A549 ko cell line | 20x Efficiency | Ubigene

The A549 cell line is a human non-small cell lung cancer cell line that was established in 1972. Scientists transferred and cultured this cell line through an explant tumor of adenocarcinomic lung tissue of a 58-year-old Caucasian male. The A549 cells found in lung tissue are squamous, are responsible for the diffusion of water and electrolytes throughout the alveoli, and can also synthesize lecithin containing highly unsaturated fatty acids through the citicoline pathway. This cell line tends to be less aggressive and spread less quickly than small cell lung carcinoma (SCLC) but proves to be more common, accounting for 85-88% of all cases of lung cancer. A549 cells have become a gene knockout cell model of type II alveolar epithelial cells, which means the A549 KO cell line is practical in studying the metabolic process of lung tissue and the possible mechanism of drug delivery to tissues. This cell line is currently used as both in vitro and in vivo models for studying lung cancer and developing drug therapies through some gene-editing technologies such as CRISPR/Cas 9, which makes A549 a suitable cell line for gene knockout/ knockin and other gene-customizing processes.

 

The rapid multiplication of A549 cells can be attributed to the significant expression of cyclooxygenase2. When A549 cells are cultured in vitro, they usually grow into a single layer of cells attached to or closely attached to the medium. When the growing time is long enough, A549 cells will go through cell differentiation. A549 cells can also be used for virus research and related protein expression changes. Additionally, since these cells are suitable transfection hosts, they have been used as a test place for paclitaxel and bevacizumab to develop new lung cancer drugs. 

Creation of NRF2-Knockout Clonal A549 Cell Lines Using a CRISPR-Directed Gene-Editing Approach

 

It is becoming increasingly apparent that CRISPR-directed gene editing will have a significant impact on the development of new therapeutic approaches to cancer and inherited diseases. With an increasing focus on the development of combinatorial approaches for cancer treatment, it is critical to establish the fact that gene-editing technology can knock out a target gene. Researchers utilized CRISPR/Cas9 to functionally disable the NRF2 gene in A549 cells, the lung cancer cells, by disrupting the NRF2 nuclear export signal (NES) domain. The protein is largely blocked from transiting into the nucleus after translation. In tissue culture, A549 cells with this gene knockout were found to have a reduced phenotype and are more sensitive to chemotherapeutic agents, such as cisplatin and carboplatin. These observations were confirmed in xenograft mouse models wherein the homozygous A549 knockout cells proliferate at a comparatively slower rate than the wild-type cells, even in the absence of drug treatment. Tumor growth was arrested for a period of 16 days, with a dramatic decrease in tumor volume being observed in samples receiving the combined action of CRISPR-directed gene editing and chemotherapy.

 

CRISPR/Cas9 gene-editing technology can identify and execute DNA cleavage, at specific sites within the chromosome, at surprisingly high efficiency and improved precision. The natural activity of CRISPR/Cas9 is to disable a viral genome infecting a bacterial cell, and subsequent genetic reengineering of CRISPR/Cas9 function in human cells presents the possibility of disabling human genes at a significant frequency. researchers utilized specific gene disruption catalyzed by CRISPR/Cas9 to improve the effectiveness of commonly used anticancer treatments, such as chemotherapy or immunotherapy.

 

In this case, researchers targeted theNRF2 gene because it is a central regulator of cellular detoxification and response to oxidative and electrophilic stresses. NRF2 expression increases when the cell enters a stressful environment, such as encountering a toxic substance. Thus, by disrupting NRF2, the result suggested that chemotherapeutic agents, such as cisplatin and carboplatin, would work more effectively and at lower dosages. In the broader sense, such an approach would ultimately lead to a reduced level of chemotherapy required to produce the same tumor-killing activity, leading to an improvement in the quality of life of a cancer patient. The well-established non-small-cell lung adenocarcinoma cell line A549 harbors a mutation in the Kelch domain of KEAP1 causing the overexpression of NRF2, and it has been used often as a gold standard for the discovery of novel therapeutic agents directed against cancer.

 

Knockout of GluIIβ using CRISPR/Cas9-mediated genome editing inhibits growth and metastatic potential of A549 cells by inhibiting receptor tyrosine kinase activities

 

Glucosidase II (GluII) plays a major role in regulating post-translation modification of N-linked glycoproteins. The expression of glucosidase II beta subunit (GluIIβ) was significantly increased in lung tumor tissues and its suppression triggers autophagy and/or apoptosis. Researchers investigated the role of GluIIβ in cell growth, metastatic potential, and receptor tyrosine kinases (RTKs) signaling activity in lung carcinoma cell lines. Therefore, CRISPR-CAS9 technology was used to knockout the GluIIβ encoding gene (PRKSH) in cell line A549, the lung carcinoma cells. These GluIIβ knockout A549 lung cancer cell lines were established by CRISPR/Cas9-mediated genome editing. 

 

GluII β knockout A549 cells exhibited drastically slower growth rates in comparison to non-target transfected cells, particularly with lower concentrations of fetal bovine serum, indicating impairment of their ability to survive under nutritional deprivation. Cell migration and anchorage-independent growth, the fundamental components of cancer cell metastasis, were significantly decreased in GluIIβ knockout A549 cells. Knockout of GluIIβ increased the sensitivity of these lung cancer cells to cisplatin but reduced their sensitivity to gefitinib. Interestingly, knocking out of GluIIβ lowered overall RTK signaling activities to less than half of those in non-target transfected cells, which could represent a novel strategy for blocking multiple RTKs in tumor cells in an effort to improve lung cancer treatment.

Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

Make genome editing easier is the goal of Ubigene. We developed CRISPR-U™ (based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. With CRISPR-U™, Ubigene has successfully edit genes on more than 100 cell lines.

Ubigene developed CRISPR-B™  which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI).

Ubigene has more than 400 types of primary cells, including epithelial cells, endothelial cells, smooth muscle cells and fibroblasts from different species, such as human, rat, and mouse. We can provide a validation report for each primary cell. Our primary cells have been widely used in many research institutes and pharmaceutical enterprises.

References:

1. A549 Cell Line: Human alveolar adenocarcinoma cell line -General Information.[2019-12-03]. 

2. Khaodee, W., Udomsom, S., Kunnaja, P. et al. Knockout of glucosidase II beta subunit inhibits growth and metastatic potential of lung cancer cells by inhibiting receptor tyrosine kinase activities. Sci Rep 9, 10394 (2019). https://doi.org/10.1038/s41598-019-46701-y

3. Pawel Bialk, Yichen Wang, Kelly Banas, and Eric B. Kmiec1. Functional Gene Knockout of NRF2 Increases Chemosensitivity of Human Lung Cancer A549 Cells In Vitro and in a Xenograft Mouse Model.

 

 

iPSC Related Services | Ubigene

Many serious diseases cannot be cured by medicines, such as heart failure, Late Stage Diabetes, hemophilia, myeloma, End-Stage Cirrhosis, etc. The best method is allogeneic transplantation. However, due to the limited donors and the risk of immune rejection, researchers are dedicated to finding more efficient and safer treatment besides allogeneic transplantation. Induced pluripotent stem cells (iPSCs) can be derived from the body cells of the patients themselves, which eliminates the risk of immune rejection, and has the potential of differentiation into different cells. Transplantation of cells derived from iPSC, such as cardiomyocytes, hepatocytes, neurocytes, T cells, hematopoietic stem cells (HSCs), and pancreatic cells, is possible to solve many medical problems.

Hepatocyte

The differentiation of liver cells induced by iPSC can alleviate the shortage of sources in liver transplantation and hepatocyte transplantation, which is more conducive to basic and clinical research. In addition, the induced hepatocyte could be used as a tool to simulate and study liver diseases and screen the hepatotoxicity of drugs in the future.

Neural stem cell and neuron

Neural stem cells differentiated from iPSC can be used to generate cell models of nervous system diseases. This approach avoids ethical problems and immune rejection and is an ideal way to obtain NSC in vitro.

iPSC can differentiate into neurons under appropriate conditions. For example, differentiation into motor neurons (MN) provides the possibility for the treatment and research of MN injury diseases such as Amyotrophic lateral sclerosis (ALS) and Spinal muscular atrophy (SMA).

T cell

iPSC can differentiate into T cells. The CAR-T cell therapy developed on the basis of iPSC has a safer and more effective pharmacological activity. iPSCs based CAR-T cells can be used in T cell immunotherapy without the limitation of Allograft rejection.

Hematopoietic stem cell

The limited number of hematopoietic stem cells (HSC), the difficulty of expansion and culture in vitro, and graft versus host disease (GVHD) limit the HSC transplantation. iPSC can proliferate and differentiate into transplantable HSCs in vitro, which brings a bright future for the treatment of malignant blood diseases.

Cardiomyocyte

iPSC derived cardiomyocytes provide a new way for the study of disease-specific and individual-specific pathogenesis of cardiovascular diseases, which has become an effective tool in the field of cardiovascular research and also brings new hope for clinical treatment. 

Pancreatic cell

iPSC can differentiate into pancreatic β-cells in vitro, which can be used in the research of disease mechanism, drug development, and cell therapy for diabetes. Using this source of pancreatic β-cells for transplantation in the treatment of diabetes can better solve the ethical, limited source problems faced by the previous islet transplantation.

By CRISPR/Cas9 technology, the mutations that simulating diseases could be introduced into iPSC. Using CRISPR/Cas9 to repair the mutations in iPSC disease models is also a popular application.

 

CircRNA Editing | Ubigene

circRNAs are circular noncoding RNAs formed by reverse splicing of pre-mRNAs. circRNAs were firstly found in viruses in the 1970s. However, due to the extensive use of the method for enrichment of poly (A) (no 5 'and 3' ends of circRNA) in the early RNA library preparation, and the calculation algorithm that RNA-seq reading requires linear alignment with the genome, a large number of circRNA information was omitted, which led to the belief that circRNA is just a byproduct of miss splicing.

With the development of high-throughput sequencing technology and bioinformatics, thousands of circRNA have been found, and more and more basic researches related to circRNA have been done. A large number of studies have shown that circRNA is endogenetic, abundant, conservative, and stable in mammalian cells, and often shows tissue or space-time specificity. It can participate in the regulation of cell growth and development, as well as the occurrence and development of diseases through a variety of mechanisms. Therefore, in recent years, circRNA has become popular in the field of non-coding RNA research

According to the origin of circRNA, it can be sorted into three types:

1) Exonic circRNAs: All sequences are derived from exons.

2) EIciRNAs: Sequences are derived from exons and introns.

3) ciRNAs: All sequences are derived from introns.

circRNA is formed by the pre-mRNA by back splicing. At present, there are three kinds of mechanisms reported as follows:

1) Intron reverse complementary sequence

The flanking introns at both sides of the exon contain many pairs of reverse complementary sequences. The reverse complementary sequence promotes the intron sequence pairing, making the Splice-Donor in the downstream close to the Splice-Acceptor in the upstream, so as to form a circRNA. (Fig 1. Left)

2) RNA binding protein

The flanking introns at both sides of the exon contain the motifs recognized by RNA binding proteins (RBPs). RBP, when combined with the specific motifs of the two flanking introns, will form dimers, promote the two flanking introns close to each other, and then connect to form a ring. 

3) Lariat-driven circularization

When the pre-mRNA is spliced, exon skipping occurs, which results in the formation of a lariat intermediate containing exon and intron. Then the intermediate is back spliced to form a circRNA. 

The most common function of circRNA is to bind to miRNA as miRNA sponge, thus affecting the regulation of miRNA on genes.

Many circRNAs contain protein binding sites, which can be used as protein sponges.

In addition to being miRNA and protein sponge, circRNAs can also be used as a scaffold protein to promote the co-location of the enzyme, to inhibit the target gene expression by binding transcription factors, to participate in the regulation of parent gene expression, and to translate polypeptides under specific circumstances. According to the different functions, the locations of circRNAs are different. For example, as a miRNA or protein sponge, circRNA needs to be transported from the nucleus to the cell-matrix to play a role. When participating in the regulation of parent gene expression or binding transcription factor to inhibit the target gene, circRNA often plays a role in the cell nucleus.

Reference：

Kristensen, L. S., Andersen, M. S., Stagsted, L. V., Ebbesen, K. K., Hansen, T. B., & Kjems, J. (2019). The biogenesis, biology and characterization of circular RNAs. Nature Reviews Genetics, 20(11), 675-691.

Relationships between circRNAs and diseases

At present, the most studied is the relationship between circRNAs and tumors. Some circRNAs promote tumor formation, such as circPvt1 in squamous cell carcinomas of the head and neck, cirs-7 (CDr1as) in colorectal cancer, esophageal squamous cell carcinoma and hepatocellular carcinoma. Some circRNAs suppress tumors, such as circsMARCA5 and circ-SHPRH in glioblastoma. Some circRNAs may play different roles in different tissues or cells, such as circHiPK3, which is a proto-oncogene in rectal cancer but suppresses cancer cells in bladder cancer.

In addition to cancers, circRNA has been found to be closely related to diabetes, cardiovascular disease, chronic inflammation and nervous system diseases. It is believed that with the development of biotechnology and more in-depth researches on circRNA, the formations and mechanisms of circRNAs can be identified. 

circRNAs can play important roles in disease prevention, diagnosis and treatment discovery.

circRNA related custom services:

circRNA involves many complex functions, so how to study its functions? Similar to protein-coding genes, the most common methods are knockout, knockdown (RNA interference) or overexpression of the circRNA. 

circRNA knockout

circRNA knockout refers to editing at the level of DNA to achieve the purpose of a complete knockout. gRNA and Cas9 would be transferred into cells by virus transduction or nucleofection. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

The most common startegies for circRNA knockout：

Strategy 1：The most commonly used method is to design two gRNAs at both ends of the circRNA exon to knockout the whole cyclized exon sequence. Although this strategy can knockout the circRNA, it will also affect the parent gene encoding the protein, and the study on its function is not ideal.

Strategy 2：The ideal method is to knockout the loop forming elements (Alu) in the flanking intron of the exon, so as to destroy the circRNA loop forming without affecting the expression of the coding gene.（Fig 4.）

Ubigene is experienced in designing the strategy of a knockout the loop forming elements in the flanking intron of circRNA exon, to achieve the purpose of knockout circRNA without affecting the expression of the coding gene. Combined with CRISPR-UTM technology, the positive clones of circRNA knockout can be generated 10x faster than other common methods.

Case study：

circ-HIPK3 is a kind of circRNA rich in human cells, which can combine with a variety of miRNAs as a regulator of cell growth and affect the formation of tumors. In order to verify how circ-HIPK3 forms into a circle, it is necessary to find the loop forming elements in flanking intron. A pair of sgRNA is designed for the two Alu elements predicted at upstream and downstream respectively. The predicted loop forming elements are knockout by CRISPR/Cas9 system to detect whether the expression of circRNA changes. After PCR and RT-qPCR verification, it was found that the expression of circ-HIPK3 was significantly down-regulated after knockout of the downstream loop forming elements, while the expression of circ-HIPK3 was not decreased but increased after knockout of the upstream loop forming elements. It was speculated that there were too many loop forming elements in the upstream and the prediction was not accurate. In order to further verify the RNA circulation driven by other elements, the large fragment of the intron in the upstream of the element was knockout by co-injection of gRNA3 or gRNA4 with gRNA5 or gRNA6. RT-qPCR results showed that the expression of circ-HIPK3 decreased, indicating that other loop forming elements of circ-HIPK3 exist.

Reference：

Zheng, Q., Bao, C., Guo, W., Li, S., Chen, J., Chen, B., ... & Liang, L. (2016). Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nature communications, 7(1), 1-13.

circRNA knockdown (RNAi)

Among the methods to study the function of circRNA, the most classical way to inhibit circRNA is to knockdown it by RNAi (shRNA or siRNA). In order to avoid affecting mRNAs of coding genes, the shRNA should be designed at the back splicing site (BSS).

Ubigene can design high-score shRNA and use lentivirus to transfer the RNA interference vector into the cells. Cells were screened according to the drug screening, and the stable cell lines with circRNA knockdown were obtained

Case study：

siRNA was used to interfere with circ-HIPK3, and whether the knockdown of circ-HIPK3 would affect cell proliferation or apoptosis was observed. First, three groups of experiments targeting the linear transcript of HIPK3 mRNA, circ-HIPK3 circular transcript, and both two transcripts. The designed siRNA interfered with the corresponding transcripts was verified on the HEK-293T cell line. 

Cell proliferation and apoptosis were detected by CCK-8 and EdU assays. The results showed that the knockdown circ-HIPK3 significantly inhibited cell proliferation.

Reference：

Zheng, Q., Bao, C., Guo, W., Li, S., Chen, J., Chen, B., ... & Liang, L. (2016). Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nature communications, 7(1), 1-13.

Gene-Editing Liver Cancer Cell Line HepG2 - An Artifact for Research in Drug, Hepatotoxicity and Can

As one of the five internal organs in the human body, the liver is vital to the body's metabolism, detoxification, blood coagulation, and other essential processes. Participating in the body's immune system, the liver is an indispensable organ for maintaining the body's normal operation. In the study of liver diseases, one of the most useful tools is to create a suitable cell model. However, due to the difficulties in the obtainment and culturing of conventional liver cells, the development of liver disease research has been confined in some ways.  Moreover,  the relatively high cost of culturing in conventional liver cells also makes it less likely to carry out the research. Therefore, selecting liver cell lines with simple culture and stable genetic background has become an alternative in liver research, and HepG2 is one of the most commonly used liver cancer cell lines.

 The application of HepG2 cell line

HepG2 was established in 1979 by Knowles et al. It is a hepatoblastoma derived from a 15-year-old Caucasian male liver cancer specimen. HepG2 cells have an epithelial-like morphology, with a typical chromosome number of 55.

1. Hepatitis virus research: HepG2 does not contain the hepatitis B virus (HBV) or hepatitis C virus (HCV), so HepG2 is a commonly used model for studies in HBV and HCV cell lines.

2. In vitro HCC model: HepG2 is a cell line derived from liver tissue of patients with hepatocellular carcinoma (HCC) and it is a common in vitro HCC model. There is no mutation in p53 tumor suppressor gene in HepG2, so it can be used to study the close relationship between p53 and hepatocellular carcinoma (HCC), as well as the onset, diagnosis, treatment and prevention of HCC.

3. Drug research: The liver is the most essential organ for detoxification and drug metabolism in the human body. HepG2 cell line is closer to human liver tissue both in terms of morphology and functions. Therefore, this cell line is often used in drug metabolism and liver toxicity studies.

The combination of liver cell line and CRISPR / Cas9 helps to study medical problems such as hepatitis, genetic diseases and cancer

Applying CRISPR / Cas9 to construct HepG2 knock-out HBV infected cell models provides new ideas for the complete cure of hepatitis B

Hepatitis B virus (HBV) is a DNA virus. The genomic DNA of HBV outside the cell is double-stranded relaxed circular DNA (rcDNA). When the virus infects the cell, the HBV genome will enter the nucleus of the host cell. Then the rcDNA will be converted into covalently closed circular DNA (cccDNA) and exist stably in the cell. At present, an important reason for not being able to completely cure hepatitis B lies in the difficulties in clearing the stable cccDNA in the liver cell nucleus. The conversion of HBV rcDNA to cccDNA is a necessary step for the establishment of persistent infection of hepatitis B virus. Therefore, the study of the molecular mechanism of HBV cccDNA formation has far-reaching significance for the treatment of hepatitis B.

In order to explore the mechanism of HBV cccDNA formation, researchers used HepG2 to express the HBV receptor, the sodium ion-taurocholic acid co-transporter (NTCP). They established an efficient HepG2-NTCP cell model for HBV infection, which could detect the formation of cccDNA after only 24 hours. Afterward, the experiments using viral DNA polymerase inhibitors and siRNA (targeting the host DNA polymerase) showed that HBV viral polymerase was not a critical factor in the conversion of rcDNA to cccDNA. However, the knockdown of POLK, POLL, and POLH can effectively reduce the expression levels of viral nucleocapsid protein (HBeAg) and 3.5 kb RNA. Among the three genes, the knockdown of POLK showed the most significant influence in the inhibition of HBV infection.

Furthermore, using CRISPR / Cas9 to knock out POLK in HepG2-NTCP cells can greatly decrease the formation of cccDNA, confirming that the formation of cccDNA in HBV requires the participation of POLK. POLK replenishment in POLK knock-out HepG2-NTCP cells can restore HBV cccDNA synthesis and the expression of viral protein.

This study strongly indicated that POLK is a key cell molecule for HBV cccDNA formation in the HBV-infected HepG2-NTCP cell model, which provides new ideas for research in the molecular mechanism of cccDNA formation and new treatments for chronic hepatitis B.

CRISPR-U ™ can transfer CRISPR / Cas9 into Hep-G2 cells 10 times more efficiently, and select suitable cells for monoclonal culture after screening. Different clones were selected for target site amplification and sequencing verification separately, after which the positive clones with the knockout gene were selected.

Building HepG2 model of missing mutant cells by CRISPR / Cas9 revealed the causes of Wilson disease (hepatolenticular degeneration)

Hepatolenticular degeneration (Wilson disease) is a recessive genetic disorder caused by pathogenic loss-of-function variants in the ATP7B gene. It is characterized by disrupted copper homeostasis resulting in liver disease and/or neurological abnormalities. The variant NM_000053.3:c.1934T > G (Met645Arg) has been reported as compound heterozygous, and is highly prevalent among Wilson disease patients of Spanish descent. Accordingly, it is classified as pathogenic by leading molecular diagnostic centers. However, functional studies suggest that the amino acid change does not alter protein function, leading some researchers to question its pathogenicity. Here, we used a minigene system and gene-edited HepG2 cells to demonstrate that c.1934T > G causes ~70% skipping of exon 6.

The CRISPR / Cas9 and ssODN were co-transformed into HepG2 cells by nuclear transfection and then selected for monoclonal culture after screening. The target sites were amplified and sequenced separately for the monoclonal, and the following 4 clones were screened for research.

Compared to the transcription in wild-type cells, compound heterozygous HepG2 cells (2F3 clone) express only 15% of the transcript containing exons 5, 6 and 7. Compared to wild-type cells,  and homozygous cells (clones 1E8 and 1F6) express only 31–33%.

In addition, protein lysates were extracted from wild-type and edited HepG2 cells to determine ATP7B protein expression by western blot. Compared to wild-type cells, compound heterozygous (2F3) and homozygous (1E8, 1F6) cells express reduced levels of ATP7B, with increased ATP7B expression in 1E8 and 1F6 homozygous cell lines compared to 2F3 cells, as expected. A control 2A1 ATP7B knock-out cell line has no detectable levels of ATP7B.

Moreover, the study suggested that c.1934T > G causes the skipping of exon 6, which results in frameshift and stop-gain, which leads to loss of ATP7B function. The outcome clarified the mechanism role of this point mutation in hepatolenticular degenerative diseases, which is helpful for the development of genetic drugs to restore correct splicing.  

mPGES-1 is a terminal rate-limiting enzyme responsible for inflammation-induced PGE2 production. The inhibition of mPGES-1 has been considered as a safe and effective target for the treatment of inflammation and cancer. However, a specific, efficient, and simple method for high-throughput screening of mPGES-1 inhibitors is still lacking. In this study, we developed a fluorescence imaging strategy to monitor the expression of mPGES-1 via CRISPR/Cas9 knock-in system.

By using nuclear transfection to efficiently transfer gRNA, Cas9 and Donor into HepG2 and other hepatocyte cell lines. After performing drug screening and monoclonal culture, the positive clones went through immunofluorescence co-localization, sequencing, RNAi and IL-1β treatment, which confirmed the successful construction of mPGES-1 reporter cells.

Fluorescent protein KI cells were treated with 4 common mPGES-1 inhibitors, after which the flow cytometry detected a significant attenuation of the fluorescent signal intensity. The result suggested that using the fluorescent protein KI cells is a high-efficiency and handy method in screening and optimization mPGES-1 inhibitor. Meanwhile, the KI cells provide new technical support for anti-inflammatory and tumor treatments targeting small molecule compounds.

CRISPR-U™ efficiently modify genes in liver cell lines

CRISPR-U ™ is an exclusive technology independently developed by Ubigene Bioscience for gene editing cell lines. By optimizing gene editing vectors and processes, the efficiency in gene-cutting and recombination of CRISPR-U ™ is 10 times higher than the conventional CRISPR / Cas9 technology. We are capable of customizing genetically-modified liver cell lines as you desire and satisfying various needs in gene editing.

Reference：

Qi, Yonghe, et al. "DNA polymerase κ is a key cellular factor for the formation of covalently closed circular DNA of hepatitis B virus." PLoS pathogens 12.10 (2016).

Merico, Daniele, et al. "ATP7B variant c. 1934T> G p. Met645Arg causes Wilson disease by promoting exon 6 skipping." NPJ Genomic Medicine 5.1 (2020): 1-7.

Chen, Zhangfei, et al. "CRISPR/Cas9-based liver-derived reporter cells for the screening of mPGES-1 inhibitors." Journal of enzyme inhibition and medicinal chemistry 34.1 (2019): 799-807.

Is your method right for studying non-coding RNA？

Single-cell cloning has gained increasing importance as CRISPR/cas9 genome editing technique has entered routine laboratory practice. However, the success of positive single-cell cloning is technically challenging. The long growing time and the extremely low efficiency of obtaining a positive single-cell clone are the major challenges. The rate of single-cell clone formation may be affected by many factors; two of which are particularly important, one is the survival rate of inoculated cells, and the other is the cell proliferation ability.

1 How to improve the survival rate of inoculated cells?

1) Maintain a stable pH

Most of the cell culture media are formulated with CO2 carbonate buffer which is suitable for the partial pressure of CO2 in incubators. However, when this buffer is in atmospheric conditions, carbon dioxide will evaporate from the medium, which will cause the pH of the medium to rise to the alkaline range (in the medium containing phenol red, the color will become more purple). This will significantly affect the viability of cells. Therefore, the HEPES buffer system can be used instead of the CO2 carbonate system. It is a better and safer choice.

2) Keep everything at the right temperature

Before digesting and plating cells to 96-well plate, the culture medium, trypsin and PBS should be warmed up to 37 ℃, to keep cells under a relatively stable condition.

3) For some sensitive or fragile cell lines, the number of inoculated cells can be increased correspondingly, for example, from 50 cells per 96-well plate to 80 cells per plate.

2 How to improve the proliferation of cells?

1) Passage appropriately

Cells were inoculated with limited dilution and cultured to form microcolonies (∼ 50-100 cells). Transfer a microcolony to a fresh well of a 96-well plate. When the cells in the new well grow to 80% confluence, the cells are transferred to a well of a 48-well plate. Allow the cells to proliferate until a sufficient number of cells can be harvested for validation.

2)Adding supplements

The universal factors that are required in nearly all cell lines, which have been identified in the literature include insulin, transferrin, and selenium. For certain cell lines, Ethanolamine may also be critical, and in some cases, attachment factors such as fibronectin, laminin, vitronectin, and growth factors may be beneficial.

Start your year off right by only $2020 on CRISPR cell lines, as fast as 6 weeks！

Ubigene Biosciences has rich experience in gene editing. 

Through continuous testing and exploration, we have built up a set of unique experimental methods and processes, which can achieve the efficiency of single-cell clone formation 3-5 times faster than the traditional ways. Combined with our CRISPR-U^TM, the positive rate is greatly improved!
 

Quick Review CRISPR v.s. RNA

Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

Make genome editing easier is the goal of Ubigene. We developed CRISPR-U™ (based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. With CRISPR-U™, Ubigene has successfully edit genes on more than 100 cell lines.

Ubigene developed CRISPR-B™  which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI).

Ubigene has more than 400 types of primary cells, including epithelial cells, endothelial cells, smooth muscle cells and fibroblasts from different species, such as human, rat, and mouse. We can provide a validation report for each primary cell. Our primary cells have been widely used in many research institutes and pharmaceutical enterprises.

Leap into Efficient Gene-editing Cell Lines

Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

Make genome editing easier is the goal of Ubigene. We developed CRISPR-U™ (based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. With CRISPR-U™, Ubigene has successfully edit genes on more than 100 cell lines.

Ubigene developed CRISPR-B™  which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI).

Ubigene has more than 400 types of primary cells, including epithelial cells, endothelial cells, smooth muscle cells and fibroblasts from different species, such as human, rat, and mouse. We can provide a validation report for each primary cell. Our primary cells have been widely used in many research institutes and pharmaceutical enterprises.

Gene-editing Cell UFC Winner: CRISPR-U™

Gene-editing Cell UFC Winner: CRISPR-U™！

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 knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. With CRISPR-U™, Ubigene has successfully edit genes on more than 100 cell lines！Subscribe us and stay tuned for a more interesting comic about gene-editing!

The peak of a researcher's life

The peak of a researcher's life! #Ubigene developed #CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial #geneknockout (KO), #pointmutation (PM) and #knockin (KI). Follow us for more fun comic about #crispr!