Functional Genomics & RNAi

RNAi Glossary


Antisense molecules
Argonaute
Cosuppression
Drug target
Dicer
Expression
Functional Genomics
Gag
Gene silencing
Gene target
Genomics
Interferon response
Knock-down
Long terminal repeat (LTR)
MicroRNA (miRNA)
Multiplicity of infection (MOI)
Off-target effects
Packaging plasmid
Phenotype
Pol
Pol III promoter
Post-Transcriptional Gene Silencing
Pseudotype
Quelling
Regulatory viral protein (Rev)
Replication incompetent
Ribozyme
RNA-Directed DNA Methylation
RNA-Induced Silencing Complex
RNAi
Self-inactivating (SIN) long terminal repeat
shRNA
Small Interfering RNA (siRNA)
Specificity
TAT
Titer
Transfection
Transduction
U6 promoter
Vesicular stomatitis virus G glycoprotein (VSV-G)
Viral infectivity factor (Vif)
Viral protein r (Vpr)
Viral protein u (Vpu)


Antisense molecules
A sequence of nucleic acids, typically created in the lab, whose sequence is exactly complementary/opposite to an mRNA molecule made by an organism. mRNA molecules made by an organism serve as templates for the synthesis of protein. Since the "antisense" mRNA molecule binds tightly to its mirror image, it can prevent a particular protein from being made.


Argonaute
A family of proteins containing multiple domains that is involved in RNA interference (RNAi). Argonaute is the main component of the siRNA induced silencing complex (RISC). It facilitates target recognition and cleavage during RNAi.


Cosuppression
Refers to the specific case of gene silencing in which RNA from a transgene and a homologous endogenous gene are suppressed at the same time.


Drug target
A gene or gene product (protein) that small molecule drugs will be screened and developed against.


Dicer
Dicer is a member of the RNase III family of nucleases which specifically cleave double-stranded RNAs. Dicer processes long dsRNA into siRNA of 21-23 nucleotides.


Expression
The process by which the information in a gene is used to create proteins.

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Functional Genomics
The analysis of genetic information and its biological function.


Gag
A lentiviral packaging element that encodes for a structural precursor protein.


Gene silencing
Targeting or interfering with a specific gene and preventing its expression


Gene target
A gene or its product (protein) which plays a critical role in disease.


Genomics
The systematic study of genes and their function.


Interferon response
A cellular response induced by the introduction of dsRNA longer than 30 nucleotides. It triggers a small and highly potent molecule that functions in an autocrine and paracrine manner, causing cells to resist viral replication.

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Knock-down
Reduction of gene expression.


Long terminal repeat (LTR)
Homologuous regions, consisting of 600-900 nucleotides, in the lentiviral vector that are required for replication, integration, and expression of viral RNA.


MicroRNA (miRNA)
Micro-RNAs are single-stranded RNAs of 22-nucleotides that are processed from ~70-nucleotide hairpin RNA precursors by the Rnase III nuclease, Dicer. Similar to siRNAs, miRNAs can silence gene activity through destruction of homologous mRNA in plants or blocking its translation in plants and animals.


Multiplicity of infection (MOI)
Represents the average number of viral particles per single cell. The MOI is calculated by dividing the total number of transducing units by the number of cells plated.


Off-target effects
Effects seen when one or several genes, not specifically targeted by the siRNA or shRNA, show loss of gene expression following transfection or transduction. The sense strand of an siRNA or shRNA may be able to initiate a loss of function response from an unrelated gene. Off target effects may also refer to a secondary effect of the antisense strand of a specific siRNA or shRNA where it has the appropriate homology to knockdown the expression of another related gene that carries the same sequence.


Packaging plasmid
Lentiviral vectors that contain all necessary elements to efficiently generate active viral particles. For improved safety, third generation packaging plasmids have necessary elements separated between 2 or 3 plasmids, eliminating the possibility of homologous recombination and generation of wild-type virus.

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Phenotype
The observable physical characteristics of an organism or cell.


Pol
A lentiviral packaging element that encodes for a structural precursor protein.


Pol III promoter
pol III promoters, typically U6 or H1, are used to drive the expression of shRNA. U6 and H1 pol III promoters have necessary elements required for the initiation of transcription upstream of a defined start site and terminate transcription at 4 or more T's.


Post-Transcriptional Gene Silencing
Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation pathway designed to act as an anti-viral defense mechanism. A form of PTGS triggered by transgenic DNA, called co-suppression, was initially described in plants and a related phenomenon, termed quelling, was later observed in the filamentous fungus Neurospora crassa.


Pseudotype
A heterologous viral envelope protein replacing the wild-type lentiviral envelope. Used to increase tropism or convey target cell specificity.


Quelling
Cosuppression as described in Neurospora crassa. The term is used to describe gene silencing in fungi.

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Regulatory viral protein (Rev)
A lentiviral packaging element that binds to Rev Response Element (RRE) sequences, allowing the cytoplasmic export of viral RNAs.


Replication incompetent
Lentiviral particles that are incapable of producing additional viral particles, due to the elimination of wild-type enhancers in the long terminal repeat region.


Ribozyme
Ribozymes are RNA molecules that act as enzymes in the absence of proteins.


RNA-Directed DNA Methylation
RNA-directed DNA methylation (RdDM) is an RNA directed silencing mechanism found in plants. Similar to RNA interference (RNAi), RdDM requires a double-strand RNA that is cut into short 21-26-nucleotide fragments. DNA sequences homologous to these short RNAs are then methylated and silenced.


RNA-Induced Silencing Complex
RNA-induced silencing complex (RISC) is an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the mRNA target.


RNAi
RNA Interference (RNAi) is a phenomenon that small double-stranded RNA (referred as small interference RNA or siRNA) can induce efficient sequence-specific silence of gene expression.

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Self-inactivating (SIN) long terminal repeat
The basal/enhancer control elements from wild-type lentiviral promoters were replaced in the long terminal repeat by transfcriptional control elements from heterologous viral or celluar promoters. This replacement renders the virus replication incompetent.


shRNA
shRNA or short hairpin RNA is an RNA molecule that contains a sense strand, antisense strand, and a short loop sequence between the sense and antisense fragments. Due to the complementarity of the sense and antisense fragments in their sequence, such RNA molecules tend to form hairpin-shaped double-stranded RNA (dsRNA). shRNA is cloned into a vector, allowing for expression by a pol III type promoter. The expressed shRNA is then exported into the cytoplasm where it is processed by dicer into siRNA which then get incorporated into the siRNA induced silencing complex (RISC).


Small Interfering RNA (siRNA)
Small Interfering RNA (siRNA) are 21-23 nucleotide double-stranded RNA molecules. Once incorporated into RISC they facilitate the cleavage and degradation of its recognized mRNA.


Specificity
The ability of an siRNA or shRNA to distinguish between highly similar target mRNA.


TAT
A lentiviral packaging element required for the efficient elongation of nascent viral transcripts.

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Titer
The number of transducing units per mL.


Transfection
The alteration of the genetic code within a cell by the addition of exogenous genetic sequences, usually refers to plasmid DNA or siRNA introduced through lipid or chemical carriers through the cell membrane.


Transduction
The integration of exogenous genetic sequences into a host genome through viral particles.


U6 promoter
A pol III promoter that allows for the expression of shRNA with a defined termination.


Vesicular stomatitis virus G glycoprotein (VSV-G)
A commonly used envelope protein for pseudotyping lentiviral particles. It is highly fusogenic allowing for broad tropism in mammalian cells.


Viral infectivity factor (Vif)
A lentiviral packaging element that blocks a cellular inhibitor of viral replication.

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Viral protein r (Vpr)
A lentiviral packaging element that participates in the migration of the viral RNA into the nucleus for chromosomal integration.


Viral protein u (Vpu)
A lentiviral packaging element that is localized at the cell membrane, facilitating the viral release.


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