Last data update: 05 March 2021 04:23 CET
Plasmid name: SHC001 (empty vector) (LMBP 7271)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
|Cloned DNA:||Human immunodeficiency virus pol gene; central polypurine tract (HIV-1 cPPT)|
|Promoter:||Human U6 small nuclear RNA gene promoter
Human phosphoglycerate kinase 1 promoter (PGK1)
Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 5' LTR)
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Escherichia coli lac operon promoter
|Terminator:||Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 3' LTR); modified HIV-1 3' LTR with deleted U3 region (ΔU3/3' LTR)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
Phage f1 origin
|Host range:||Escherichia coli
Mammalian cells; SV40 permissive cells
|Further information:||Product No: SHC001
SHC001 (empty vector) is a lentiviral vector without a shRNA insert and is useful as a negative control in experiments using the MISSION shRNA library clones. This allows one to examine the effect of transfection on gene expression and interpret the knockdown effect seen with shRNA clones.
This plasmid allows for transient or stable transfection of the shRNA as well as the production of lentiviral particles.
The human U6 promoter is a polymerase III promoter which is optimal for producing shRNAs due to precise initiation and termination of transcription.
Ampicillin and puromycin antibiotic resistance genes provide selection in bacterial or mammalian cells respectively.
In addition, self-inactivating replication incompetent viral particles can be produced in packaging cells (HEK293T) by co-transfection with compatible packaging plasmids.
The U3 region of the HIV-1 3' LTR is deleted (ΔU3 or dU3) and facilitates self-inactivation of the HIV-1 5' LTR after transduction to enhance the biosafety of the vector.
The element also contains a polyadenylation signal for transcription termination and polyadenylation of mRNA in transduced cells.
The vector contains the psi packaging signal, allowing viral packaging, and the rev response element (RRE) from HIV-1, permitting Rev-dependent nuclear export of unspliced viral mRNA.
Unlike adenovirus or mouse-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, SHC001 (empty vector) provides solutions for long-term knockdown and phenotypic observation, and transduction of difficult or sensitive cell lines (non-dividing cells or primary cells).
SHC001 (empty vector) does not contain the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).
Grow for 20-22 hours in TB-8gly medium (modified Terrific Broth medium, containing 8 ml/l glycerol instead of 4 ml/l) with 100 μg/ml carbenicillin, resulting in an increased plasmid stability as compared to ampicillin.
BCCM/GeneCorner uses the 'Terrific Broth, modified' from Sigma (cat. nr. T0918, Fluka). You can also prepare the medium yourself:
1. add the following to 800 ml distilled H2O:
- 12,0 g tryptone (enzymatic casein digest)
- 24,0 g yeast extract
- 8 ml glycerol
2. adjust to 900 ml with distilled H2O
3. sterilize by autoclaving (20 minutes at 121°C)
4. allow to cool to room temperature
5. adjust volume to 1000 ml with 100 ml of a filter-sterilized solution of 0,17M KH2PO4 and 0,72M K2HPO4
HOST STRAIN INFORMATION
Sigma recommends the host strain Escherichia coli K12 GC5 (catalogue number: G3169) for transformation with this shRNA control vector.
According to Sigma, the genotype of this GC5 strain is identical to the genotype of the Escherichia coli K12 DH5αT1R strain, which is a T1 bacteriophage resistant DH5α derivative.
Genotype: Φ80lacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 relA1 gyrA96 hsdR17(rk-, mk+) phoA supE44 thi-1 tonA (Ref.: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/shclngbul.Par.0001.File.tmp/shclngbul.pdf)
The nucleotide sequence of the human U6 promoter corresponds with the EMBL Nucleotide Sequence database accession number AY623053.1, except for one nucleotide: the A-residue at position 303 in the EMBL record is replaced by a G-residue (position 67 of the human U6 promoter sequence in the SHC001 (empty vector)).
Other names of the plasmid are pLKO.1-puro and MISSION pLKO.1-puro Control Vector.
|EMBL Accession number:||AY623053.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||04/04/2011|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: EcoRI, HindIII and PvuII.|
|History of deposit:||This plasmid was purchased from Sigma-Aldrich(1) and deposited in the frame of the Hercules project 'RNAi collection and associated facilities' by Prof. Dr R. Beyaert(2)(3).
(1) Schnelldorf, Germany, www.sigmaaldrich.com
(2) Inflammation Research Center, VIB, Ghent, Belgium
(3) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Related plasmid reference:||Stewart et al., RNA 9 (2003), 493-501 [PMID: 12649500]
|Restricted distribution:||- Acknowledge BCCM/GeneCorner (supplier) and the Hercules Foundation (funder) in any publication resulting from the use of a shRNA clone
- Restricted to project-related users
- The buyer cannot sell or transfer this product, its components nor materials made using this product or its components to another research group.
- Visit www.sigma.com/shRNA for more, up-to-date info on the limited use of this product.
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 GC5|
|Cultivation medium:||TB-8gly + ampicillin (100 μg/ml)|
|Biosafety level:||L1 in E. coli; L2 in mammalian cells|
|Other culture collection numbers:||LMBP 07132|
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.