Regular Plasmid Inducible Gene Expression Vector (Low Leak)

The Tet-On inducible gene expression system is a powerful tool to control the timing of expression of the gene(s) of interest (GOI) in mammalian cells. Our Tet-On inducible gene expression vectors are designed to achieve nearly complete silencing of a GOI in the absence of tetracycline or its analogs (e.g. doxycycline), and strong, rapid expression in response to the addition of tetracycline or 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. In the absence of tetracycline, the tTS protein derived from the fusion of TetR (Tet repressor protein) and KRAB-AB (the transcriptional repressor domain of Kid-1 protein) binds to the TRE promoter, leading to the active suppression of gene transcription. The rtTA protein, on the other hand, derived from the fusion of a mutant Tet repressor and VP16 (the transcription activator domain of virion protein 16 of herpes simplex virus), binds to the TRE promoter to activate gene transcription only in the presence of tetracycline.

While our standard Tet-On vector system expresses tTS and rtTA as a fusion protein, which acts as a gene activation switch, the low leak version of our Tet-On vector is designed to allow tissue-specific induction of target transgenes in the presence of tetracycline, while minimizing leaky expression in non-target tissues in the absence of tetracycline. There are three expression cassettes in this vector: 1) the GOI driven by the TRE promoter, 2) the tTS gene driven by a ubiquitous promoter, and 3) the rtTA gene driven by a user-selected tissue-specific promoter. In the absence of tetracycline, the tTS, ubiquitously expressed in all tissues, binds to the TRE promoter with high affinity thereby suppressing the GOI expression in all tissues. In the presence of tetracycline, the rtTA which is specifically expressed in the target tissue, can bind to the TRE promoter to activate the GOI expression only in the target tissue.

The regular plasmid Tet-On vector can be delivered to a wide variety of mammalian cells by conventional transfection. Delivering plasmid vectors into mammalian cells by conventional transfection is one of the most widely used procedures in biomedical research. While several more sophisticated gene delivery vector systems have been developed over the years such as lentivirus, AAV, adenovirus 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 below.

Switch-like gene activation: Unlike Tet-On vectors expressing only rtTA 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.

Minimized leaky expression in non-target tissues: The low leak version of our Tet-On vector incorporates a ubiquitous CBh promoter to drive the expression of the tTS protein, which binds to TRE in the absence of tetracycline thereby inhibiting transgene expression in non-target tissues.

Tissue-specific induction: The low leak version of our Tet-On vector utilizes a user-selected tissue-specific promoter for driving tetracycline-induced gene expression specifically in the target tissues of interest.

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 occupies only about 5.2 kb, including the Tet-On components, leaving plenty of room to accommodate the user's GOI and promoter for driving Tet-regulatory proteins.

High-level expression: The TRE promoter can drive very high levels of expression of the GOI 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 promoter: Tetracycline-responsive element promoter. 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.

rBG pA: Rabbit beta-globin polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

Promoter: The tissue-specific promoter chosen to drive expression of the rtTA protein.

rtTA: Reverse tetracycline responsive transcriptional activator M2. 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.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream rtTA protein.

CBh promoter: CMV early enhancer fused to modified chicken β-actin promoter. This drives the expression of the downstream tTS protein.

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).

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream tTS protein.

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.