When host cells are transfected with an expression vector but not subjected to selection, the vector DNA is gradually lost as the cells divide, resulting in a relatively short duration of target protein expression. Compared to stable expression systems, transient expression systems do not require the integration of exogenous genes into the genome, thereby avoiding the effects of positional effects that occur during exogenous gene integration.

After the vector is introduced into the host cell, the target gene integrates into the cell’s genome, where it persists through cell passages and enables long-term, stable production of the target protein.

Transcription of the target gene is induced by exogenous small molecules, allowing it to be expressed at specific times or in specific tissues or cell types; upon induction, the expression of the target gene can be significantly enhanced. Currently, regulation systems based on tetracycline or doxycycline, as well as the estrogen receptor-tamoxifen system, are widely used.

Advantages: Short expression cycle, high expression levels, etc.

Disadvantages: High technical requirements, such as plasmid purity and transfection efficiency.

Advantages: Sustained and stable expression of the target protein.

Disadvantages: Stable expression is relatively time-consuming and labor-intensive due to the need for steps such as resistance selection and even forced amplification.

There is no interaction between the factors involved in induction regulation and endogenous cellular factors; therefore, on the one hand, the expression of the target gene itself is not affected by changes in the intracellular environment, and on the other hand, the inducer has no effect on the expression of endogenous genes, thereby ensuring high precision and specificity.

HEK293 cells, derived from the 293 cell line, are cultured in serum-free suspension; CHO-S cells, derived from a cloned isolate of Chinese hamster ovary cells, have been adapted for serum-free suspension culture.

CHO-S and CHO-K1 are derived from CHO; these cell lines have simple culture conditions, moderate adhesion strength, and are relatively easy to transfect.

Including prokaryotic replicons; antibiotic resistance genes; polyclonal sites

Virus sources: SV40 (green monkey vaciovirus); CMV (cytomegalovirus); RSV (rhabdovirus); ADV (adenovirus); LTR (long terminal repeat of retroviruses); Cell sources: HSP (heat shock protein)

Enhancers are typically 100–200 bp in length and, like promoters, consist of several components. Their basic core component is usually 8–12 bp in length and can exist as a single copy or in a series of multiple copies.

Commonly used enhancers: SV40 enhancer; CMV enhancer; RSV enhancer; LTR enhancer

Its function is to enable post-transcriptional cleavage of mRNA and the addition of a polyA tail—that is, polyadenylation. PolyA enhances the stability of mRNA. The two sequences required for polyadenylation are: (1) a GU-rich or U-rich region downstream of the polyA tail; and (2) a 5’–AAUAAA sequence located 11–30 bp upstream of the polyA tail.

Thymidine kinase gene (tk), dihydrofolate reductase gene (dhfr), neomycin resistance gene (neo), and chloramphenicol acetyltransferase gene (cat)

Ad virus particles have a diameter of 70–90 nm and exhibit icosahedral symmetry; the capsid surrounding the core consists of 252 capsomer subunits. The G+C content of the DNA varies among different Ad virus subtypes and is related to the strength of their tumorigenic potential.

A linear, single-stranded DNA virus; a defective, nonpathogenic human parvovirus not associated with human disease

They are a class of RNA animal viruses; their genome consists of two identical positive-strand RNA molecules, and the viral particles contain components such as tRNA primers, reverse transcriptase, RNase H, and integrase. These viruses can be divided into two groups: the foovirus group (e.g., human foovirus (HFV)) and the lentivirus group (e.g., HIV types I and II and oncogenic viruses).

RNA viruses; currently, the most commonly used lentiviral vectors are primarily derived from modified feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) lentiviruses. At present, lentiviral vector systems generally consist of a four-plasmid expression system, comprising two packaging plasmids, a capsid plasmid, and a lentiviral expression plasmid.

Advantages: High safety, high titer, large capacity (36 kb), capable of infecting undifferentiated cells, suitable for direct in vivo application, and capable of infecting tissues in situ; it is currently the most commonly used vector in gene therapy.

Advantages: Wide range of host cells; can infect cells in both the mitotic and post-mitotic phases; wild-type virus can integrate at specific sites; 90% exists in an attached form, while 10% is integrated; low immunogenicity; allows for repeated infection.

Advantages: Broad host range, strong promoter, high infection efficiency, and the ability to continuously pass infected or transformed cells through successive passages.

Advantages: It can infect not only dividing cells but also non-dividing cells; it has a broad host range; it has a large capacity (7–8 kb); it actively infects host cells through transfection and integrates into the genome to maintain long-term stability.

Disadvantages: High immunogenicity and short expression duration; lack of tissue specificity; screening in eukaryotic cells is complex and time-consuming.

Disadvantages: Low insertion capacity for exogenous genes (≤4.7 kb); lack of efficient packaging cells; complex preparation process; random integration; low titer; and potential for immune reactions.

Disadvantages: It cannot integrate into non-dividing cells; it can only package exogenous genes smaller than 10 kb; recombination between the vector and endogenous retroviral sequences can result in the formation of replicating retroviruses; and there is a possibility of carcinogenesis due to random viral integration.

Disadvantages: Integrates only into proliferating cells; random integration carries a risk of mutagenesis; lacks tissue or cell specificity.