Last data update: 05 March 2021 04:23 CET
Plasmid name: SHC009 (sh-ARHGDIA) (LMBP 7278)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
|Cloned DNA:||Short hairpin RNA sequence targeting the human Rho GDP dissociation inhibitor α transcript (sh-ARHGDIA)
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
|Parental clone:||SHC001 (empty vector)|
|Further information:||Product No: SHC009
SHC009 (sh-ARHGDIA) is a lentiviral plasmid that contains a shRNA sequence targeting the human ARHGDIA transcript.
The shRNA insert is expressed under control of the human U6 promoter.
ARHGDIA is an ubiquitously expressed protein that acts on human Rho GTPases including RhoA, Rac1 and Cdc42, by keeping these proteins locked in an inactive state. Complete understanding of ARHGDIA's roles is still being elucidated, but it is believed to be involved in various signal transduction pathways and cellular cytoskeleton functions.
SHC009 (sh-ARHGDIA) is useful as a positive knockdown control in experiments using cell lines expressing ARHGDIA.
The specific clone number for the ARHGDIA control is TRNC0000008004 and was chosen from the available complete target set (GenBank Accession Number NM_004309). The control has demonstrated consistent knockdown of 90% compared to normal gene expression in the human epithelial lung carcinoma cell line A549 via quantitative RT PCR and Western blot analysis (Antibody Product No. R3025).
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)
Other names of the plasmid are MISSION shRNA Human Positive Control Vector #2 Purified DNA and MISSION Rho GDP Dissociation Inhibitor (GDI) alpha (ARHGDIA) shRNA Control Vector.
|EMBL Accession number:||NM_004309, view at GenBank|
|Latest sequence update:||10/06/2011|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: EcoRI, EcoRI/NcoI, HindIII, NcoI and PvuII.
The EcoRI site could not be experimentally confirmed.
|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 07138|
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.