Regular Plasmid Gene Expression Vector

レンチウイルスベクター概要

Delivering plasmid vectors into eukaryotic cells by conventional transfection is one of the most widely used procedures in biomedical research. While several other sophisticated gene delivery vector systems have been developed over the years, such as lentiviral and transposon-based vectors, conventional plasmid transfection remains the workhorse of gene delivery in many labs. This is largely due to its technical simplicity as well as efficiency in a wide range of cell types. A key feature of transfection with regular plasmid vectors is that it is transient, with a very low fraction of transfected cells stably integrating the plasmid into the genome (typically less than 1%).

For further information about this vector system, please refer to the papers below.

レンチウイルスベクターの特長

Our vector is optimized for high copy number replication in E. coli and high-efficiency transfection. Cells transfected with the vector can be selected and/or visualized based on the marker gene expression chosen by the user.

レンチウイルスベクターのメリット

Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward and 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.

レンチウイルスベクターのデメリット

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 depending on the 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, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and is rarely used in vivo.

Non-uniformity of gene delivery: Although a successful transfection can result in a 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 carry very few or none. This is unlike transduction by virus-based vectors, or transfection using transposon vectors, which tend to result in relatively uniform gene delivery into cells.

ベクタービルダーのレンチウイルスプラスミドベクターの基本コンポーネント

Promoter: The promoter that drives your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed 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.

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 transduced with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation. 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.