Introduction of Vero cell line:
Vero cells are derived from the kidney of a normal adult African green monkey on March 27 in the 1960s, and are one of the more commonly used mammalian continuous cell lines in microbiology and molecular and cell biology research, including studies that involve gene-editing such as gene knockout/knock-in that require CRISPR/Cas9 technology. At its 93rd passage, the cell line was brought to the National Institute of Allergy and Infectious Diseases at the National Institutes of Health in the United States and was provided to the American Type Culture Collection (ATCC) in 1966.
Vero cell line, which is also anchorage-dependent cell line, has been used extensively in virology studies but has also been used in many other applications, including the propagation and study of intracellular bacteria and parasites, and assessment of the effects of chemicals, toxins, and other substances on mammalian cells at the molecular level. Therefore gene-edited Vero cells, such as Vero knockout cell lines, are getting more and more popular in studies in these fields. Besides, Vero cells have been licensed in the United States for production of both live (rotavirus, smallpox) and inactivated (poliovirus) viral vaccines, and throughout the world, Vero cells have been used for the production of several other viruses, including Rabies virus, Reovirus, and Japanese encephalitis virus. These studies are mostly related to Vero CRISPR/cas9 mammalian cells, Vero knockdown cell line, virus packaging, and Vero point mutation cell line. Vero cells can also be used as host cells for eukaryotic parasites such as Trypanosoma.
Figure 1. Vero 76 cells (ATCC #CRL-1587), approximately 95% monolayer. These cells will need to be subcultured within one day.
Vero cells grew well during subculture and found clear cell membrane boundaries and good cytoplasmic transparency. The morphology of Vero cells is relatively complete, and the rate of cell proliferation is relatively fast. Vero cells can be adherent after 24 hours of cell culture after being transferred to flasks. Cells can reach a relatively stationary phase after three days of culture. Cells can grow into a monolayer on the fifth day of culture and cells can be cultured until the twelfth day. It was found that the growth was dense, and the cells began to age until the fourteenth day. The cell growth rate after spinning was slower than before spinning, but the monolayer cells lasted longer than before spinning. No mycoplasma growth and contamination were found during mycoplasma inspection. The results of cell type analysis showed that there were no obvious abnormalities in the karyotype of Vero cells, and there was no significant change in chromosome number.
Applications of Vero cell line:
Vero cells can be used for a variety of research purposes. Vero cells are widely used in the study of molecular mechanisms of viral infections, the production of vaccines, and recombinant proteins. Vero cells were found to be highly sensitive to many types of viruses shortly after they were established, including simian vacuole virus, measles virus, rubella virus, arthropod-borne virus, and adenovirus. It was later found to be susceptible to bacterial toxins, including diphtheria toxin, heat-labile enterotoxins, and Shiga-like toxins. Therefore, Vero cells are suitable for being a gene-customizing model such as CRISPR knockout cell that involves virus infection.
1. Enhancing viral vaccine production using engineered knockout vero cell lines
The global adoption of vaccines to combat disease is hampered by the high cost of vaccine manufacturing. The work described herein follows two previous publications that report a strategy to enhance poliovirus and rotavirus vaccine production through genetic modification of the Vero cell lines used in large-scale vaccine manufacturing. CRISPR/Cas9 gene editing tools were used to knockout Vero target genes previously shown to play a role in polio- and rotavirus production. Subsequently, small-scale models of current industry manufacturing systems were developed and adopted to assess the increases in polio- and rotavirus output by multiple stable knockout cell lines. Unlike previous studies, the Vero knockout cell lines failed to achieve desired target yield increases. These findings suggest that additional research will be required before implementing the genetically engineered Vero cell lines in the manufacturing process for polio- and rotavirus vaccines to be able to supply vaccines at reduced prices.
2. Gene-edited vero cells as rotavirus vaccine substrates
Rotavirus (RV) is the main cause of severe gastroenteritis worldwide and can lead to a large number of gastroenteritis-related illnesses in children under 5 years of age. Oral-attenuated live attenuated RV vaccine can prevent disease, but the high manufacturing cost and maintenance of cold chain identified a subset of viral cell host genes by siRNA. When knocking down RV replication, these antiviral host genes are used alone CRISPR -Cas9 deleted. The results showed that Vero cells with EMX2 gene deletion had higher RV replication and antigen production than other tested Vero cell substrate components, which provided the possibility of improving the production of RV vaccines.
CRISPR/Cas9 knockout Vero cell line strategies
Workflow of knockout Vero cell line
1. E A Govorkova, G Murti, B Meignier, C de Taisne, and R G Webster. African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses.
2. Growth and Maintenance of Vero Cell Lines Nicole C. Ammerman* , Magda Beier-Sexton, and Abdu F. Azad.
3. Nichole Orr-Burks, Jackelyn Murray, Carl d D.Kirkwood. Gene-edited vero cells as rotavirus vaccine substrates.
4. Nichole Orr-Burks, Jackelyn Murray, Weilin Wu, Carl D.Kirkwood, Kyle V. Todd, Les Jones, Abhijeet Bakre, Houping Wang, Baoming Jiang, Ralph A.Tripp. Gene-edited vero cells as rotavirus vaccine substrates