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MISSION® LentiExpress Human Kinases

Pre-arrayed lentiviral particles targeting human kinome

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Description

General description

Small interfering RNAs (siRNAs) expressed from short hairpin RNAs (shRNAs) are a powerful way to mediate gene specific RNA interference (RNAi) in mammalian cells. The MISSION product line is a viral-vector based RNAi library targeting annotated mouse and human genes. shRNAs that generate siRNAs intracellularly are delivered by amphotropic lentiviral particles, allowing screening in a wide range of mammalian cell types. In these cell lines, MISSION shRNA clones permit rapid, cost efficient loss-of-function and genetic interaction screens.

Protein kinases are among the largest and most well studied gene families. Protein phosphorylation plays an essential role in intercellular communication in eukaryotic organisms by mediating signal transduction during development, transcription, immune response, metabolism, apoptosis, and cell differentiation. Aberrant regulation of kinases plays a causal role in many diseases, and the study of these proteins and their functions will contribute to the discovery and development of new therapeutics.

MISSION LentiExpress Human Kinases contains ∼3200 lentiviruses carrying shRNA sequences targeting ∼501 human kinase genes allowing for quick, high throughput loss-of-function screens. These kinase genes were chosen based on the 2002 paper by Manning, G., et al. and The RNAi Consortium. Each gene is represented by a target set that consists of ∼6 individual constructs, targeting different regions of the gene sequence. Each shRNA construct has been cloned and sequence verified to ensure a match to the target gene.

The shRNA sequences are delivered to cells by replication incompetent lentiviral particles. Unlike murine-based MMLV or MSCV retroviral systems, lentiviral-based particles permit efficient infection and integration of the specific shRNA sequence into differentiated and non-dividing cells, such as neurons and dendritic cells. Self-inactivating replication incompetent viral particles are produced in packaging cells by co-transfection with compatible packaging plasmids.

In addition, the lentiviral transduction particles are pseudotyped with an envelope G glycoprotein from Vesicular Stomatitis Virus (VSV-G), allowing transduction of a wide variety of mammalian cells.

Lentiviral particles are provided in a pre-arrayed, ready-to-use format in 96-well plates. The Human Kinases consists of 41 plates, wherein each plate contains 80 different kinase-specific viruses. The particles are pre-diluted to approximately 5,000 viral particles per well in a single reaction volume of 30μL In addition to the virus generated from the human kinases, each plate includes negative controls to monitor transduction efficiency.

Linkage

To view data and protocols for the LentiExpress kinase panel visit the links below.
Protocols
Applications

Other Notes

Screens can be quickly carried out using LentiExpress by simply adding cells of interest into each well of the 96-well plate. Pre-arrayed transduction-ready particles allow for rapid lentiviral shRNA screening in a variety of mammalian cell types.

To achieve the best transduction efficiency, especially in hard to transduce cell lines, it is strongly recommended that MISSION LentiExpress Optimization Plate, Catalog No. SHXC01, be used prior to screening. This product is intended for rapid determination of the optimal cell number and/or MOI to use for LentiExpress assays.

Packaging

MISSION LentiExpress Human Kinases consist of forty-one individual 96-well plates. In each plate, 88 wells contain 30 μL of shRNA Lentiviral Particles and controls and 8 wells contain 30 μL of media only. A CD contains detailed clone position, RefSeq, locus link, gene description, gene symbol, clone ID, and hairpin sequence.

Legal Information

Use of this product is subject to one or more license agreements. For details, please see http://sigmaaldrich.com/missionlicense .

3M is a trademark of 3M Company

LentiExpress is a trademark of Sigma-Aldrich Co. LLC

MISSION is a registered trademark of Sigma-Aldrich Co. LLC

Price and Availability

Safety & Documentation

Safety Information

RIDADR 
UN3373 - class 6.2 Biological substance, Category B
WGK Germany 
3

Documents

Certificate of Analysis

Protocols & Articles

Protocols

Optimal Cell Density Determination for Transduction Using MISSION LentiExpress Optimization Plate

Day 0 0.1 Thaw LentiExpress optimization plate at room temperature while completing steps 0.2 through 0.7. 0.2 Fill and label four 1.5 ml tubes with 300 µl of complete media. 0.3 Trypsinize KHOS cell...
Keywords: Microscopy, PAGE

Peer-Reviewed Papers
15

References

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