Viral Vector Transcription Using Cell Lines


intro music Viral vectors, such as the HIV-1 derived lentiviral vectors gives you stable integration of your gene of interest into the host genome. into the host genome. The lentivirus vector plasmid is constructed by flanking the genes to be transferred with viral sequences. to be transferred with viral sequences. transferred with viral sequences. The viral sequences can be recognised by viral proteins in a packaging cell line. The viral sequences can be recognised by viral proteins in a packaging cell line. The packaging cell line makes competent viruses from the helper plasmid DNA that you transfected, making viral particles Cell lines that express viral proteins such as the HEK293T, Cell lines that express viral proteins such as the HEK293T, Cell lines that express viral proteins such as the HEK293T, Chinese Hamster Ovaries (CHO ) and NIH3T3 allow you to create virions allow you to create virions capable of transducing a target cell line. capable of transducing a target cell line. target cell line Reagents are used because the cell surface is negatively charged and the nucleic acids are also negatively charged. negatively charged. This means an inherent repulsion between the two surfaces exist. repulsion between the two surfaces exist. two surfaces exist. Specific reagents such as cationic lipids or cationic polymers enables you to neutralise the charges and to form a complex with the DNA complex with the DNA so that it can enter cells via endocytosis. When you incubate the plasmids with an appropriate cell line such as 293Ts, such as 293Ts, the cells read the plasmid DNA sequence and make an RNA copy of it. the cells read the plasmid DNA sequence and make an RNA copy of it. plasmid DNA sequence and make an RNA copy of it. and make an RNA copy of it. The cells also transcribe & make proteins from the packaging genes to make infectious but non-replicative virions. non-replicative virions. virions. These virions, which are secreted into the media in which the cells are grown, which the cells are grown, grown, the cells are grown, contain RNA copy of the gene that you want transferred into the cell type that you are studying. When the viral supernatant viral supernatant is is introduced and incubated in the target cells – called transduction, called transduction, the virus will reverse transcribe the RNA into DNA. the virus will reverse transcribe the RNA into DNA. It will then insert it into the genome of every cell that it infects. insert it into the genome of every cell that it infects. every cell that it infects. note: not all viral vectors insert into the host genome. some like the adenoassociated vectors adenoassociated vectors are expressed episomally. vectors are expressed episomally. To summarise: Particular cell lines such as 293T allow you to transcribe you to transcribe and translate transcribe and translate viral proteins into virions that contain your gene of interest. viral proteins into virions that contain your gene of interest. viral proteins into virions that contain your gene of interest. viral proteins into virions that contain your gene of interest. viral proteins into virions that contain your gene of interest. viral proteins into virions that contain your gene of interest. viral proteins into virions that contain your gene of interest. viral proteins into virions that contain your gene of interest. viral proteins into virions that contain your gene of interest. viral proteins into virions that contain your gene of interest. you can then introduce the viral supernatant into cells and have the gene be transcribed and translated in those cells. you can then introduce the viral supernatant into cells and have the gene be transcribed and translated in those cells. you can then introduce the viral supernatant into cells and have the gene be transcribed and translated in those cells. you can then introduce the viral supernatant into cells and have the gene be transcribed and translated in those cells. you can then introduce the viral supernatant into cells and have the gene be transcribed and translated in those cells. All the best with your experiments.

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