Mouse Kinase Activity GIPZ lentiviral shRNAmir gene family collection (Open Biosystems Cat.# RMM4948)
For further information please see: https://www.openbiosystems.com/RNAi/shRNALibraries/
The complete library of Mouse Kinase Activity GIPZ lentiviral shRNA mir genes was rearranged and organized according to the gene definitions by ourself and is now available in the Toolbox. Please see the spreadsheet and use the Accession number for search of your shRNA.
shRNA library (Open Biosystems).xls
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.
The library is provided in 8% glycerolstocks with carbenicillin (100 μg/ml) and Zeozin (μg/ml) in a special LB medium(low salt, NaCl 5g/l).
All shRNAmir sequences are cloned into the transfer vector pGIPZ, a replication incompetent lentiviral vector.
The pGIPZ vector contains a TurboGFP cassette (lincensed by Evrogen) for marking an efficient shRNAmir expression, puromycin resistance as a mammalian selectable marker and ampicillin/zeozin resistance as a bacterial selectable marker.
We recommend to verify all requested clones by sequencing, for example with T7 primer.
https://www.openbiosystems.com/RNAi/shRNAmirLibraries/GIPZLentiviralshRNAmir/
A tetracycline inducible lentiviral vector pTRIPZ can be used and is available in the Toolbox Plasmid Repository. The pTRIPZ vector contains a TurboRFP casette (selectable marker same as pGIPZ) and Tet-On® or Tet-Off®configurations for regulation.
https://www.openbiosystems.com/RNAi/shRNAmirLibraries/TRIPZlentiviralinducibleshR/
The shRNA mir sequences from the library could be easily transferred. A transfer of the whole library from the pGIPZ into the pTRIPZ format is planned.
Scientific application
The shRNA transfer vector (pGPIZ/pTRIPZ) has to be co-transfected with a mix of packaging plasmids into the HEK 293 T cell line to produce replication incompetent shRNA containing lentiviruses (Shimada et al.1995). After virus production in HEK 293 cells the replication incompetent lentiviruses can be transduced into mammalian cell line of interest.
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 recommend 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 he transfer vector into TLA HEK 293 producer cells needs the S2 safety requirements (GenTSV).
The use of lentiviral replication incompetent vectors (pGIPZ and pTRIPZ) without Viral Packaging Mix underlie the S1 guidelines (Gen TSV).