gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Regular Plasmid Inducible Gene Expression Vector (Tet-On)
The Tet-On inducible gene expression system is a powerful tool to control the timing of expression of the gene(s) of interest in mammalian cells. Our Tet-On inducible gene expression vectors are designed to achieve nearly complete silencing of a gene of interest in the absence of tetracycline and its analogs (e.g. doxycycline), and strong, rapid expression in response to the addition of tetracycline or one of its analogs (e.g. doxycycline). This is achieved through a multicomponent system which incorporates active silencing by the tTS protein in the absence of tetracycline and strong activation by the rtTA protein in the presence of tetracycline.
Delivering plasmid vectors into mammalian cells by conventional transfection is one of the most widely used procedures in biomedical research. While a number of more sophisticated gene delivery vector systems have been developed over the years such as lentiviral vectors, adenovirus vectors, AAV vectors and piggyBac, conventional plasmid transfection remains the workhorse of gene delivery in many labs. This is largely due to its technical simplicity as well as good efficiency in a wide range of cell types. A key feature of transfection with regular plasmid vectors is that it is transient, with only a very low fraction of cells stably integrating the plasmid in the genome (typically less than 1%).
For further information about this vector system, please refer to the papers listed below.
|Science 268:1766-1769 (1995)||Development of rtTA.|
|J Gene Med. 1:4-12 (1999)||Development of tTS.|
Our Tet-On inducible gene expression vectors are designed to achieve nearly complete silencing of the gene(s) of interest in the absence of tetracycline, and strong, rapid expression in response to the addition of tetracycline. Our vector is optimized for high copy number replication in E. coli and high-efficiency transfection in many mammalian cell lines.
Switch-like gene activation: Unlike rtTA only Tet-On systems that usually have significant leaky expression in the absence of induction, our Tet-On gene expression vectors act as true tetracycline-regulated on-and-off switch for controlling gene expression, which can minimize the background expression without induction and result in high sensitivity and high dynamic range of the tetracycline induction.
Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of viral vector plasmids into live virus.
Very large cargo space: Our regular plasmid vectors can accommodate ~30 kb of total DNA. The plasmid backbone only occupies about 4.6 kb, including Tet-On components, leaving plenty of room to accommodate the user's gene(s) of interest and promoter for driving Tet-regulatory proteins.
High-level expression: The TRE promoter can drive very high levels of expression of the gene(s) of interest in its induced state. Additionally, conventional transfection of plasmids often results in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the gene(s) carried on the vector.
Non-integration of vector DNA: Conventional transfection of plasmid vectors is also referred to as transient transfection because the vector stays mostly as episomal DNA in cells without integration. However, plasmid DNA can integrate permanently into the host genome at a very low frequency (one per 102 to 106 cells depending on cell type). If a drug resistance or fluorescence marker is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection or cell sorting after extended culture.
Limited cell type range: The efficiency of plasmid transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo.
Non-uniformity of gene delivery: Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others may carry very few or none. This is unlike transduction by virus which tends to result in relatively uniform gene delivery into cells.
TRE: Tetracycline-responsive element promoter (2nd generation). This element can be regulated by a class of transcription factors (e.g. tTA, rtTA and tTS) whose activities are dependent on tetracycline or its analogs (e.g. doxycycline).
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of interest is placed here.
SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
Promoter: The promoter chosen to drive expression of the tTS/rtTA cassette.
tTS: Tetracycline-controlled transcriptional silencer. This protein binds to TRE promoter to actively suppress gene transcription only in the absence of tetracycline and its analogs (e.g. doxycycline).
T2A: Self-cleaving 2A peptide from thosea asigna virus that allows multiple proteins to be made from a polycistronic transcript containing multiple ORFs separated by T2A. The cleavage occurs through a putative “ribosomal skipping” mechanism.
rtTA: Reverse tetracycline responsive transcriptional activator M2 (2nd generation). This protein binds to TRE promoter to activate gene transcription only in the presence of tetracycline or its analogs (e.g. doxycycline). It has higher sensitivity to the inducing drug and lower leaky activity in the absence of the drug compared to its predecessor.
BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the tTS/rtTA cassette.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.