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Functional Genomics & RNAi

Vector Maps

MISSION RNAi

The MISSION research and development team, in collaboration with the RNAi consortium (TRC) scientists, have generated a series of vectors to enable successful implementation of your RNAi experiments. The base vector pLKO.1-puro was developed1 at the Broad Institute as part of TRC. Our contribution to TRC is a diverse set of vectors for critical control experiments, as well as vectors for custom shRNA production. This page contains information pertaining to these vectors.

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TRC1.5 Vector: pLKO.1-puro
TRC1.5 Control Vectors
TRC1.5 Alternate Vector Backbones
TRC2 Vector: TRC2-pLKO-puro
Inducible shRNA VectorsNEW
Lentiviral Packaging Vectors
References

TRC1.5 Vector: pLKO.1-puro Vector and Use

TRC1.5 clones are in the exact same vector backbone as the TRC1 clones

Features of the pLKO.1-puro vector allow for transient or stable transfection of the shRNA as well as production of lentiviral particles.1 Stable gene silencing is selected using the puromycin selectable marker while self-inactivating replication incompetent viral particles can be produced in packaging cells (HEK293T) by co-transfection with compatible packaging plasmids.2,3 TRC1.5 is exclusive to Sigma Life Science and contains almost 200,000 clones including more than 49,000 validated clones. It combines all of the content from TRC1 plus an additional 39,212 clones targeting 2,661 new human genes and 2,395 mouse genes. Unlike adenovirus or murine-based MMLV or MSCV retroviral systems, lentiviral-based particles permit efficient infection and integration of the specific shRNA construct into differentiated and non-dividing cells, such as neurons and dendritic cells, overcoming low transfection and integration difficulties when using these cell lines. Compared to siRNA and other vector-based systems, pLKO.1-puro provides solutions for long-term knockdown and phenotypic observation, transduction of difficult or sensitive cell lines (non-dividing cells or primary cells), and is an economical renewable resource.

With an shRNA insert, the length of the pLKO.1-puro plasmid is 7,086 bp, as indicated in the vector map below. Without an shRNA insert, pLKO.1-puro vector has a length of 7,052 bp. The sequence file provided is the empty pLKO.1-puro vector (7,052 bp).

pLKO.1-puro sequence (without shRNA insert, 7052bp) (8 Kb txt file)

TRC1.5 Vector Map (pLKO.1-puro)
pLKO.1-puro Vector Map

TRC1.5 Vector Description and Features

Name Description
cppt Central polypurine tract
hPGK Human phosphoglycerate kinase eukaryotic promoter
puroR Puromycin resistance gene for mammalian selection
SIN/LTR 3' self inactivating long terminal repeat
f1 ori f1 origin of replication
ampR Ampicillin resistance gene for bacterial selection
pUC ori pUC origin of replication
5' LTR 5' long terminal repeat
Psi RNA packaging signal
RRE Rev response element

TRC1.5 Control Vectors

Sigma offers a variety of controls for conducting the most rigorous and informative shRNA experiments possible. Visit the shRNA controls web page to learn more.

Product Purpose Vector Map Vector File
SHC001 Empty vector (no shRNA insert) Control SHC001
(186 Kb PDF)
SHC001
(8 Kb Text File)
SHC002 Non-Mammalian shRNA Control SHC002
(189 Kb PDF)
SHC002
(8 Kb Text File)
SHC003 TurboGFP Control SHC003
(295 Kb PDF)
SHC003
(9 Kb Text File)
SHC004 TurboGFP shRNA Control SHC004
(189 Kb PDF)
SHC004
(8 Kb Text File)
SHC005 eGFP shRNA Control SHC005
(187 Kb PDF)
SHC005
(8 Kb Text File)
SHC007 Luciferase shRNA Control SHC007
(188 Kb PDF)
SHC007
(8 Kb Text File)
SHC008 B2M shRNA Control SHC008
(190 Kb PDF)
SHC008
(8 Kb Text File)
SHC009 ARHGDIA shRNA Control SHC009
(188 Kb PDF)
SHC009
(8 Kb Text File)
SHC010 CMV-TagCFP™ Control SHC010
(186 Kb PDF)
SHC010
(8 Kb Text File)
SHC011 CMV-TagYFP™ Control SHC011
(185 Kb PDF)
SHC011
(8 Kb Text File)
SHC012 CMV-TagRFP™ Control SHC012
(183 Kb PDF)
SHC012
(8 Kb Text File)
SHC013 CMV-TagFP635™ Control SHC013
(186 Kb PDF)
SHC013
(8 Kb Text File)
SHC014 UbC-TurboGFP™ Control SHC014
(186 Kb PDF)
SHC014
(9 Kb Text File)
SHC015 UbC-TagFP635™ Control SHC015
(186 Kb PDF)
SHC015
(9 Kb Text File)
SHC016 Non-Target shRNA Control SHC016
(236 Kb PDF)
SHC016
(7 Kb Text File)

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TRC1.5 Alternate Vector Backbones

 

Sigma offers a variety of alternate vector backbones as part of our standard shRNA offering. With our customizable MISSION shRNA ordering interface, it is possible to order TRC shRNAs in these vector backbones directly online on the clones detail pages for Plasmid DNA or Lentiviral Transduction Particles. For custom hairpins, please visit our custom cloning page for details on ordering.

Product Purpose Vector Map Vector File
pLKO.1-CMV-Neo Alternate vector backbone pLKO.1-CMV-Neo
(30 Kb PDF)
pLKO.1-CMV-Neo
(8 Kb Text File)
pLKO.1-Neo Alternate vector backbone pLKO.1-Neo
(30 Kb PDF)
pLKO.1-Neo
(8 Kb Text File)
pLKO.1-CMV-tGFP Alternate vector backbone pLKO.1-CMV-tGFP
(179 Kb PDF)
pLKO.1-CMV-tGFP
(8 Kb Text File)
pLKO.1-Neo-CMV-tGFP Alternate vector backbone pLKO.1-Neo-CMV-tGFP
(193 Kb PDF)
pLKO.1-Neo-CMV-tGFP
(8 Kb Text File)
pLKO.1-puro-CMV-tGFP Alternate vector backbone pLKO.1-puro-CMV-tGFP
(32 Kb PDF)
pLKO.1-puro-CMV-tGFP
(9 Kb Text File)

 

Sigma also offers several alternate vector backbones with fluorescent proteins or the UbC promoter. These constructs are available only as custom options. Please visit our custom cloning page for details on ordering.

Product Purpose Vector Map Vector File
pLKO.1-puro-CMV-TagCFP™ Alternate vector backbone pLKO.1-puro-CMV-TagCFP
(32 Kb PDF)
pLKO.1-puro-CMV-TagCFP
(8 Kb Text File)
pLKO.1-puro-CMV-TagYFP™ Alternate vector backbone pLKO.1-puro-CMV-TagYFP
(187 Kb PDF)
pLKO.1-puro-CMV-TagYFP
(8 Kb Text File)
pLKO.1-puro-CMV-TagRFP™ Alternate vector backbone pLKO.1-puro-CMV-TagRFP
(188 Kb PDF)
pLKO.1-puro-CMV-TagRFP
(8 Kb Text File)
pLKO.1-puro-CMV-TagFP635™ Alternate vector backbone pLKO.1-puro-CMV-TagFP635
(188 Kb PDF)
pLKO.1-puro-CMV-TagFP635
(8 Kb Text File)
pLKO.1-puro-UbC-TurboGFP™ Alternate vector backbone pLKO.1-puro-UbC-TurboGFP
(188 Kb PDF)
pLKO.1-puro-UbC-TurboGFP
(9 Kb Text File)
pLKO.1-puro-UbC-TagFP635™ Alternate vector backbone pLKO.1-puro-UbC-TagFP635
(189 Kb PDF)
pLKO.1-puro-UbC-TagFP635
(9 Kb Text File)

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TRC2 Vector: TRC2-pLKO-puro Vector and Use

The TRC2-pLKO-puro vector also allows for transient or stable transfection of the shRNA as well as production of lentiviral particles. The only change between the TRC1.5 vector and the TRC2 vector is the addition of the WPRE (or the Woodchuck Hepatitis Post-Transcriptional Regulatory Element).4 This allows for enhanced expression of transgenes delivered by lentivirus.5 As with TRC1.5, stable gene silencing is achieved by using the puromycin selectable marker. Self-inactivating replication incompetent viral particles can be produced in packaging cells (HEK293T) by co-transfection with compatible packaging plasmids. TRC2-pLKO-puro provides solutions for stable long-term knockdown and phenotypic observation, transduction of difficult or sensitive cell lines (non-dividing cells or primary cells), and enhanced expression of transgenes.

With an shRNA insert, the length of the TRC2-pLKO-puro plasmid is 7,518 bp, as indicated in the vector map below. Without an shRNA insert, the TRC2-pLKO-puro vector has a length of 7,484 bp. The sequence file provided is the empty TRC2-pLKO-puro vector (7,484 bp).

TRC2-pLKO-puro sequence (without shRNA insert, 7484bp) (8 Kb text file)

TRC2 Vector Map (TRC2-pLKO-puro)

TRC2 Vector Description and Features

Name Description
cppt Central polypurine tract
hPGK Human phosphoglycerate kinase eukaryotic promoter
puroR Puromycin resistance gene for mammalian selection
WPRE Woodchuck Hepatitis Post-Transcriptional Regulatory Element
SIN/LTR 3' self inactivating long terminal repeat
f1 ori f1 origin of replication
ampR Ampicillin resistance gene for bacterial selection
pUC ori pUC origin of replication
5' LTR 5' long terminal repeat
Psi RNA packaging signal
RRE Rev response element



TRC2 Control Vectors

Product Purpose Vector Map Vector File
SHC201 Empty TRC2 vector (no shRNA insert) Control SHC201
(222 Kb PDF)
SHC201
(7 Kb Text File)
SHC202 Non-Target shRNA Control SHC202
(226 Kb PDF)
SHC202
(7 Kb Text File)
SHC203 TurboGFP Control SHC203
(234 Kb PDF)
SHC203
(8 Kb Text File)
SHC204 TurboGFP shRNA Control SHC204
(225 Kb PDF)
SHC204
(7 Kb Text File)

Please note: At this time, there are no alternate vector backbones available for TRC2.

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Inducible shRNA Vectors

The pLKO vector has been redesigned to contain a LacI (repressor) and a modified human U6 shRNA promoter with LacO (operator) sequences. In the absence of IPTG (isopropyl-β-D-thio-galactoside), an analogue of lactose, LacI binds to LacO preventing expression of the shRNA. When IPTG is present, the allosteric LacI repressor changes conformation, releasing itself from lacO modified human U6 promoter, and subsequently allows expression of the shRNA.

We are proud to offer two different IPTG inducible vectors for your research. The preferred inducible vector, pLKO-puro-IPTG-3xLacO (399 KB PDF), contains three lac operon sequences (two in the U6 promoter and one 3' of the promoter) affording both tight regulation and great gene silencing. Whereas, the pLKO-puro-IPTG-1xLacO (95 Kb PDF) vector contains a single lac operon sequence in the U6 promoter, which allows for an advantage to shRNA expression, but looser control of the promoter when not induced.

 

Product Purpose Vector Map Vector File
pLKO-puro-IPTG-1xLacO Alternate vector backbone pLKO-puro-IPTG-1xLacO
(95 Kb PDF)
pLKO-puro-IPTG-1xLacO
(8 Kb Text File)
pLKO-puro-IPTG-3xLacO Alternate vector backbone pLKO-puro-IPTG-3xLacO
(399 KB PDF)
pLKO-puro-IPTG-3xLacO
(8 Kb Text File)

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Lentiviral Packaging Vectors

Finally, for those wanting to package their own lentivirus, we offer the optimized packaging mix. Visit the Lentiviral packaging mix web page to learn more.

 

Product Purpose Vector Map Vector File
SHP001
pMISSIONgagpol
Packaging Vector #1 pMISSIONgagpol
(192 Kb PDF)
pMISSIONgagpol
(13 Kb Text File)
SHP001
pMISSIONvsvg
Packaging Vector #2 pMISSIONvsvg
(158 Kb PDF)
pMISSIONvsvg
(9 Kb Text File)

 


References

  1. Stewart, S. A. et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 2003, 9, 493–501.
  2. Zufferey, R. et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol 1997, 15, 871–85.
  3. Zufferey, R. et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 1998, 72, 9873–80.
  4. Donello J. E. et al. Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. J. Virol. 1998, 72, 5085-92.
  5. Zufferey, R. et al. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 1999, 73, 2886-92.

 


Contact Us

For questions about the library, pricing and quotes or other concerns, please e-mail us at: MISSIONRNAi@sial.com.

MISSION is a registered trademark of Sigma-Aldrich Co. LLC Label License.
TurboGFP, TagCFP, TagYFP, TagRFP, and TagFP635 are trademarks of Evrogen Co.

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