Lentiviral vectors are derived from human HIV virus, which belong to retrovirae family. Currently the most commonly used LV vectors are the third generation self-inactivated vector with a cloning capacity of 6-7kb. The LV vectors are pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) to broaden cellular tropism. The lentiviral vectors are not only efficient to transduce non-dividing and dividing cells, but also can give stable expression by inserting the transgene into host chromosomes. In particular, Lentivirus can be used to generate transgenic mice and rats. Lentivirus also has been using for gene knockdown studies.
Lentiviral vector for gene overexpression
The VVC has the third generation self-inactivated lentiviral vectors available for gene overexpression.
The puromycin selection marker gene in this lentiviral vector can be used for selection to improve the transduction efficiency and establish the stable cell lines. The promoters in the lentiviral vectors include CMV, UBC, EF1Î±. The gene can be cloned into our lentiviral vector for ubiquitous expression. The lentiviruses produced by VVC have been successfully used on campus. The VVC tested lenti-EGFP in several cell lines, which showed high transduction efficiency, such as IEC6, MEF and primary VSMCs (See Fig.1,2,3.). The VVC also has a second generation doxcycline inducible lentiviral vector system available (Tet-on). The reverse transactivator rtTA-M2 is driven by a separate promoter, such as EF1a, UBC or Rosa26 in a lentiviral vector. The tet-regulated promote is used to drive the genes of interest. We transduced mouse embryonic stem cells with lenti-tet-on system (Fig.4). Recently the VVC introduced a third generation of rtTA3, which greatly improved the inducibility. We are working on to replacing our second generation system. The lentiviral vector can also be used to efficiently generate transgenic mice and rats (Fig.5).
Lentiviral shRNA mediated gene knockdown
The VVC has a lentiviral shRNA vector for gene knockdown. This vector contains a U6 promoter driving shRNA and a PGK-puromycin cassette for selection. A 21 mer oligo pairs need to be chemically synthesized and the shRNA structures following anneal will be subcloned into the AgeI and EcoRI sites for expression. The shRNA hairpin is driven by polymerase III promoter U6, the selection marker cassette is driven by polymerase II promoter PGK. We have knocked down autotaxin gene expression using lenti-shRNA following puromycin selection in mouse B16 melanoma cells (Fig.6).
Lentiviral miRNA based gene knockdown
The VVC has a miR-30 based lentiviral gene knockdown vector systems (a gift from HHMI), which includes a ubiquitin knockdown and a tetracycline inducible knockdown system. Compared with shRNA mediated knockdown, miRNA expression is driven by polymerase II promoter, which can be used for a tissue specific knockdown or inducible knockdown. It can also be used to trackdown the silenced cells. aThe knockdown library is already available from Openbiosystems for human and mouse genes. The VVC can help to design the miR-30 based gene knockdown lentiviral vector for ubiquitous knockdown, or doxcycline inducible knockdown. MiRNA based gene knockdown has the advantage in tracking knocking down cells, for example, These cells expressing EGFP showed a knockdown of nuclear protein lamin A/C (Fig.7).
Since the lentiviral vector is a human HIV based vector. It is very important to detect the replication competent virus. The VVC has an established assay to detect replication competent virus. We are approved by IBC to use a core antigen p24 ELISA assay to test the replication competent virus from each batch of viruses produced by VVC.Back to Viral Vector Core Laboratory