virus in gene therapy, scientists have designed a modified of this virus called Self-complementary adeno-associated virus (scAAV). AAV is a single-stranded DNA to tinker and need to synthesis the second strand; however, scAAV is synthesizing both strands simultaneously which together form a double-stranded DNA. Considering that scAAV do not have the second stage of DNA synthesis, the expression of the gene in the cell would be rapid. Therefore, scAAV carries many features of its counterpart, AVV.

The nano-engineered materials: In addition to viral vector, nano-engineered materials like Ormosil  has also been used as a DNA vectors and can transfer the high amount of DNA to the target cells (Ormosil  is abbreviated of modified silica which is as organic.

Taken together and considering the precise explanation by Ezequiel, you could use both viral systems. However, I also think employing the lentiviral vectors can be easier as you are not going to work on gene therapy because as Ezequiel said due to normal viral cycle, lentivirus can be collected from the supernatant of HEK293T cell after transfection and could be infected to SH-SY5Y cells, but lysis of host cells in respect to AVV is required and so more complex purification is needed.

Hope these would be useful.

Farhang

Thank you

Hi Dina

Lentiviral vectors (LV) are very efficient on SH-SY5Y cells. At Vectalys, a biotech company specialized in Lv production and cell transduction we have transduced these cells for many projects. The first think you can do is to use a fluorescent protein-expressing LV to monitor your transduction experiment. Clontech sells very efficient ZsGreen1- or mCherry-expressing LV: http://www.clontech.com/FR/Products/Viral_Transduction/Lentiviral_Particles/Fluorescent_Proteins?sitex=10023:22372:US

Find in the attached file the data we obtained with such vectors when transducing SH-SY5Y. Feel free to contact us at: [email protected]

Pascale

http://www.clontech.com/FR/Products/Viral_Transduction/Lentiviral_Particles/Fluorescent_Proteins?sitex=10023:22372:US