The establishment of stable expression cell lines requires analysis of the function and expression of transformation. DNA needs to be stably transfected into the host cell chromosome. After the foreign gene enters the cell, part of it can enter the cell nucleus through the cytoplasm. Depending on the cell type, up to 80% of the foreign DNA entering the nucleus is transiently expressed. In rare cases, the foreign DNA entering the cell is connected to the nucleus through a series of non-homologous molecular recombination, forming a huge structure that is finally integrated into the cell chromosome. The cell genome is partially expressed, so integration does not necessarily mean expression. Only genes integrated into the expression region will be expressed, and the amount of expression of foreign genes integrated into different chromosome segments is also different. Since the uptake, integration, and expression of foreign genes are rare events, stable transfectants are usually screened based on the new phenotype. In general, this new phenotype is provided by the co-transfected antibiotic resistance gene. The aminoglycoside phosphotransferase carried by the bacterial Tn5 transposon sequence (neo resistance gene) can convert G418 into a non-toxic form. G418 (also known as Geneticin) is an aminoglycoside antibiotic with a structure similar to neomycin, gentamicin, and kanamycin. Aminoglycosides bind to ribosomes irreversibly and cause premature termination or mistranslation of translation by destroying their proofreading ability, thereby inhibiting protein synthesis and preventing the growth of sensitive cells. The structure of G418 contains a dibasic cyclool and 2-deoxystreptamine, which are connected to amino sugars through glycosidic bonds. The structural uniqueness of G418 stems from the hydroxyl function at the C-6 position instead of the amino function. This difference enables it to bind specifically to the 80S ribosome complex. Compared with other aminoglycosides that bind non-specifically to eukaryotic cell ribosomes, G418 has a stronger effect in inhibiting eukaryotic cell protein synthesis. The aminoglycoside modifying enzyme, aminoglycoside phosphotransferase (APH), can covalently modify the amino or hydroxyl functions of antibiotics (such as G418), weakening the drug-ribosome interaction, thereby preventing G418 from functioning, which manifests as a drug resistance effect. G418 is toxic to both prokaryotic and eukaryotic cells, including bacteria, yeast, plant and mammalian cells, as well as protozoa and worms. It is the most commonly used selection reagent for stable transfection. When the neo gene is integrated into the appropriate place of the eukaryotic cell genome, it can start the transcription of the sequence encoded by the neo gene into mRNA, thereby obtaining the efficient expression of the resistance product aminoglycoside phosphotransferase, so that the cells acquire resistance and can grow in the selective medium containing G418. This selection property of G418 has been widely used in gene transfer, gene knockout, resistance screening and transgenic animals.
Figure 1. G418 inhibits the eukaryotic ribosomal machinery. (Garreau de Loubresse N, et al.; 2014)
Taking genetic engineering as an example, G418 is often used in the following three directions: 1. Gene transfection screening: In gene transfection experiments, G418 is often used to screen cells that have been successfully transfected with drug-resistant genes. The transfected cells are cultured in a medium containing G418, and cells that have not been successfully transfected will be eliminated. 2. Gene knockout experiments: G418 is also used in gene knockout experiments to study gene function by screening cells carrying specific mutations or deletions. 3. Establishment of stable cell lines: In order to establish cell lines that stably express exogenous genes, researchers will use G418 for long-term screening to ensure that only cells that have been successfully transfected and stably express drug-resistant genes survive. It should be noted that the effective killing concentration of antibiotics varies depending on the cell type, culture medium, growth conditions, cell density, cell metabolic rate, and cell cycle. In order to overcome these variables and ensure success, ideally, the working concentration for each specific culture system should be determined experimentally, as well as the working concentration when new variables are introduced.
Alternate Names:
Geneticin
G-418
G418 sulfate salt
Gentamicin analog
References:
1. Garreau de Loubresse N, et al.; Structural basis for the inhibition of the eukaryotic ribosome. Nature. 2014, 513(7519):517-22.
Highly efficient expression and secretion of human lysozyme using multiple strategies in Pichia pastoris
Biotechnol J.
Authors: Wang Y, Wang B, Gao Y, Nakanishi H, Gao XD, Li Z.
Abstract
Background: Human lysozyme (hLYZ), an emerging antibacterial agent, has extensive application in the food and pharmaceutical industries. However, the source of hLYZ is particularly limited.
Results: To achieve highly efficient expression and secretion of hLYZ in Pichia pastoris, multiple strategies including G418 sulfate screening, signal sequence optimization, vacuolar sorting receptor VPS10 disruption, and chaperones/transcription factors co-expression were applied. The maximal enzyme activity of extracellular hLYZ in a shaking flask was 81,600 ± 5230 U mL-1 , which was about five times of original strain. To further reduce the cost, the optimal medium RDMY was developed and the highest hLYZ activity reached 352,000 ± 16,696.5 U mL-1 in a 5 L fermenter.
Conclusion: This research provides a very useful and cost-effective approach for the hLYZ production in P. pastoris and can also be applied to the production of other recombinant proteins.
Development of a high-efficiency gene knockout system for Pochonia chlamydosporia
Microbiol Res.
Authors: Shen B, Xiao J, Dai L, Huang Y, Mao Z, Lin R, Yao Y, Xie B.
Abstract
The nematophagous fungus Pochonia chlamydosporia, which belongs to the family Clavicipitaceae (Ascomycota: Pezizomycotina: Sordariomycetes: Hypocreales), is a promising biological control agent for root-knot and cyst nematodes. Its biocontrol effect has been confirmed by pot and field trials. The genome sequence of the fungus was completed recently; therefore, genome-wide functional analyses will identify its infection-associated genes. Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150μgml(-1) G418 sulfate, representing a new selectable marker for P. chlamydosporia. The neomycin-resistance gene (neo), which was amplified from the plasmid pKOV21, conferred G418-resistance on the fungus; therefore, it was chosen as the marker gene. We subsequently developed a gene knockout system for P. chlamydosporia using split-marker homologous recombination cassettes with resistance selection and protoplast transformation. The split-marker cassettes were developed using fusion PCR, and involved only two rounds of PCR. The final products comprised two linear constructs. Each construct contained a flanking region of the target gene and two thirds of the neo gene. Alkaline serine protease and chitinase were confirmed to be produced by P. chlamydosporia during infection of nematode eggs and could participate in lysis of the eggshell of nematode eggs. Here, we knocked out one chitinase gene, VFPPC_01099, and two protease genes (VFPPC_10088, VFPPC_06535). We obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC_01099), 13% (VFPPC_10088) and 15% (VFPPC_06535). This efficient and convenient technique will accelerate functional genomic studies in P. chlamydosporia.
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