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 .

Friday, August 6, 2021

Gold standard of biological production -- CHO cell line | Ubigene Biosciences

 




Background


CHO cell line (Chinese hamster ovary cell) is derived from the ovaries of the female Chinese hamster acquired by Theodore Puck in 1957. CHO cell line belongs to epithelial cell lines, usually adherent growth, but also suspension growth. Among other special factors (e.g.using γ-ray mutducing CHO cell line, a new cell line CHO-DG44, is produced. These DHFR deficient strains require glycine, hypoxanthine, and thymidine nucleoside to grow), CHO cell line is easy to grow in large-scale cell culture under specified conditions.And it allows post-translational modification of human biosimilar, these characteristics make CHO cell line a powerful biological factory. Its unique combination of beneficial quality characteristics makes it an ideal cell line for producing many proteins. CHO cell line is commonly used in biological and medical research and is commercially used to produce therapeutic proteins. CHO cell line is currently widely used in genetics, toxicity screening, as well as nutritional aspects and gene expression, particularly CHO cell line for recombinant protein therapeutics and the most commonly used mammalian hosts used in industrial production. CHO cell line is the gold standard for the production of biological agents. In addition, the CHO cell line does not synthesize proline and express the epidermal growth factor receptor (EGFR), so the CHO cell line is ideal for studying various EGFR mutations.[1]

Applications of CHO cell line


1. CHO cell line is the most common mammalian cell line used for mass production of therapeutic proteins.

Protein therapy produced by mammalian cell lines changes the pattern of human medical levels. Mammalian cell-based biological processes have been used to manufacture viral vaccines, diagnose and treat proteins. In the production of protein agents, cell lines are the main host of protein production. The most widely used host mammalian cell lines are the Chinese hamster ovary (CHO) cell line and mouse myeloma cell line. After long-term domestication and transformation, the CHO cell line has now developed several CHO cell lines, common with CHO-K1, CHO-S, CHO-DXB11, CHO-DG44, etc. CHO-S cell line is also a wild-type cell strain, both with CHO-K1 from the original e CHO cell line. CHO cell line is the preferred manufacturing host cell line for more than 70% of protein drugs in the market because the CHO cell line is able to grow at high density in serum-free suspension culture and can maintain high levels of protein expression during a prolonged fermentation cycle. In addition, the CHO cell line can be mixed with complex glycans in externally expressed proteins, typical glycoproteins containing only a few N-linked oligoglycans, aiding in protein folding and intracellular transport. When these glycans terminate in sialic acid residues, they increase resistance to proteolhydrolysis and prolong serum half-life in the body. Due to these physical and pharmacokinetics advantages, recombinant glycoprotein expression focuses on maximizing the number of complex glycans containing sialic acid in each molecule. Thus, the CHO cell line also becomes a host cell line widely used for recombinant protein expression[2].


2.CHO cell line allows post-translational modifications to recombinant proteins

CHO cell line, as mammalian cell line, is closer to human cell protein compared to post-translational modification of yeast cell line and Drosophila cell line S9 in the eukaryotic expression system. CHO cell line not only has the characteristics of adherent growth but also can conduct high-density suspension culture in serum-protein-free medium, suitable for large-scale industrial production. CHO cell line is subordinate to fibroblasts, with less endogenous protein secretion, and can efficiently isolate and purify recombinant proteins. In addition, the CHO cell line has high expression and amplification of recombinant proteins. At the same time, the CHO cell line also has its own inevitable genetic defects, its intracellular non-free proline synthesis gene, resulting in the inability to convert glutamate into glutamate-γ-semilaldehyde, and the addition of assisted-growing L-proline to the corresponding medium [2].

The Application of CRISPR/Cas9 Technology in CHO Cell Line


CRISPR/Cas9 technology has been widely used for fast, simple, and efficient mammalian genome engineering. CRISPR/Cas9 has become an efficient tool for generating gene breaks in CHO cell line, and CRISPR/Cas9 plays a very important role in expanding product diversity, controlling and improving product quality, and production of recombinant proteins in CHO cell line engineering. The high efficiency, stability, ease of use, and low cost of CRISPR/Cas9 systems make it an attractive gene-editing tool in academia and industry. The introduction of CRISPR/Cas9 systems in CHO cell line and combined with CRISPy design tools will significantly accelerate the pace of CHO cell line gene-editing and can increase the improvement rate of CHO cell line, thus improving the production and quality of biological drugs [3].

Ubigene has successfully modified genes in the CHO-K1 cell line, and Ubigene’s technical team has developed our CRSIPR-UTM technology via 12 years of experience in gene editing. Our KO CHO cell line service comes with a knockout efficiency as high as 80%. Ubigene provides gene-editing services worldwide, including KO, KI, Point mutation cell lines, plasmid construction, and virus packaging.


Figure 1

Detailed Applications of gene-editing in CHO cell line


1.Gene knockout in CHO cell line

CHO cell line is the preferred host for the biological pharmaceutical industry to produce complex pharmaceutical proteins, and the genomic engineering of the CHO cell line will benefit product production and stability. To verify the effects of Dnamt3a KO on the stability of transgene expression, Long et al. tested the knockout efficiency of Dnmt3a, encoding a protein involving DNA methyltransferase, polycolonies of the 3a-30 and control CHO-K1 cells stably transfected with CMV or EF1α were passaged under selection pressure in the presence (G418+) or absence (G418−) of G418 for 60 passages. The MFIs were detected to evaluate the intensity values of the expressed eGFP at 10, 20, 30, 40, 50, and 60 passages. The Dnmt3a-deficient 3a-30 cell line transfected with CMV exhibited the most stable and the highest expression levels regardless of the presence or absence of G418 (Fig.1). The transgene expression levels in the 3a-30 and normal CHO cells stably transfected with EF1α exhibited a loss of productivity during long-term culture. The results indicated that the Dnmt3a KO in the CHO cell line can enhance the long-term stability of recombinant protein expression by using CMV for at least 50 passages (Fig.1 B)[4].


Figure 2 Level of Dnmt3a expression in wild-type (WT) and knockout (KO) CHO-K1 cell line.


2.Gene knock-in in CHO cell line

In the CHO cell line, programmable nuclease-mediated gene knock-in has mainly been performed by NHEJ-dependent methods. Sakuma et al. reported the purposeful examples of knocking-in the large DNA using transcription activator-like effector nuclease (TALEN) and the PITCh system (carrying a single-chain Fv-Fc (scFv-Fc), along with DsRed and puromycin resistance gene (PuroR))[5].

To estimate the capacity and efficiency of gene knock-in at the HPRT1 locus in CHO-K1 cell line, Sakuma et al. first performed whole plasmid integration using the TALEN-mediated PITCh system, which was previously proven to work well in human cell lines. DsRed and PuroR gene cassettes, driven by elongation factor 1α (EF-1α) and Simian virus 40(SV40) promoters, respectively, were independently placed in the donor plasmid, to easily screen the donor-incorporated cell lines (Fig. 4a). However, these gene cassettes can work even if the plasmid is integrated into the genome via random integration. Subsequently, a modified TALEN target sequence, enabling MMEJ-mediated PITCh, was added to the donor plasmid. The modified TALEN site contains a different spacer sequence from the original target site on the genome, as shown in Figure 4b. After the occurrence of DSBs at each target site. Ideally, 9-bp microhomologies can be utilized for MMEJ-mediated integration (Fig. 4b) [5].


Figure 3 The fluorescent image


Figure 4 The 5 'and 3' connection sequences of the Knock-in cell clones


CRISPR/Cas9 technology can also achieve the above experimental objectives. In addition, CRISPR/cas9 technology is simpler and more efficient than traditional technologies(e.g.TALEN)! The CRISPR-UTM technology of Ubigene is exclusively developed based on traditional CRISPR/Cas9 technology. And gene knockout efficiency is up to 60% -80%, other gene-editing experiments can also be completed efficiently.


3.Case of overexpression of Human-source protein in CHO cell line

Mutations in the hGHR gene are the main cause of Laron syndrome in order to construct genes hGHR associated with stably expressing congenital GH insensitive syndrome as well as CHO cell line of mutants (hGHR-E42K, hGHR-H56R). Lihua et al. used PUC-hGHR plasmid, fixed mutations obtaining two mutants (hGHR-E42K, hGHR-H56R), restricted enzyme cutting cloned the target gene to the eukaryotic carrier pcDNA3.1/ (zeo +), the recombinant cell lines were then transfected to CHO cell line with Lipofectamine2000, stable cell groups expressing hGHR and mutants through Zeocin resistance screening, and the hGHR, STAT5-P expression levels were confirmed with RT-qPCR and Western Blot. The results showed that sequencing verified the successful introduction of E42K and H56R missense mutations in hGHR and cloned to eukaryotic expression carriers, successfully detected mRNA and proteins of hGHR and mutants in CHO cell line after transfection and a lower phosphorylated STAT5 protein expression in the mutant cell line (hGHR-E42K, hGHR-H56R) than in wild cell strains. Therefore, at phosphorylation STAT5 levels after GHR mutations, it was found that both phosphorylation STAT5α, STAT5βprotein expression was significantly lower than wild-type GHR, explained the intrinsic mechanism of growth retardation in patients with GHR mutations [6].


Figure 5 Peak sequencing map of hGHR-E42K and hGHR-H56R

Figure 6 Detecting the expression of WT hGHR and mutant in CHO cell line by using RT-qPCR


So far, Ubigene has utilized CRISPR-U ™ technology to successfully modified genes from more than 100 cell lines, including the CHO cell line. And Ubigene provides gene-editing services worldwide.

Wednesday, July 28, 2021

Gene Point Mutation/Knock-in Cell Lineas low as $8480 | Ubigene

 

Thursday, July 22, 2021

Ubigene Lentivirus Packaging/Stable Cell Line service as low as $559, 500 USD off on every 5K spent









Monday, July 19, 2021

Red Cotton™ Gene Knockout Project | Ubigene





Friday, July 16, 2021

Well-known SW480 cell line is not “simple” | Ubigene

 

Background

SW480 cell line was isolated from a 50-year-old male patient with in-situ rectal adenocarcinoma. SW480 cell line was passaged to the 91st generation when A·Leibovitz submitted it to ATCC. So far, SW480 cell line is a relatively complete cell characterization strain in many colon cancer cell lines, and its biological and genetic characteristics have been reported in many literatures. SW480 cell line belongs to adherent culture and presents epithelioid (Fig. 1), it has the following features:

(1)Carcinoembryonic antigen is positive

(2)Keratin immunoperoxidase staining is positive

(3)G of 273 codon of p53 gene → Arg is caused by A mutation →His replacement, C of 309 codon → T mutation leads to Pro → Ser replacement;

(4)The expression of oncogenes ( c-Myc, Kras, Hras, Nras, Myb, Sis, and Fos) were positive. The 12 codon missense mutation of Kras oncogene can be used as a positive control for PCR detection;

(5)No expression of cytolytic enzyme.

Figure 1

Reference: https://www.atcc.org/products/ccl-228#detailed-product-information


Applications and Prospect

Colorectal cancer (CRC) is one of the most common digestive system malignant tumors. The incidence rate of colorectal cancer places third in malignant tumors, and it has the second-highest mortality rate. According to GLOBOCAN data, there are 1.85 million new cases of colorectal cancer in 2018, and about 880,000 people died, which brought great threat to human health. Both Kras mutation and P53 mutation carried by SW480 cell line are pathogenic mutations with high frequency. Therefore, SW480 cell line can be used as a typical colon cancer cell model for studying the carcinogenic molecular mechanism of Kras and P53 mutations and establish drug screening platform. In addition, it can be used as a positive control for PCR identification of these mutations.

Detailed Applications

It is used to establish 3D culture model to study the mechanism of tumorigenesis

miRNAs are widely involved in tumorigenesis and biological regulation of tumor stem cell lines, and they are potential therapeutic targets. Masazumi Sakaguchi et al. used SW480 cell line in 3D culture to simulate the growth of colon cancer cells, and used this model to test the effect of miR-137 on tumor growth. This study found that miR-137 regulates the growth of tumor cells by targeting DCLK1, which reveals that miR-137 may become a drug target.(Fig.2)


Figure 2

Biomarkers for potential tumor diagnosis:

Omentin-1 is a protein associated with colorectal cancer. Epidemiological and clinical studies have shown that the level of omentin-1 in blood is significantly increased in patients with CRC. However, the reason for the up regulation of Omentin-1 and whether it is expressed in colorectal cancer cells have not been determined. Yaqin Zhang et al. used SW480 cell line and HCT116 cell line as colorectal cancer models to explore this problem at the transcription, translation and secretion levels. The results showed that Omnitin-1 was up-regulated in tumor cells and secreted to extracellular (Fig. 3).



Figure 3


Study the effect of Kras mutation on Correlation and potential strategy of drug resistance in tumor

In colorectal cancer, the activation of RAS-RAF and PIK3CA-AKT pathway is related to the drug resistance of EGFR mAbs. In order to study the molecular mechanism of drug resistance, Stefania Napolitano et al. used several colorectal cancer cell lines with Kras mutation, such as SW480O, LOVO, HCT116, to explore the antitumor effect of related drugs. The results showed that cetuximab combined with regorafenib could overcome the resistance of anti EGFR antibody (Fig. 4).


Figure 4

Case study of CRISPR/Cas9 mediated SW480 modification

CRISPR/Cas9 has been widely used in disease modeling, drug target intervention and treatment development. A number of studies have used CRISPR/Cas9 to modify the gene in SW480 cell line and other colorectal cancer cell lines to explore the biological functions and potential intervention targets of Ras, P53 and other signaling pathways in the development of colorectal cancer.

Ubigene has successfully generated KO SW480 cell line for many customers, and guarantees that 100% refund if there is protein residue. Now Ubigene has time-limited discount of gene KO cell line service, as low as 3780 USD. There is only a week left. Click here for more information >


To study the role of m6A modification of RNA in colorectal cancer, METTL3 knockout SW480 cell line is generated.

Jihao Xu et al. generated METTL3 knockout SW480 cell line with CRISPR/Cas9 technology, and found that METTL3 knockout resulted in the slowing down of SOCS2 mRNA degradation rate and up-regulation of its expression, this results to a significant inhibition of cell proliferation.(Fig.5) The study well exemplified the m6A modification of RNA as an important participant in the tumorigenic process of colorectal cancer.


Figure 5


CRISPR/Cas9 technology introduces SNP rs6854845G>T mutation into SW480 , to explore the effect of the mutation on chromosome and gene expression:

Single nucleotide polymorphisms (SNPs) are closely associated with diseases, including cancer. Zhu Cong et al. used CRISPR/Cas9 technology to introduce SNP rs6854845 G>T mutation in primary colon epithelial cell lines, SW480 cell line and HCT116 cell line, and detected the effect of this mutation on chromosome high-level structure and gene expression. The results showed that this mutation only affected the advanced chromatin structure and gene expression of normal colonic epithelial cell lines, which was tolerated by SW480 and HCT116 colonic cancer cell lines. It is suggested that carcinogenesis could escape the regulation of chromatin conformation and gene expression by normal polymorphism.(Fig.6)


Figure 6

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CRISPR/Cas9 technology has played a key role in the researches of cancer treatment. As a new gene-editing tool, it does have unique advantages. Therefore, Ubigene independently developed CRISPR-U™ Technology, which is 10-20 times more efficient than traditional gene editing.

So far, Ubigene has used CRISPR-U ™ technology to successfully modified genes from more than 100 cell lines, including the SW480 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 3780 USDKI/Point mutation cell line service, as low as 8480 USD gRNA plasmids, as low as 80 USD


Last week to join us, enjoy this great deal >;


Wednesday, July 7, 2021

Gene editing in BEAS-2B cell line facilitates respiratory researches | Ubigene Biosciences

1.Background


Human bronchial epithelium cell line (BEAS-2B) was derived from normal bronchial epithelium obtained from the autopsy of non-cancerous individuals. This cell line comes from the ATCC CRL-9609, it is also known as bronchial epithelial cell line. The BEAS-2B cell line was initially infected by an adenovirus 12-SV40 hybrid virus, and the immortalized cell line of BEAS-2B established by continuous cell passage. BEAS-2B cell retains the ability to differentiate from serum response, this ability can be used to screen chemical or biological agents that induce or affect differentiation and carcinogenesis. Therefore, BEAS-2B cells are the ideal cell model for the study of drug metabolic activation and respiratory tumors and molecular mechanisms.


2.Applications of BEAS-2B cell line


2.1 The BEAS-2B cells shared a similar potential with hMSCs on both osteogenic and adipogenic differentiation

After culturing for 21 days under each differentiation induction condition, Oil Red O was used to stain lipid vacuoles in the differentiated adipocytes and Alizarin Red S was used to stain calcium deposits in the differentiated osteocytes. The result shows that both BEAS-2B and hMSC1 cells showed an almost equivalent positive staining for either osteogenesis or adipogenesis. These results indicate that, similar to hMSC1, BEAS-2B exhibited a strong capability of differentiating into osteocytes and adipocytes after each differentiation induction .


2.2 The BEAS-2B can be used to screen chemical and biological agents.

BEAS-2B has been extensively used to study cellular and molecular mechanisms involved in lung carcinogenesis, including the role of epithelial-mesenchymal transition (EMT) in lung carcinogenesis, as well as pneumococcal infections. In addition, the BEAS-2B cell line has been utilized as an in vitro cell model for assaying or screening various chemicals and biological agents with potential pulmonary toxicity or lung carcinogenicity.


3.CRISPR-U ™ mediated Gene editing in BEAS-2B cell line


Genome editing and the creation of cell models can advance researches in the area of functional genomics, signaling pathways, metabolism, cell death, drug discovery, drug response, and cancer research, etc. BEAS-2B is the ideal cell model for the study of drug metabolic activation and respiratory tumors and molecular mechanisms. So far, CRISPR/Cas9 technology has been used by many researchers to study cancer characteristics, disclosure of drug resistance mechanism, cancer treatment, cell death research, functional genomics, signaling pathway, drug discovery, drug response, and cell therapy, etc. CRISPR-U™ (based on CRISPR/Cas9 technology), developed by Ubigene, is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve gene editing in vitro and in vivo. It has great advantages for gene editing of BEAS-2B cells.

Figure 1 Gene editing in BEAS-2B cells with CRISPR-U™


3.1 Gene knockout in BEAS-2B cell line with CRISPR-U™

gRNA and Cas9 would be transferred into cells by virus transduction, liposome transfection, or nucleofection. After drug screening, single clones would be isolated. Positive clones would be validated by sequencing. Specific case is as shown below:

Figure 2 Knockout Strategies of BEAS-2B cell line


P53 gene is an important factor in the malignant transformation of BEAS-2B cells by radon. The P53 gene was knocked out in human normal lung epithelial cells (BEAS-2B) and radon was treated for gene knockout BEAS-2B cells and normal BEAS-2B cells, respectively. The researchers found earlier malignant transformation of BEAS-2B cells from radon after knocking the P53 gene. The BEAS-2B cell (P53 KO-Rn) proliferate faster (BEAS-2B <0.05), earlier cloning (P <0.05), more invasive (P <0.05), and earlier changes in mRNA expression levels of epithelial-interstitial transformation (EMT) -related genes (P <0.05). The results show that radon irradiation leads to malignant transformation of BEAS-2B cells and that P53 plays an important role in radon gas-induced lung cancer.

Reference :http://www.paper.edu.cn/releasepaper/content/201910-45



3.2  Gene KI/point mutation BEAS-2B cell line

BEAS-2B cells would be co-transfected with gRNA, Cas9 and donor vector by electroporation, and then screened. After drug screening, single clones would be generated. Positive clones would be validated by sequencing. Specific cases are as shown below:

Figure 3 Knock-in Strategies of BEAS-2B cell line


Figure 4 Point mutation Strategies of BEAS-2B cell line



(1)Case of point mutation cell line-1

To study the specific influence of NRASQ61K mutant on angiogenesis, NRASQ61K  was knocked into normal (non-neoplastic) human bronchial epithelial cells (BEAS-2B). Compared to wild type (WT) cells, NRASQ61K expressing cells growing on CAM were characterized by a disorganized vasculature with several hemorrhagic areas. To confirm the proangiogenic effects of NRASQ61K in vivo, a Matrigel plug assay was performed in athymic nude mice. As shown in Figure 5B, NRASQ61K -expressing cells demonstrated a marked increase in angiogenesis in the plugs, as indicated by a bright red color. However, angiogenesis was negligible and hemoglobin levels were lower in the WT cells. As shown in Figure 5C, cells spontaneously form capillary-like structures on Matrigel, which is a critical step in tumor angiogenesis. Therefore, NRASQ61K expression has the effect on tubulogenesis.


Figure 5 Knock-in of NRASQ61K into BEAS-2B



(2)Case of point mutation cell line-2

Arsenite Exposure Led to Activation of NFAT but Not AP-1 and NFκB in Beas-2B Cells, the COX-2 gene promoter region contains NFκB, AP-1, and NFAT binding sites, which can be recognized by these transcription factors and in turn lead to COX-2 transcription. To determine whether NFAT regulated COX-2 expression through direct binding to the COX-2 promoter region, point mutation of the two NFAT binding sites in the promoter region of COX-2-Luc reporter was carried out by CRISPR/Cas9. The results demonstrated that this mutation resulted in impairment of the COX-2 transcription induced by arsenite exposure. And the researchers found that it may be noted that NFAT activation by arsenite in the time course studies reached a peak earlier, as compared with that of COX-2 induction. Because the exposure of arsenite only causes NFAT activation in BEAS-2B cells, not AP-1 and NFκB activation. Thus, the activation of NFAT by arsenite is the reason for COX-2 induction in BEAS-2B cells.


In addition, to confirm the important role of NFAT3 in arsenite-induced COX-2 expression in BEAS-2B cells, the researchers built and used the siNFAT3. They used the cells for 10 μm NFAT selective inhibitors were pretreated and then exposed to 20μm arsenite. The results demonstrated that stable transfection of siNFAT3 in Beas-2B cells resulted in a dramatic reduction of NFAT3 protein expression in Beas-2B cells (Fig. 6b). Specific knockdown of NFAT3 expression by siNFAT3 blocked arsenite-induced COX-2 transcription and protein expression (Fig. 6, e and h).

These results distinctly demonstrate that NFAT3 is a major mediator for COX-2 induction by arsenite in Beas-2B cells.

Figure 6 Point mutation cases of BEAS-2B cell line

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