Hello Juwon Lee

Generally, lentiviral plasmids cannot be used in direct transfections for transient expression of a transgene like a bacterial plasmid. They are designed to produce and package viral particles that are then used to infect (transduce) target cells, leading to stable, genomic integration of the gene of interest. The lentiviral plasmids contain elements (LTRs, RRE, ψ) required for viral replication and packaging, which are not expressed effectively for transient expression in typical transfection scenarios. Instead, the vector DNA is transfected into packaging cells to produce viral particles, which are then harvested and used to transduce target cells for gene delivery.

The right method of using the lentiviral vector is by following the below steps.

1. Transfect the lentiviral vector plasmid and packaging plasmids into a packaging cell line, such as HEK293T cells.

2. Over a couple of days, the packaging cells will produce viral particles containing the gene of interest, which are then released into the cell culture supernatant.

3. The harvested supernatant, containing the lentiviral particles, is then used to infect or transduce the desired target cells.

4. After entering the target cells, the viral RNA is reverse transcribed into DNA and integrates into the host cell's genome, resulting in stable, long-term expression of the transgene.

LTRs are powerful regulatory regions that control gene expression. Their presence in transfected DNA, especially in lentiviral plasmids, can significantly impact the efficiency and level of transgene expression. Lentiviral vectors rely heavily on LTRs for their natural function in gene integration and expression. When used in a direct transfection for transient expression, these LTRs can severely limit the expression of the transgene because the full lentiviral replication cycle, which typically involves viral production and transduction, is not complete. However, some lentiviral plasmids (mostly third generation) can be used in transient transfections, but the expression will be greatly limited due to the LTRs.

Best,

Thank you, Malcolm Nobre

I appreciate your answer.

Best Regards,

The lentivirus plasmid alone (without a packaging system) will act the same as a regular plasmid from bacteria; they both will stay inside cells with a drug against the selectable marker gene as an episome. In the long term of the drug, the plasmid will integrate into the cell genome with a very low ratio (randomly). Once the drug is removed from the cell culture, the positive cells will be diluted quickly as the cells grow. Regarding the transfection efficiency, usually the lentivirus plasmid is bigger than bacteria-derived plasmid and the transfection efficiency is relative lower, but it depends.

Thank you, Feng Wang

Best Regards,