In the construction of genetically engineered mouse models, the plasmid is the most critical delivery vector. It is not only the delivery tool for introducing editing machinery into cells but also the key determinant of editing efficiency and stability. This article, starting from the practical needs of mouse model construction, systematically deconstructs the design logic and essential elements of plasmids.
 

I. What is a Plasmid?
 

A plasmid is an extrachromosomal, double-stranded circular DNA molecule capable of autonomous replication in a host cell (e.g., E. coli, ES cells). In gene editing, it performs two core tasks:
 

In vitro amplification and storage:​ Utilizing bacterial systems (e.g., E. coli) for rapid replication to obtain high-purity DNA.
 

Intracellular delivery and expression:​ Following transfection into ES cells or zygotes, it drives the expression of editing tools to accomplish knockout, knock-in, or point mutation.
 

II. Main Types of Plasmids
 

Plasmids can be classified by host type into prokaryotic vectors, eukaryotic vectors, and viral vectors; or by function into cloning vectors, expression vectors, shuttle vectors, and reporter gene vectors. The core structural elements of different plasmid types are designed based on "functional requirements" and "host adaptability."

III. Core Structural Elements of a Plasmid
 

In constructing genetically engineered mouse models, the plasmid's core structural elements need to integrate two key modules: mammalian expression elements (e.g., CMV promoter, Kozak sequence, polyA signal) and bacterial replication/selection elements (e.g., pUC ori, ampicillin resistance), facilitating in vitro amplification and sequencing verification.
 

1. Origin of Replication (Ori):​ The site where plasmid DNA replication initiates. Its primary function is controlling the initiation of plasmid replication. The pUC ori is a high-copy-number origin, allowing rapid plasmid amplification in E. colifor easy large-scale preparation. If a stable cell line in ES cells is required, the plasmid should contain the SV40 ori, which, combined with the SV40 large T antigen, enables high-copy maintenance.

 

Key features:
 

Sequence Structure:​ The Ori region has high A-T content; its low hydrogen bond stability facilitates DNA unwinding, thereby initiating replication.
 

Copy Number Control:​ The type of Ori (e.g., pUC, ColE1, pSC101) is the primary factor determining plasmid copy number, thereby affecting amplification efficiency.
 

Host Specificity:​ The host range of a plasmid is determined by the compatibility between the Ori sequence and host replication proteins (e.g., E. coliDnaA), categorized as narrow-host-range or broad-host-range.
 

Incompatibility:​ Plasmids carrying identical or highly homologous Oris belong to the same incompatibility group; they compete for replication resources within a cell and cannot stably coexist.
 

Replication Mechanism:​ Ori is the starting point for bidirectional (theta-type) replication. Prokaryotic plasmids typically have a single origin, while eukaryotic chromosomes have multiple replication origins.
 

2. Selection Marker:​ A specific gene sequence on the plasmid that confers antibiotic resistance to the host cell. By culturing in antibiotic-containing media, cells successfully transformed with the target plasmid can be efficiently identified and selected.

Selection markers are divided into "bacterial selection" and "eukaryotic selection":
 

A. Prokaryotic Selection
 

Ampicillin Resistance Gene (AmpR):​ Encodes β-lactamase, which hydrolyzes ampicillin. Used to select E. colicolonies containing the plasmid.
 

Kanamycin Resistance Gene (KanR):​ Encodes aminoglycoside phosphotransferase, inactivating kanamycin.
 

B. Eukaryotic Selection
 

Neomycin Resistance Gene (NeoR):​ Confers resistance to G418. Most commonly used for stable selection in ES cells or mouse fibroblasts to establish stable clones after gene editing.
 

Puromycin Resistance Gene (PuroR):​ Confers resistance to puromycin. Acts rapidly, often used for quick enrichment of successfully transfected cells.
 

Hygromycin B Resistance Gene (HygR):​ Confers resistance to hygromycin. Often used as a second selection marker to distinguish cells in multi-plasmid co-transfection.
 

3. Multiple Cloning Site (MCS):​ Contains recognition sites for multiple restriction endonucleases, allowing a foreign DNA fragment to be inserted into the vector via restriction enzyme digestion and ligation. When a foreign gene is successfully inserted into the MCS, it will be transcribed under the control of the promoter, leading to the synthesis of the target protein within the host cell.
 

4. Gene Editing-Specific Elements

1) Promoters:​ Can be divided into two categories: constitutive and inducible promoters. For editing tools requiring strong, widespread expression, strong constitutive promoters (e.g., CMV, CAG) are chosen. For situations requiring stable, long-term expression in specific cell types or precise spatiotemporal control, cell-type-specific or inducible promoters are selected.
 

CMV Promoter:​ Human cytomegalovirus immediate-early promoter. One of the strongest promoters, widely used for driving high-efficiency expression of Cas9 protein, suitable for transient transfection to achieve high editing efficiency.
 

CAG Promoter:​ Exhibits extremely strong transcriptional activity in the vast majority of mammalian cells and is less prone to epigenetic silencing compared to CMV. It is the preferred promoter for driving high-intensity, broad-spectrum gene expression.
 

EF1α Promoter:​ Human elongation factor 1α promoter. Shows more stable expression, less prone to epigenetic silencing in ES cells and stem cells, suitable for long-term stable expression.
 

2) Translation and Processing Optimization Elements
 

Kozak Sequence:​ Located around the start codon (ATG), optimizes ribosome binding, enhancing the translation efficiency of Cas9 protein.
 

Intron:​ Such as the first intron of EF1α. Studies show that the presence of an intron can significantly enhance mRNA stability and nuclear export, thereby increasing gene expression levels.
 

Polyadenylation Signal (Poly(A) Signal):​ Most nascent eukaryotic mRNAs have a poly(A) tail at their 3' end, e.g., bGH pA (bovine growth hormone polyA) or SV40 pA. Ensures proper mRNA polyadenylation, preventing degradation, and is an essential element for expression vectors.
 

3) Homology Arms and Repair Template (HR Donor)
 

For gene knock-in (KI) or conditional knockout (cKO) models, the plasmid must contain homology arms (500-1000 bp) flanking the edit site. These guide the cell to perform homology-directed repair (HDR) rather than the error-prone NHEJ repair.
 

References:

Makrides SC. Vectors for gene expression in mammalian cells. New Comprehensive Biochemistry. 2003;38:9–26. doi: 10.1016/S0167-7306(03)38002-0. Epub 2004 Jan 7. PMCID: PMC7147855.