Lentiviral vectors for gene overexpression
The Vector Core provides third generation self-inactivating Lentiviral vectors for gene overexpression.
Vector Core staff will insert your gene into a lentiviral vector that suits your needs. Choose from these features:
- Consitutive expression using highly active promoters from CMV (cytomegalovirus), UBC (ubiquitin), and EF1α (elongation factor-1α).
- Regulated expression using advanced doxycycline-inducible promoter (Tet-ON) and reverse transactivator rtTA3 combinations.
- Tissue-specific expression using a promoter active in the tissue of your choice.
- Marker genes EGFP, dsRed, luciferase and puromycin resistance for use in visualizing infected cells and selecting stable cell lines for extraordinarily high levels of expression.
Core staff will also produce Lentiviral vectors from a construct that you made or purchased.
Lentiviral vectors produced by the UTHSC Vector Core have been successfully used on campus. Our Lenti-EGFP vector was tested in several cell lines and gave very high infection efficiency. Figures 1, 2 and 3 show typical infections of intestinal epithelial IEC6 cells, MEF (murine embryonic fibroblasts) and primary VSMC (vascular smooth muscle cells)
Fig. 1: Lenti-E GFP transduced IEC6
Fig. 2: Lenti-EGFP transduced MEF
Fig. 3: Lenti-EGFP transduced VSMC
The Vector Core also offers an advanced doxcycline-inducible Tet-ON promoter system for your gene so that you can turn its expression on or off at times you choose. The reverse transactivator rtTA is driven by a separate promoter of your choice, including the ubiquitous EF1a, UBC or Rosa26 promoter. Choose from the second generation rtTA-M2 if low basal levels of expression are desired or third generation rtTA3 for almost undetectable basal levels and extremely tight regulation.
We have transduced mouse embryonic stem cells with Lenti-Tet-ON-EGF (Fig.4). The Lentiviral vectors can also be used to efficiently generate transgenic mice and rats (Fig.5).
Fig.4: Lentivirus transduced mouse embryonic stem cells using Tet-ON
Fig. 5: Transgenic rats generated using Lentiviral vectors
Lentiviral shRNA and miRNA mediated gene knockdown
The Vector Core offers Lentiviral shRNA and miRNA vectors for gene knockdown in cell culture and in animals.
Fig.6: Lentiviral shRNA vector
The shRNA vector contains a constitutively active U6 promoter driving the shRNA that contains your target gene specific sequence and a puromycin resistance cassette driven by the PGK promoter for selection of cells expressing the shRNA.
We have knocked down autotaxin gene expression using our Lenti-shRNA following puromycin selection in mouse B16 melanoma cells (Fig. 6).
Fig.7: Lamin A/C knockdown using miR-30 based Lentiviral vector.
Cells transduced by the Lenti-EGFP-miR30 lamin vector fluoresce green (A), cells expressing Lamin A/C are stained red (B) and all cell nuclei are stained with DAPI (D). The merged micrograph (C) shows that none of the transduced cells express detectable Lamin A/C.
The miRNA vectors are based on a miR-30 Lentiviral gene knockdown vector system (a gift from HHMI). Expression of your gene is knocked down by insertion into miR-30 of sequences specific to the gene. miRNA vectors offer the versatility of choosing between constitutive, inducible and tissue-specific knockdown of your gene.
We offer constitutive knockdown using the UBC ubiquitin promoter, a regulated knockdown using the doxycyxline-inducible Tet-ON system, or a tissue-specific knockdown using a promoter active only in the tissue of your choice.
Have the Vector Core design your miRNA-30 based gene knockdown vector. Or purchase an existing gene-specific miRNA-30 Lentiviral vector from the human and mouse gene knockdown library offered by OpenBiosystems.
In adition to the versatility of constitutive, inducible and tissue-specific knockdown, miRNA based gene knockdown has the advantage of tracking cells with knocked down expression. Figure 7 shows knockdown of nuclear protein Lamin A/C.
Since Lentiviral vectors are derived from pieces of HIV, it is very important to test the vector productions for the presence of molecular recombinants that may have reconstituted replication competent lentivirus (RCL). The Vector Core has established a highly sensitve core antigen p24 ELISA assay for detection of RCL. Every production of Lentiviral Vectors is tested for RCL using this assay and we guarantee delivery to you of Lentiviral vectors that are free of RCL.
Steven R. Goodman, Ph.D.
Office of Research
910 Madison, Suite 608
Memphis, TN 38163
Phone: (901) 448-7125
Fax: (901) 448-7133