Cluster of Excellence –
University of Freiburg

Mouse Kinase Activity GIPZ lentiviral shRNAmir gene family collection (Open Biosystems Cat.# RMM4948)

The complete library of Mouse Kinase Activity GIPZ lentiviral shRNA mir genes was rearranged and organized according to the gene definitions by ourself. All clones are available from the BIOSS Toolbox upon request only for research members of the University of Freiburg and the University Medical Center Freiburg.

Please see the spreadsheet and use the Accession number for the search of your shRNA.

shRNA library (Open Biosystems).xls

Please note:

  • This collection includes 1614 constructs of mouse specific shRNAmir sequences, which can affect appr. 500 mouse specific kinases. Most kinases are represented on average by two to three independent shRNAmir constructs.
  • All shRNAmir sequences are cloned into the transfer vector pGIPZ, a replication incompetent lentiviral vector.
  • The pGIPZ vector contains a TurboGFP cassette (licensed by Evrogen) for marking an efficient shRNAmir expression, puromycin resistance as a mammalian selectable marker and ampicillin/zeocin resistance as a bacterial selectable marker.
  • Bacterial culture need to grow in low salt LB Medium (5 g/l NaCl) with 100 µg/ml ampicillin and 25 µg/ml zeocin.
  • We recommend checking all requested clones by sequencing, for example with T7 primer.

For further information please see:  

 dharmacon.gelifesciences.com/shrna/gipz-lentiviral-shrna/

  • Alternatively,  the tetracycline inducible lentiviral vector pTRIPZ can be used to express shRNAmir sequences. The pTRIPZ vector contains a TurboRFP casette and Tet-On® or Tet-Off® configurations for regulation. The pTRIPZ vector is available in the Toolbox Plasmid Repository.

For further information please see:

dharmacon.gelifesciences.com/shrna/tripz-lentiviral-shrna-controls/

Scientific application

The shRNA transfer vector (pGPIZ/pTRIPZ) has to be co-transfected with a mix of packaging plasmids into the HEK293T cell line to produce replication incompetent shRNA containing lentiviral particles (Shimada et al.1995). The supernatant containing the lentiviral particles can be used directly to transduce target cells.

Please keep in mind that the infection efficiency is depending on used cell lines, it has been shown that the infection efficiency can be decreased in mouse cell lines.

It is recommended to use the Thermo Scientific Open Biosystems TransLenti Viral Packaging system for delivering and expressing the shRNA of interest in either dividing or non-dividing mammalian cells.

TransLenti_Viral_GIPZ_Packaging_System_Technical_Manual.pdf

Please pay attention that all work with packaging plasmids to facilitate viral packaging of the transfer vector into HEK293T producer cells need the S2 safety requirements (GenTSV).

The use of lentiviral replication incompetent vectors (pGIPZ and pTRIPZ) without Viral Packaging Mix underlies the S1 guidelines (Gen TSV).

 

Purchase Order Regulation:

As a non-profit repository, the Toolbox properly stores and maintains the Mouse Kinase Activity GIPZ lentiviral shRNAmir gene family collection in the BIOSS Signalhaus and thereby makes it available for all BIOSS members.

  • On request the Toolbox team will provide shRNAmir clones as bacterial plate.
  • To support operation of the Toolbox Repository and to maintain the high quality standard of our work we charge a fee for shRNAmir clones. The fee is used to cover consumables and operating cost. (The transfer of material contemplated herein shall not be considered as a "sale of goods" for any purposes.) Any request of material will be covered under a Toolbox-MTA between the RECIPIENT and the BIOSS Toolbox.

     

      BIOSS Members

    Members of the University of Freiburg & the University Medical Center Freiburg

      shRNAmir clone                 

       15,00 €

       25,00 €

Please address all requests for shNRAmir clones ONLY to the following Email addresss: toolbox@bioss.uni-freiburg.de