Regular Plasmid Tet Regulatory Protein Expression Vector

Overview

VectorBuilder’s regular plasmid Tet regulatory protein expression vector can be used for simple transfection-based delivery of Tet regulatory protein(s) (tTS, rtTA, etc.) into mammalian cells to help you achieve tetracycline-inducible expression of target genes placed downstream of a tetracycline responsive-element (TRE) promoter.

The Tet-On inducible 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 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. 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 regular plasmid Tet regulatory protein expression vector includes an expression cassette consisting of the Tet regulatory protein(s) driven by a user-selected promoter, the GOI driven by the TRE promoter must be provided using a separate helper vector to achieve tetracycline induced gene expression in the presence of tetracycline, while minimizing leaky expression in the absence 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 below.

References Topic
Science. 268:1766-9 (1995) Development of rtTA
J Gene Med. 1:4-12 (1999) Development of tTS

Highlights

Our regular plasmid Tet regulatory protein expression vector allows highly efficient, transfection-based delivery of Tet regulatory protein(s) into mammalian cells. Using this vector to coexpress the Tet regulatory proteins tTS and rtTA along with the TRE driven GOI can achieve nearly complete silencing of the GOI 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.

Advantages

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 live virus.

Very large cargo space: Our vector can accommodate ~30 kb of total DNA. The plasmid backbone only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.

High-level expression: Conventional transfection of plasmids can often result in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the genes carried on the vector.

Disadvantages

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.

Key components

Promoter: The promoter driving the downstream Tet regulatory protein(s) is placed here.

Kozak: Kozak translation initiation sequence. Facilitates translation initiation of ATG start codon downstream of the Kozak sequence.

ORF:  The open reading frame of the Tet regulatory protein(s) is placed here.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcription termination of the upstream ORF.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transfected with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.