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Human shRNA Resource (NKI) Knockdown Resource

Human shRNA Resource (NKI) Knockdown Resource

The NKI Human shRNAi Library is a short-hairpin RNA (shRNA) library constructed by The Netherlands Cancer Institute (NKI). This fully sequence verified library has substantial advantages over many similar resources including knockdown validation.

The shRNA constructs have been designed and cloned into a retroviral vector and can easily be utilised for gene silencing experiments. Multiple shRNA expression vectors represent many of the genes, each with at least one shRNA construct covering a unique region of the target gene. Researchers can study the function of specific genes and also search the human genome for novel therapeutic targets for many diseases.

As part of our sequence verification service, individual clones are end sequenced before they are sent to you. If the requested clone does not match the expected sequence, Source BioScience will not apply any charge and will contact the purchaser to confirm if they wish to cancel the order.This risk free option ensures that you have the right clone, removing the chance of receiving one of the wrong clones that are inherent in some libraries and can also save you time from having to sequence the clone yourself.

PLEASE NOTE –that Source BioScience excludes any warranty pertaining to the accuracy of the sequence for clone orders where the client chooses to reject the offered sequence verification option, as the clones are supplied as-is, direct from the original library.

Library Specifications:

  •  Library consists of ~ 13,500 clones
  •  Targets ~ 5,000 cancer related genes
  •  Up to three 19-mer target sequence constructs per gene
  •  Controlled quantities of silencing reagent are delivered to the cell
  •  Backbone plasmid allows for the shRNA cassettes to be packaged in retroviruses
  •  Unique barcode to expedite screening in high-throughput experiments

Each clone has:

  •  pRSC vector containing an H1 promoter
  •  Unique hairpin-loop knockdown cassette
  •  Two bacterial resistance markers (chloramphenicol & ampicillin)
  •  Puromycin-selectable marker for mammalian cells
  •  H1 promoter + shRNA insert can be shuttled between different vectors using EcoRI & XhoI sites

Protocols / Clone Handling

Please note that the clone(s) are tested as negative in our phage contamination assay prior to shipping. However, these clones should still be handled with care as no phage assay can be guaranteed to be 100% accurate.

Plates contain LB broth with 8% glycerol, ampicillin and chloramphenicol, are sent on Dry Ice. These should be stored at -70°C.

Individual clone pools have been streaked onto LB agar containing ampicillin (50µg/ml) and chloramphenicol (20µg/ml). Please store them at 4°C (not in a freezer). As soon as possible, transfer the culture to LB broth containing ampicillin /chloramphenicol + 8% glycerol and incubate overnight at 37°C (do not try to transfer single colonies, instead scoop out the whole bacterial culture using a disposable inoculation loop). Incubate overnight at 37°C for subsequent freezing at -70°C.

Background strain: DH5 alpha genotype: fhuA2 ∆(argF-lacZ)U169 phoA glnV44 F80 ∆(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17

These clones are for research purposes only.

References

We are grateful to Dr. Roderick Beijersbergen (NKI) for the preparation of this library and Dr. Róisín NicAmhlaoibh (CRUK) for the commercialization.
Please acknowledge the NKI, Cancer Research UK and Source BioScience LifeSciences in any resulting publications.

Please also cite the following paper:

Bernards R, Brummelkamp TR, Beijersbergen RL. shRNA libraries and their use in cancer genetics. Nat Methods. 2006 Sep;3(9):701-6. Review.


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