Viral Vector Core Laboratory
UTHSC Viral Vector Core Laboratory (VVC) was established with support from a Department of Defense grant awarded in May 2007. Located in the Cancer Research Building on the Memphis campus of UTHSC, the VVC provides services to the Memphis, Knoxville and Chattanooga campuses. Viral Vector Core services include lentiviral, adenoviral and adeno-associated viral vector, as well as traditional retroviral vectors. Vectors services are available to all UTHSC investigators to support their research efforts on a fee-for-service basis.
Director:
Junming Yue, Ph.D.
Assistant Professor of Pathology
Rm 266 Cancer Research Bldg.
19 S. Manassas St.
Memphis, TN 38163
(901) 448-2091
email: jyue@uthsc.edu
The most advanced Lentiviral vectors with capacity to deliver 7 kbp of your gene sequence.
Lentiviral vectors are derived by deletion of the replication genes from human HIV,
a member of the Retroviridae virus family. The deletions make the vectors safe and provide space for gene inserts.
Lentiviral vectors from the Vector Core offer:
- Extraordinarily efficient gene delivery – 100% of cells are infected in most cases.
- Infection of all mammalian species and cell types using the Vesicular Stomatitis Virus glycoprotein (VSV-G) to coat the vectors.
- Gene delivery to non-dividing and dividing cells.
- Regulated, tissue-specific and constitutive overexpression of your gene.
- Immediate stable expression because your genes are inserted into host cell chromosomes within hours after infection.
- Create gene knock-downs using shRNA or miRNA in animals and in cell culture – these can also be regulated or tissue-specific!
- Generate transgenic mice and rats using Lentiviral vectors.
- Third generation self-inactivating (SIN) vectors for increased safety.
The most advanced Adenoviral vectors with capacity to deliver up to 7 kbp of your
gene sequence.
Adenoviral vectors are derived from human adenovirus serotype 5 by deletion of the
early gene E1 and E3 regions. The gene of interest is usually inserted in the E1 region
of adeno-genome.
Adenoviral vectors from the Vector Core offer:
- Extraordinarily efficient gene delivery – 100% of cells are infected in most cases.
- Infection of many types of human and rodent cells and many cell types in vivo.
- Gene delivery to non-dividing and dividing cells.
- Very high levels of gene expression that can last for three to six months.
- Regulated and constitutive overexpression of your gene.
- Create gene knock-downs in cell culture using miRNA – these can also be regulated!
State-of-the-art Adeno-associated vectors with capacity to deliver up to 4.5 kbp of
your gene sequence.
Adeno-associated viral vectors are derived from naturally replication deficient parvoviruses,
which have not been associated with any human diseases. This parent parvovirus cannot
replicate without a helper virus such as adenovirus or herpesvirus.
The Viral Vector Core offers a helper-free production system based on human adeno-associated virus-2 (AAV-2) with the following features:
- Superior biosafety rating- helper free virus system based on AAV-2
- Broad range of infectivity- infect non-dividing and dividing cells
- High titer
- Long-term expression
- Gene overexpression or inducible expression
- shRNA or miRNA based knockdown
Resources
1. Adenoviral vector for gene overexpression
The Vector Core uses the AdenoEasy system from OD260, Inc for our clients. This system
requires insertion of your gene into a shuttle vector under control of the CMV promoter.
Homologous recombination is then used to incorporate the shuttle vector containing
your gene into the adeno-genome in place of the E1 gene region.
In addition to constitutive expression using the CMV promoter, you can also choose inducible expression using a doxycycline-inducible Tet-ON system similar to the one available in our Lentiviral vectors. In the Adenoviral vector Tet-ON, vector core staff eliminated the need for a second vector by replacing the E3 region of the adeno-genome with the the reverse transactivator rtTA. A phage packaging system is used to obtain the recombinant adeno-genome with a 100% recombination rate. We have tested this system using EGFP reporter gene and shown that it gives an almost undetectable basal level in the absence of doxycycline and excellent expression upon induction that increases with the amount of doxycycline used
2. Adenoviral shRNA mediated adenoviral vector knockdown
The Vector Core staff constructed an AdenoEasy based shRNA vector, which can be used for gene knockdown studies. The hairpin structure is driven by the Polymerase III promoter H1.
3. Detection of replication competent adenoviral vector
The Vector Core uses a PCR-based method approved by the UTHSC IBC for detecting replication
competent virus.
Adenoviral Associated Vector
1. Adenoviral-associated vector for gene overexpression and inducible expression
Vector core has a AAV-2 based helper free vector system available. The gene of interest
can be subcloned into a shuttle vector pCMV-MCS or pTet-MCS to package the virus
in AAV-293 cells.
2. Adenoviral-associated vector for gene knockdown using shRNA or miRNA
Available soon: The vector core is building polymerase III based shRNA and polymerase
II based miRNA gene knockdown vectors in the AAV system and will make them available
soon.
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.
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. The Lentiviral vectors can also be used to efficiently generate transgenic mice and rats.
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.
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.
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.
Biosafety evaluation
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.
The UTHSC Viral Vector Core provides the following services to UTHSC faculty members:
1. Design Lentiviral and Adenoviral vectors for overexpression and knockdown of gene expression. Adenoviral Associated vectors designed for overexpression are available now and knockdown versions will be available soon.
2. Production of Lentiviral, Adenoviral and Adeno-associated vectors.
3. Concentration and purification of vectors.
4. Training in viral vector manipulation.
5. Detection of replication competent virus.
6. Distribution of recombinant reporter vectors for EGFP and the reverse transactivator rtTA.
7. Assistance in IBC and IACUC approval for use of viral vectors.
Lawrence Pfeffer, Ph.D
Professor
Department of Pathology
Director of the Center for Cancer Research
Email: lpfeffer@uthsc.edu
Viral Vector Core Laboratory, Director/Manager
Junming Yue, Ph.D.
Assistant Professor
Department of Pathology
Email: jyue@uthsc.edu
Fax: 901-448-3910
Phone: 901-448-2091
Viral Vector Core Scientific Advisory Committee
Dr. Lawrence Pfeffer, Ph.D (Chair)
Professor
Department of Pathology
Director of the Center for Cancer Research
Dr. Kui Li, M.D./Ph.D
Associate Professor
Department of Molecular Sciences
Email: kli1@uthsc.edu
Dr. Tiffany Seagroves, Ph.D
Associate Professor
Department of Pathology
Email: tseagro1@uthsc.edu