Thursday, September 16, 2021

A powerful weapon to defeat COVID-19 -- Vero cell line | Ubigene

Background


Vero cell line, also known as green monkey kidney cell line, is a non-doubled renal cell line of African green monkey (belongs to Chloroebus). In 1962, Chiba University in Japan isolated the renal epithelial cells of normal adult African green monkeys and obtained the Vero cell line. Vero cell line is a continuous aneuploid cell line, which means that its chromosome number is abnormal. As a continuous cell line, Vero cell line can go through many division cycles without aging. Vero cell line has defective interferon secretion function. Unlike normal mammalian cell lines, they do not secrete interferon when infected by virus α/β. However, they still have interferon- α/β. Therefore, when recombinant interferon is added to its culture medium, they can still respond. So far, Vero cell line is widely used in the study of the molecular mechanism of virus infection and the production of vaccines and recombinant proteins, and are regarded as an ideal cell model for cultivating influenza vaccines and studying the molecular mechanism of virus infection [1]. WHO approved Vero cell line as a vaccine production cell and recommended it as an alternative matrix for influenza vaccine production.

Detailed applications


1.Vero cell line is the most commonly used cell line for the production of viral vectors and vaccines.

Vero cell line is considered to be non-tumorigenic within a limited generation, so it can be used as a matrix for vaccine research. In 2005, Vero cell line was approved by the World Health Organization as a cell line for the production of human vaccines. Vero cell line has been used to produce polio vaccine, rabies vaccine and ACAM200 smallpox vaccine [2].


2.Vero cell line can perform extensive cell characteristic research and the creation of large cell bank

After Vero cell line was isolated, several subcellular lines were derived, such as Vero81, Vero76 and VeroE6. Genomic analysis showed that these cell lines originated from a female African green monkey. Because Vero cell line can be subcultured indefinitely, it can be used for a wide range of cell characteristics and the creation of a large cell bank[3].


3.Vero cell line is widely used to study the molecular mechanism of virus infection

The insufficient expression of interferon in Vero cell line is considered to be one of the reasons why Vero cell line is very sensitive to many viruses, including simian vacuolar virus, measles virus, rubella virus, arthropod carrying virus and adenovirus. Later, it was found that it was also easy to be infected with bacterial toxins, including diphtheria toxin, Heat Intolerant enterotoxin and Shigella like toxin. They do not secrete signal peptide interferon after virus infection, so the antiviral defense mechanism of cells is damaged[3]. This characteristic makes Vero cell line widely used in virology, bacteriology, parasitology and toxicology [3].

Application of CRISPR/Cas9 technology in Vero cell line


Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the pathogen of COVID-19 (COVID-19) in 2019. It has triggered the most influential public health crisis in the 21st century. At this time, more than 7.5 million people around the world had been infected and more than 420,000 people died from infection. And the data is still growing. An effective vaccine is a powerful “weapon” for human to win this “fierce battle”, and determining the host factors necessary for infection is very important for understanding the pathogenesis of COVID-19, revealing the changes of host susceptibility and determining new host oriented therapy, which may be effective for current and future pandemic Coronavirus. CRISPR/Cas9 technology has become one of the main research methods for the precise editing of genome.


Ubigene also has successfully used CRISPR-UTM technology to modify the gene in Vero cell line. Because the gene knockout efficiency of CRISPR-UTM technology is leap to 80%, gene knockout in Vero cell line using CRISPR-UTM technology has a higher success rate than using traditional CRISPR/Cas9 technology. Vero-E6 cell line is highly sensitive to SARS-COV-2 infection and virus induced cytopathic effects. Wei, Jin et al. conducted two independent genome-wide CRISPR screening in Vero-E6 cell line and used Vero-E6 cell line of two different Cas9 nuclease constructs (Cas9-v1 and Cas9-v2). They evaluated 25 of these genes, including pro-viral and antiviral genes (Fig. 1). They found that pro-viral and antiviral genes are of great significance for understanding the pathogenesis, treatment and vaccine design of COVID-19. For example, SMARCA4 can promote the proliferation of alveolar epithelial cells and prevent pulmonary fibrosis, a common disease of SARS-CoV and SARS-CoV-2[4].

Figure 1 Genome wide CRISPR screening can identify genes crucial to SARS-CoV-2 induced cell death

Case Study


1.Study invasion and infection of Vero cell line by porcine epidemic diarrhea virus with CRISPR/Cas9 system.

Porcine Epidemic Diarrhea Virus (PEDV) is a positive single chain virus, belonging to the single chain virus genus of coronavirus subfamily. Aminopeptidase N (APN or CD13) is a type II zinc metalloproteinase, which can mediate a variety of cell physiological processes, including antigen presentation, cell differentiation, cell movement and coronavirus entry. Since porcine APN (pAPN) is the main receptor of porcine enteropathogenic α coronavirus (TGEV). Ji, Chun Miao et al. found that neither hAPN nor pAPN was the functional receptor of PEDV by knocking out the endogenous expression of hAPN or pAPN in Vero cell line. In order to completely exclude potential vAPN production in Vero cell line that may be below the detection limit, they used the CRISPR/Cas9 system to generate vAPN knockout cell line, that is, targeting vAPN-CDS1 with two gRNA/Cas9 complexes and deleting the 160 bp fragment between 290-449 nucleotides downstream of the translation initiation site (Fig. 2). These knockout cells were confirmed by IFA that Vero-vAPNKO1 and Vero-vAPNKO2 lacked the expression of vAPN (Fig. 3a). The results showed that Vero-vAPNKO1 and Vero-vAPNKO2 cell lines showed similar CPE and GFP expression to normal Vero cell line after PEDV-GFP infection, indicating that vAPN knockout had no effect on PEDV entry and infection [5].

Figure 2 vAPN gene knocked out in Vero cell line via CRISPR/Cas9


Figure 3 Time resolved immunofluorescence


2.Development of enhanced RV vaccine substrates via using gene knockout Vero cell line

Rotavirus (RV) is the main cause of severe gastroenteritis worldwide. Nichole et al. used siRNAs to screen host genes that have a negative impact on RV replication, and then knocked out the selected genes through CRISPR/Cas9 gene editing. Compared with wild-type Vero cell line, fully sequenced gene knockout Vero cell line substrates can increase RV replication and RV vaccine antigen expression. The results showed that compared with other tested Vero cell line substrates, Vero cell line with EMX2 gene deletion had higher RV replication and antigen production. Gene knockout Vero cell line can promote the development of enhanced RV vaccine substrate and help to improve RV vaccine production.

Figure 4


3.CRISPR/Cas9 mediated Knock-in HSV DNA genome in Vero cell line

Bacterial Artificial Chromosomes (BACs) is the powerful tool for controlling the large genome of DNA viruses (such as herpesvirus). Because the genome of these DNA viruses (herpesviruses) is too large to be cloned into plasmids. For example, Herpes virus (HSV), a common DNA virus with a genome length of 152 kbp, may cause cheilitis, genital herpes and encephalitis. However, mutant HSV is a candidate for tumor therapy and can be used to kill tumor cells. Suenaga et al. used CRISPR/Cas9 system and Vero cell line as matrix to study mutant HSV. This method can achieve gene knockdown and gene knock-in of HSV without inserting artificial genes into the virus genome, and improve the isolation efficiency of expected mutant virus clones. In addition, CRISPR/Cas9 system can also be applied to other DNA viruses, such as Epstein Barr virus, cytomegalovirus, vaccinia virus and baculovirus. CRISPR/Cas9 system plays a very important role in the study of various types of viruses, including clinical isolated cell lines [6].


Figure 5 Gene specific mutations were introduced into HSV-1 genome by CRISPR/Cas9

So far, Ubigene has used CRISPR-UTM system to successfully modified genes from more than 100 cell lines, including the Vero cell line. And Ubigene provides gene-editing services worldwide. Now Ubigene has time-limited discount of all gene-editing services: KO cell line service, as low as 2021 USD. Ubigene provides over 600 types of in-stock KO cell lines. Over 10,000 in-stock gRNA plasmids, only 80 USD. Lentivirus Packaging, as low as 559 USD. Stable Cell Line, only 1780 USD. KI/Point mutation cell line service, as low as 8480 USD.

Versatile use of Luciferase cell line | Ubigene

Background


Luciferase (Luc) is a general term for a class of enzymes that catalyze the oxidation and luminescence of luciferin or fatty aldehyde in organisms. It comes from the organisms that can emit light in nature.he main application of luciferase in gene expression research is to insert the luciferase gene into the expected cell chromosome by molecular cloning technology, the stable expression of luciferase was obtained through the selection of monoclonal cell technology. Its luminescent principle is that luciferase and luciferase substrate consume ATP in the presence of oxygen and Mg2+ to produce an oxidation reaction, and part of the chemical energy is converted into light energy to release. Because there is no non-specific interference of excitation light, its signal-to-noise ratio is high, and its tissue penetrating ability is significantly stronger than that of green fluorescent protein (GFP).

In 1986, firefly luciferase gene was used as a reporter gene to determine gene expression.Because of its convenience, high sensitivity and high success rate, luciferase gene has been widely used as a reporter gene, including promoter activity, transcription factor, mammalian cell two hybrid tests, and in vivo animal imaging experiment. It has an important influence on many scientific research fields. Ubigene can provide stable Luc cell line to researchers. In addition to the stable expression of luciferase gene, the stable Luc cell line of Ubigene also has the advantages of strong specificity, high sensitivity, high imaging quality, accurate luminous intensity, and quantitative characteristics, which can allow researchers to track their cell lines accurately.

Detailed applications


1.Application in vivo optical imaging experiment of living animals[1]

Bioluminescence imaging technology expressing biotinidase is the main method to study in vivo imaging of living animals. In vivo imaging technology can quickly and accurately screen and identify positive cell lines. The number of positive clones increased and the amount of luminescence increased, then the relationship between the number of positive cell lines and the luminous flux of the bioluminescent imaging system was discussed. The pMX-Luc2 plasmid was constructed by the molecular cloning method and cotransfection with pMD. G plasmid into 293T gag-pol cell line to obtain the retrovirus expressing Luc2 (Fig. 1). The retrovirus was used to infect mouse colon cancer CT26, human small cell lung cancer NCI-H446, human colon cancer HT-29, human ovarian cancer SKOV3, and human hepatoma SMMC-7721 cell lines to establish and screen Luc2 positive cell lines (Fig. 2). Different numbers of cell lines were inoculated in the culture dish, four nude mice were inoculated subcutaneously at different sites (dorsal or caudal vein) with 200μl different concentrations of SKOV3-Luc2 cell line. The luminous flux of the cell line inoculated in vitro and in vivo was detected by the bioluminescence imaging system. The results showed that the retroviral infection of Luc2 was a feasible method to establish the Luc2 positive cell line. The number of Luc2 positive cell line has a significant linear relationship with the luminous flux in the bioluminescent imaging system.


Figure 1. Bioluminescence test for Luc2-positive cell line


Figure 2. Bioluminescence luminous flux in SKOV3-Luc2 cell line (A), and Luminous flux diagram (B)


2.Verify the relationship between target gene and candidate gene[2]

To verify the relationship between FN1 (target gene) and MiR-96-5P (candidate gene), the sequence containing the binding site of FN1 and MiR-96-5P was cloned into luciferase reporter vector Psi-check2. Multiple comparisons showed that compared with the NC group, transfection of 100nM MiR-96-5P inhibitor significantly up-regulated the luciferase activity of cell line transfected with wild-type vector, but there was no significant change in the fluorescence activity of mutant vector (Fig. 3). he results of luciferase analysis showed that FN1 was the direct target gene of MiR-96-5P.

Figure 3. Fluorescence activity of HK2 cell line co transfected with MiR-96-5P inhibitor and WT/ mutant vector


3.Application of the pathological research[3]

The insulin 2 promoter was inserted into the PGL3-Basic vector to construct the plasmid with the luciferase reporter gene and transfection into islets β cell line MIN6, glucose induction was performed to detect insulin secretion by dual luciferase reporter gene system [Fig 4]. In the case of using PGI3-Basic blank plasmid instead of construction plasmid as blank control, the detection value of luciferase does not change with the induction of different concentrations of glucose, but remained at a low level (Fig. 4A), indicating that plasmid transfection had no effect on the test. Renilla luciferase was used as an internal reference to removing the differences between groups caused by the experimental operation. The results showed that (Fig. 4B and C) the expression value of Renilla in each group was basically the same, and the changing trend of each experimental group was the same after removing the influence of internal reference value.

Figure 4 . Effects of different concentrations of glucose on luciferase expression


4. Application in promoter activity analysis[4]

A single secretory luciferase reporter (SSLR) analysis method was developed and validated for promoter analysis. The method uses the early expression level of secretory luciferase associated with the test promoter as an internal standardized control for subsequent analysis of the same promoter (Fig. 5). SSLR analysis was compared with dual-luciferase reporter (DLR) analysis using HMGCR (3-hydroxy-3-methylglutamyl coenzyme A reductase) and LDLR (low-density lipoprotein receptor) promoter structures (Fig. 6). Similar results were obtained by both methods. Comparison of the reactions of HMGCR promoters in SSLR transient analysis is very advantageous compared to that of the same promoter in stable cell lines.


Figure 5. Luciferase activity/per hour after standardization.The data of secreted GLuc were standardized to the initial time point to correct the difference in plasmid transfection efficiency. HeLa cell line was transfected with 250 or 500 ng luciferase reporter for 6 to 9 hours. After removing the transfectant, the secretory luciferase activity was measured every hour. (A) The luciferase activity was measured after transfection with 250 or 500 ng pGluc-promHMGCR. (B) The luciferase activity of pGluc-promHMGCR was standardized for 6 hours. (C) The luciferase activity was measured after transfection with 250 or 500 ng pGluc-promLDLR. (D) The luciferase activity of pGluc-promLDLR was normalized to 6 hours.


Figure 6. Comparison of SSLR analysis and DLR analysis. pGLuc-promHMGCR, pGL3-promHMGCR/pRL-TK, pGL3-promLDLR/pRLTK, or stable pGLuc-promHMGCR HeLa cell line were incubated under the condition of±25-OHC. For SSLR analysis samples, luciferase activity was measured 6 hours before 25-OHC treatment and 16 hours after 25-OHC treatment. (A) The promoter activity of HMGCR and LDLR of ±25-OHC was significantly lower than that of normal control. (B) The HMGCR and LDLR promoter activities of ±25-OHC were normalized to secrete luciferase at 6h and Renilla at DLR. (C) In stable pGLuc-promHMGCRHeLa cell lines A6, B2, and B3, the HMGCR promoter activity of ±25-OHC were detected.


Luciferase cell line shed light on different biological fields. Therefore, Ubigene generated a list of high-quality stable Luciferase cell lines. They can be directly applied to in vivo cell injection to detect tumorigenicity of the cell line. It also has the advantages of strong specificity and high sensitivity, high imaging quality, and accurate quantification of luminous intensity, which allows researchers to track their own cell line accurately .

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