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MLP Retroviral shRNA-mir Arrayed Focused Sets

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Pooled screening overview
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CAT#	PRODUCT	SIZE	STORAGE	소비자가	주문
TRM4011	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Epigenetic readers consolidated set	ea	-80C	-	로그인
TRM4010	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Epigenetic erasers consolidated set	ea	-80C	-	로그인
TRM4009	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Epigenetic writers consolidated set	ea	-80C	-	로그인
TRM4008	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Protein methyltransferase	ea	-80C	-	로그인
TRM4007	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Plant Homeo Domain	ea	-80C	-	로그인
TRM4006	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Methyl-lysine- and/or methyl-arginine-binding domain-containing protein	ea	-80C	-	로그인
TRM4005	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Lysine demethylases	ea	-80C	-	로그인
TRM4004	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Histone deacetylase	ea	-80C	-	로그인
TRM4003	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Histone acetyltransferases	ea	-80C	-	로그인
TRM4002	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Bromodomain-containing proteins	ea	-80C	-	로그인
TRM4001	PLATINUM Select Mouse MLP retroviral shRNA-mir focused set-Epigenetics	ea	-80C	-	로그인
TRH3043	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Epigenetic writers, readers and erasers consolidated set	ea	-80C	-	로그인
TRH3042	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Epigenetic writers consolidated set	ea	-80C	-	로그인
TRH3041	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Epigenetic erasers consolidated set	ea	-80C	-	로그인
TRH3040	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Epigenetic readers consolidated set	ea	-80C	-	로그인
TRH3039	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Protein methyltransferase	ea	-80C	-	로그인
TRH3038	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Plant homeodomains (PHDs)	ea	-80C	-	로그인
TRH3037	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Methyl-lysine- and/or methyl-arginine-binding domain-containing protein	ea	-80C	-	로그인
TRH3036	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Lysine demethylases	ea	-80C	-	로그인
TRH3035	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Histone deacetylase	ea	-80C	-	로그인
TRH3034	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Histone acetyltransferases (HAT)	ea	-80C	-	로그인
TRH3033	PLATINUM Select Human MLP retroviral shRNA-mir focused set-Bromodomain-containing proteins	ea	-80C	-	로그인

MLP Retroviral shRNA-mir focused sets

PLATINUM Select MLP Retroviral shRNA-mir sets targeting popular gene families and pathways are available and custom arrays can be created. These focused sets are available in a 96 well arrayed (bacterial glycerol stock) and pooled formats making them ideal for RNAi screening for a variety of biological assays. The flexibility of formats and the renewable nature of vector-based RNAi provide economical library options for screening or as a long-term resource suiting the needs of most RNAi applications.

Focused sets were created using resources from the National Institute of Health’s Cancer Genome Anatomy Project (CGAP) and Gene Ontology (GO). Annotation is based on electronic and manual literature-based curation as well as sequence analysis. (See Annotation Standard Operating Procedures from the Gene Ontology Consortium for more information.)

Benefits of the arrayed libraries include:

Predefined sets created across several gene families and pathways (see available focused sets here)
shERWOOD designed shRNA-mir provides potency, specificity and 100% guaranteed knockdown
Single copy knockdown minimizes off-target effects and provides more confidence in results

Don’t see your gene family or pathway of interest? Contact us at support@transomic.com to ask about custom arrayed or pooled shRNA-mir sets.

pMLP vector cartoon showing shRNA-mir and selection cassettes

pMLP Vector Design

The shRNA-mir sequences have been cloned in to the pMLP vector which has the following characteristics:

MSCV-based retroviral vector for delivery and expression in most mammalian cell lines including murine or human hematopoietic and embryonic stem (ES) cells.
Robust shRNA-mir expression from the retroviral LTR promoter
Provides puromycin resistance for removal of untransfected or untransduced cells
turboGFP provides a visual marker for retroviral integration and shRNA-mir expression

Arrayed and pooled screening methodologies.

Arrayed and pooled screening with shRNA-mir

Vector-based RNAi screens can be implemented using two different strategies. The classic arrayed strategy, adapted from the small molecule screening field, is a well-by-well screen testing one gene per well using plasmid DNA or viral particle formats. A number of possible readouts have proven successful with this technique including cell viability, reporter assays, and morphological changes. Additionally, high-content screening with RNAi is a popular way to perform phenotypic screens on a large number of genes in parallel (Hu, G and Luo Ji 2012).

The multiplexed (pooled) strategy enables hundreds to thousands of genes to be simultaneously targeted. This is only possible using viral vector-based RNAi. Multiple constructs are pooled together, packaged into virus and cells are transduced at a multiplicity of infection (MOI) ensuring single integrations per cell. From this, each transduced cell becomes its own experiment and positive or negative selection can be used to determine hits (see figure 1). shRNA-mir that induce or diminish a phenotype can be detected using next generation sequence analysis of the shRNA integrated into the target cell’s genome.

Learn more about available pooled shRNA screening libraries here.

shRNA-mir designed by the shERWOOD algorithm are uniquely suited for both arrayed and pooled screening given their consistent potency. Every shRNA-mir designed is expected to provide potent knockdown even when expressed from a single integration in the genome. The additional potent constructs per gene provide better primary hit validation in arrayed screens, and potent knockdown at single copy is required for effective pooled screening.

References:
Hu, G and Luo, Ji (2012) A primer on using pooled shRNA libraries for functional genomic screens. Acta Biochim Biophys Sin 2012, 44: 103–112.

Pooled screening

Pooled RNAi screening: The figure above depicts the concept and workflow for pooled selection screening. Positive (survival) screens select for genes that provide resistance to a selection pressure when knocked down. Negative (dropout) screens highlight genes that provide resistance or sensitize to a selection pressure when knocked down.

In pooled shRNA screening, hundreds to thousands of hairpins are combined (pooled) and interrogated simultaneously in a multiplex assay without the need for robotics or liquid handling.

The schematic to the right depicts this process.

Transduction: Cells are transduced at a low MOI ensuring a single shRNA is expressed in each cell.
Screen: The screen employs a selection process that is specific to the researcher’s assay. There are two underlying strategies for selection: Negative selection (dropout) screens include an untreated control for comparison to allow the detection of shRNA that provided resistance or sensitized the cells to the selective reagent. Positive selection (enrichment) screens only detect surviving cells and do not require an untreated control.
Analysis: Under selection, resistant cells increase in the population and sensitized cells decrease in the population. These changes in representation can be detected by sequence analysis (either Sanger sequencing or Next Generation Sequencing).

pMLP-_generic-shRNA_.docx (125,966kb)

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관련제품: MGC premier cDNA clones; MGC premier Expression-Ready cDNA clones