Lentivirus – Services and Fees
The VCF provides high- and low-titer HIV lentivirus preparation of stock viruses for fluorescent protein expression, cDNA expression, localization of subcellular expression, and for shRNA expression from a library for all human and mouse genes. Further, the VCF will help investigators in the design and development of lentiviruses for cDNA expression that may be used for cell-based studies and transgenic mouse production, cell transduction, and characterization (development of stable cell lines and analysis by qRT-PCR) as well as lentiviral titering and testing for replication competence.
Lentiviral Expression of Fluorescent Proteins
The VCF now offers a suite of lentiviral constructs for expression of fluorescent proteins to allow users to identify transduced cells both in vitro and in vivo:
| Vector | Excitation |
Emission |
|---|---|---|
| pLKO-CMV-TurboGFP Lentiviral vector expressing a Green Fluorescent Protein. Also, has a puromycin selectable marker. |
482 | 502 |
| pLVX-IRES-ZsGreen1 Lentiviral vector expressing a Green Fluorescent Protein. Also, has a puromycin selectable marker. |
493 | 505 |
| pLKO.1-puro-CMV-TagYFP Lentiviral vector expressing a Yellow Fluorescent Protein. Also, has a puromycin selectable marker. |
508 | 524 |
| pLKO.1-puro-CMV-TagCFP Lentiviral vector expressing a Blue Fluorescent Protein. Also, has a puromycin selectable marker. |
458 | 480 |
| pLKO.1-puro-UbC-TagFP635 Lentiviral vector expressing a Dark Red Fluorescent Protein. Also, has a puromycin selectable marker. |
588 | 635 |
| pLKO.1-puro-CMV-TagRFP Lentiviral vector expressing a Orange-Red Fluorescent Protein. Also, has a puromycin selectable marker. |
555 | 584 |
| pLKO.1-puro-UbC-TurboGFP Lentiviral vector expressing a Green Fluorescent Protein. Also, has a puromycin selectable marker. |
482 | 502 |
| pLKO.1-puro-CMV-TagFP635 Lentiviral vector expressing a Dark Red Fluorescent Protein. Also, has a puromycin selectable marker. |
588 | 535 |
| pLVX-Tight-Puro-Luc Control Lentiviral vector for Tetracycline-regulated expression of luciferase and a separate expression cassette for puromycin resistance. |
n/a | n/a |
| pLVX-AcGFP1-C1 Lentiviral vector expressing a Green Fluorescent Protein. Also, has a puromycin selectable marker. |
475 | 505 |
| pLVX-AcGFP1-N1 Lentiviral vector expressing a Green Fluorescent Protein. Also, has a puromycin selectable marker. |
475 | 505 |
| pLVX-DsRed-Monomer-C1 Lentiviral vector expressing a Red Fluorescent Protein. Also, has a puromycin selectable marker. |
556 | 586 |
| pLVX-DsRed-Monomer-N1 Lentiviral vector expressing a Red Fluorescent Protein. Also, has a puromycin selectable marker. |
556 | 586 |
| pCYTO-CMV-GFP-MCS-EF1-puro Lentiviral vector expressing copGFP as a cytosolic marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pCYTO-CMV-MCS-GFP-EF1-puro Lentiviral vector expressing copGFP as a cytosolic marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pCT-Tublin-GFP Lentiviral vector expressing a copGFP/alpha-tubulin fusion protein as a cytoskeleton marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pCT-Tublin-RFP Lentiviral vector expressing an mRuby/alpha-tubulin fusion protein as a cytoskeleton marker. Also, has a puromycin selectable marker. |
556 | 586 |
| pCT-Autophagosome-GFP Lentiviral vector expressing an copGFP/LC3 fusion protein as an autophagasome marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pCT-Autophagosome-RFP Lentiviral vector expressing an mRuby/LC3 fusion protein as an autophagasome marker. Also, has a puromycin selectable marker. |
556 | 586 |
| pCT-Lyso-GFP Lentiviral vector expressing an copGFP/CathepsinB fusion protein as a Lysosome marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pCT-Secretory-GFP Lentiviral vector expressing a copGFP/Secretory consensus peptide fusion protein as an ER/Golgi apparatus marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pCT-Golgi-GFP Lentiviral vector expressing a copGFP/Galactosyltransferase fusion protein as a Golgi apparatus marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pCT-ER-GFP Lentiviral vector expressing a copGFP/ER targeting sequence fusion protein as an Endoplasmic Reticulum marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pCT-Mito-GFP Lentiviral vector expressing a copGFP/CytochromeC Oxidase VIII fusion protein as a Mitochondria marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pCT-H2B-GFP Lentiviral vector expressing a copGFP/H2B fusion protein as a Nuclear marker. Also, has a puromycin selectable marker. |
475 | 505 |
| pLVX-iRFP-IRES-puro Lentiviral vector expressing iRFP. Also, has a puromycin selectable marker. |
690 | 713 |
Related Fees
Lentivirus Prep Requested |
Lentivirus Prep Requested |
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|---|---|---|---|---|---|---|
| Small-scale (4 ml) | Medium-scale (25 ml) | Large-scale (75 ml) | Small-scale (100 μl) |
Large-scale (500 μl) |
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| Lentivirus vectors expressing fluorescent proteins, luciferase, fusion proteins, or DNA provided by investigator | $200 | $500 | $1,200 | $700 | $3,200 | |
Lentiviral Expression of shRNA-Mediated RNAi
The UPCI VCF provides low and high-titer lentivirus mainly from the library of the RNAi Consortium, a shRNA library consisting of 150,000 pre-cloned shRNA constructs targeting 15,000 annotated human genes and 15,000 annotated mouse genes. Alternatively, virus is generated from vectors prepared by the VCF specifically for the user, prepared by the user, or obtained from additional commercial and academic resources. The VCF will continue to optimize the utilization of whole genome or function-specific shRNA virus pools to aid in the discovery of unbiased gene function. Once optimized, the VCF will offer new gene-discovery resources through a shared cost-collaborative basis.
- Preparation of lentiviral particles expressing shRNA or cDNA
The Mission shRNA library and RNAi Consortium is the primary source of the shRNA-expressing lentivirus vectors. This vector system is based on the pLKO.puro series of lentiviral expression vectors. A minimum of five different shRNA-expression plasmids will be generated for each gene. This is a standard approach to achieve efficient knockdown as each shRNA may have variations in ability to induce knockdown of gene expression. To package virus, the gene-specific shRNA and cDNA expression plasmids are transfected into 293-FT cells together with the packaging plasmids pMD2.g(VSVG), pRSV-REV, pMDLg/pRRE. Forty-eight hours post transfection, viral particles are collected in the culture supernatant, filtered (0.45 μM) and stored at -80°C or used immediately to transduce the target cells. Viral stocks are prepared at low titer (106 particles per ml) or high-titer (5 x 109 particles per ml), as requested. - Expression knockdown or cDNA quantification by quantitative real-time RT-PCR
The extent of expression knockdown can be determined for the investigator by the VCF by real-time quantitative PCR, using procedures we have optimized in the lab. Briefly, RNA is isolated and analyzed using the Applied Biosystems Taqman® Gene Expression Cells-to-CT™ Kit. Each sample is analyzed in triplicate and the results provided are an average of all three analyses. Analysis of mRNA expression is conducted using the ΔΔCT method using validated Applied Biosystems TaqMan® Gene Expression Assays.
Related Fees
| Service | Description | Fee |
|---|---|---|
| Lentiviral shRNA vector set | 5 unique shRNA lentivectors specific to your gene (4 x 1 ml) + GFP-expression control lentivirus (4 x 1 ml). Target shRNA available: shRNA specific to all mouse and human genes. | $850 |
| Service | Description | Lentivirus Prep Requested |
Lentivirus Prep Requested |
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|---|---|---|---|---|---|---|---|
| Small (4ml) | Medium (25 ml) | Large (75 ml) | Small (100 μl) |
Large (500 μl) |
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| Preparation of shRNA-expression lentivirus | Preparation of a single viral prep after identification of effective shRNA vector analysis | $200 | $500 | $1,200 | $700 | $3,200 | |
| Preparation of shRNA-expression lentivirus using 6 vector system compatible with GIPZ and TRIPZ. | Preparation of a single viral prep after identification of effective shRNA vector analysis or if vector provided by investigator | $300 | $600 | $1,400 | $900 | $3,500 | |
| DNA required (μg) | 10 | 40 | 120 | 40 | 120 | ||
Cell Line Development Services
| Service | Description | Fee |
|---|---|---|
| Lentiviral shRNA complete package | Preparation of 4 ml virus from lentiviral shRNA vector set, cell line transduction and characterization of stable cells by qRT-PCR (3x) (requires separate cell line optimization for puromycin resistance). Cells must be Mycoplasma-free. | $6,000 |
| Cell transduction and selection (puromycin or GFP) | Cell line provided by investigator is transduced using optimized protocols and selected by puromycin or GFP (sorting). Puromycin dose provided by investigator or tested, as below. Cells must be Mycoplasma-free. Fee is for transduction and selection of the lentivirus. (Fluorescent protein, cDNA expression, or shRNA vector set; virus not included - must be ordered separately). | $4,000 |
| Characterization of selected cells by qRT-PCR | Cell pellet is provided by the investigator and analyzed by qRT-PCR with actin as the normalizing control gene. Investigator provides cell pellets after knockdown, including GFP or non-transduced control. Cell pellets are prepared as follows: 800,000 cells pelleted, washed 2x in PBS, and stored at -80°C. One run conducted, in triplicate. | $850 |
| Cell line optimization for puromycin resistance | Cell line provided by investigator is optimized for puromycin dose using empty vector control. Cells must be Mycoplasma-free. | $1,400 |
cDNA Expression
Lentiviral vectors for cDNA expression are provided to investigators for $240 (5 μg). Lentiviral production may be ordered separately.
| Name of available lenti-vectors | Description |
|---|---|
| pLVX-puro | Expression of cDNA and separate expression cassette for puromycin resistance |
| pLVX-AcGFP1-C1 | Expression of cDNA as a C-terminal fusion with AcGFP |
| pLVX-AcGFP1-N1 | Expression of cDNA as an N-terminal fusion with AcGFP |
| pLVX-DsRed-Monomer-C1 | Expression of cDNA as a C-terminal fusion with DsRed |
| pLVX-DsRed-Monomer-N1 | Expression of cDNA as an N-terminal fusion with DsRed |
| pCYTO-CMV-GFP-MCS-EF1-puro | Expression of cDNA as a C-terminal fusion with EGFP (EGFP on the N terminus) |
| pCYTO-CMV-MCS-GFP-EF1-puro | Expression of cDNA as an N-terminal fusion with EGFP (EGFP on the C terminus) |
| pLVX-IRES-Hyg | Expression of cDNA and hygromycin resistance linked by an IRES |
| pLVX-IRES-Neo | Expression of cDNA and neomycin resistance linked by an IRES |
| pLVX-IRES-Puro | Expression of cDNA and puromycin resistance linked by an IRES |
| pLVX-IRES-ZsGreen1 | Expression of cDNA and ZsGreen1 linked by an IRES |
| pLVX-Tight-Puro | Tetracycline-regulated expression of cDNA and separate expression cassette for puromycin resistance |
Lentivirus for Development of Induced Pluripotent Stem Cells
| Service | Description | Lentivirus Prep Requested titer ~ 106 particles/m |
Lentivirus Prep Requested titer ~ 109 particles/m |
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|---|---|---|---|---|---|---|---|
| Small (4ml) | Medium (25 ml) | Large (75 ml) | Small (100 μl) |
Large (500 μl) |
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| Lentivirus preparation for iPS cell creation | Choose from pFUW-OSKM or tet-inducible pTetO-FUW-OSKM vectors | $200 | $500 | $1,200 | $700 | $3,200 | |
Pre-Made Cell Lines
Due to the nature of these cell lines, you agree that the cell lines ordered will NOT be shared with anyone else in or outside of the University of Pittsburgh Cancer Institute. These cell lines will be kept within your lab for testing and research purposes only.
| Cell Line | Description | Sample |
|---|---|---|
| MDA-MB-231/TagYFP | MDA-MB-231 cells expressing a Yellow Fluorescent Protein (Excitation: 508nm, Emission: 524nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/TagCFP | MDA-MB-231 cells expressing a Blue Fluorescent Protein (Excitation: 458nm, Emission: 480nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/TagRFP | MDA-MB-231 cells expressing an Orange-Red Fluorescent Protein (Excitation: 555nm, Emission: 584nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/TagFP635 | MDA-MB-231 cells expressing a Dark Red Fluorescent Protein (Excitation: 588nm, Emission: 535nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/Cyto-GFP | MDA-MB-231 cells expressing copGFP as a cytosolic marker (Excitation: 482nm, Emission: 502nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/Tubulin-GFP | MDA-MB-231 cells expressing a copGFP/alpha-tublin fusion protein as a cytoskeleton marker (Excitation: 482nm, Emission: 502nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/LC3-GFP | MDA-MB-231 cells expressing a copGFP/LC3 fusion protein as an autophagasome marker (Excitation: 482nm, Emission: 502nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/LC3-RFP | MDA-MB-231 cells expressing an mRuby/LC3 fusion protein as an autophagasome marker (Excitation: 558nm, Emission: 605nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/Secretory-GFP | MDA-MB-231 cells expressing a copGFP/Sectretory concensus peptide fusion protein as an ER/Golgi marker (Excitation: 482nm, Emission: 502nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/Golgi-GFP | MDA-MB-231 cells expressing a copGFP/Galactosyltransferase fusion protein as a Golgi apparatus marker (Excitation: 482nm, Emission: 502nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/ER-GFP | MDA-MB-231 cells expressing a copGFP/ER targeting sequence fusion protein as an Endoplasmic Reticulum marker (Excitation: 482nm, Emission: 502nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/Mito-GFP | MDA-MB-231 cells expressing a copGFP/CytochromeC Oxidase VIII fusion protein as a Mitochondria marker (Excitation: 482nm, Emission: 502nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/H2B-GFP | MDA-MB-231 cells expressing a copGFP/H2B fusion protein as a Nuclear marker (Excitation: 482nm, Emission: 502nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/iRFP | MDA-MB-231 cells expressing iRFP, a near-infrared Fluorescent Protein (Excitation: 690nm, Emission: 713nm): co-expressed with a puromycin selection casette. |  |
| MDA-MB-231/LUC | MDA-MB-231 cells expressing Luciferase: co-expressed with a puromycin selection casette. | |
*For all cells, suggested cell culture media is: RPMI 1640 with 10% FBS (HI), 10 µg/ml Gentamycin and Puromycin (0.5 µg/mL) |
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