Lentiviral Transgenesis

Lentiviral transgenesis can be an alternative technology to the classical method of DNA microinjection.

Lentiviruses belong to the large family of retroviruses.They are characterized by a complex genome and morphology. Lentiviruses have already been isolated from many species, for instance sheep, cattle, goat, monkeys and humans. The best studied lentivirus is the human lentivirus, the HIV (Human Immunodeficiency Virus).

Transgenic animal production by lentiviral vectors most often based on HIV vectors. Our company also uses integrating recombinant HIV vectors during animal transgenesis.

The current lentiviral vector system is composed of an expression vector and packaging constructs, and the vector encoding the envelope protein. The pathogenic genes, as the viral enhancer and promoter sequences within the LTRs (long terminal repeats) are deleted for biosafety considerations. The expression vector harbours the transgene of interest, and the necessary cis-acting element between the LTR sequences. The viral proteins, as gag, pol, tat, rev ^[1] are essential for production of infectious particles. They are expressed in trans by the packaging cell line. The envelope protein (env) ensures the infection of a broad spectrum of target cells.

After entry into the host cell, the viral RNA genome is reverse transcribed into DNA, which is randomly integrated into the host genome (provirus) and serves as a template for the production of progeny virions. Integration into the host genome is important, because it is the basis for persistent infection and transmission of the integrated provirus to offspring after infection of germ cells (i.e. vertical transmission)^[3].

The the lack of viral promoters in the vector system requires other promoters to be incorporated to drive transgene expression in the target cells. Tissue-specific expression can be achieved by incorporating specific promoter sequences.

Lentiviruses can transduce non-dividing cells, because the lentiviral genome is actively transported into the nucleus ^[4]. This feature of lentiviruses makes suitable them for efficient transgenesis.

The embryo micromanipulation with lentiviral vectors is easier, and less damaging for the embryos than DNA pronuclear injection. Lentiviral vectors should be microinjected under the zona pellucida, into the perivitellinar space ^[5].

This method has several advantages over common DNA microinjections. Invasiveness is much lower with higher yield of transgenic animals-40-80% efficiency in rodents. High-level transgene expression can be obtained. Single transgene integration is typical to multiple loci. Lentiviral vectors has some important limitations. A transgene should not exceed 8 kb in size to be carried by a lentiviral vector, while the volume of genetic material is virtually unlimited in the case of DNA microinjection. This method is not efficient in rabbits.^[6]