HuH-7 knockout cell line | Ubigene

Definition of HuH-7 cell:

HuH-7 is a type of human liver cell line cancer cell line, originally taken from a liver tumor in a 56-year-old Japanese male in 1982. HuH-7 is an Adult hepatocellular carcinoma cell line, which has been used extensively in hepatitis C and dengue virus research, sometimes by using CRISPR/Cas9 technologies to create CRISPR knockout cells. These years, HuH-7 cells are primarily grown in the laboratory for research purposes, especially in research that involves hepatitis C knockout. The introduction of the Huh7 cell line permitted the screening of drug candidates against laboratory-cultured hepatitis C virus and permitted the development of new drugs against hepatitis C.

HuH-7 is an immortal cell line composed of epithelial-like and tumorigenic cells. The majority of HuH-7 cells are highly heterogeneous, with a chromosome number between 55 to 63. The cells are adherent to the surface of flasks or plates. According to previous studies, it is assumed that HuH-7 cells can produce alpha-fetoprotein, pancreatin alpha antibody, plasma ceruloplasmin, fibrinogen, fibronectin, etc. Therefore gene-editing knockout cells HuH-7 is helpful when it comes to research that involves these kinds of proteins.

Application of gene-editing in HuH-7 cell line:

1. CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line

The HuH-7 cell line was derived from a hepatic carcinoma that can convert the substrate MDZ in cell culture to its metabolite products; however, HuH-7 cells are not very efficient at MDZ metabolism because they are homozygous for the slow metabolizing CYP3A5 *3 allele. Thus, there is a need to develop a gene knockout and point mutation liver cell line that mimics the rapid drug metabolism associated with the CYP3A5 *1 genotype in cell culture. Researchers hypothesized that by genetically modifying the HuH-7 cell line to the more metabolically active CYP3A5 *1/*1 or *1/*3 genotypes, the cells would have increased MDZ and Tac metabolic activity. To test the hypothesis, they used CRISPR/Cas9 bioengineering to develop and characterize new cell lines and then phenotypically evaluate the genotypes’ effects on MDZ and Tac metabolism. These newly engineered cells can be used as a parental cell line in future studies to assess the association of additional genetic variants with drug metabolism and the metabolism of other drugs.

2. Coexpression of HBV-specific gRNAs and Cas9 suppressed the production of HBV proteins in vitro

To examine whether the clustered regularly interspaced short palindromic repeats CRISPR/Cas9 system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in HuH-7 cells transfected with an HBV-expression vector, which created an overexpression HuH-7 cell line. Among the eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, researchers further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in a reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

3. Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication

The destruction of HBV-expressing vector was examined in HuH-7 cells co-transfected with dual-gRNAs and HBV-expressing vector using PCR and sequencing method, and the destruction of cccDNA was examined in HepAD38 cells using KCl precipitation, PSAD digestion, rolling circle amplification and quantitative PCR combined method. The cytotoxicity of these gRNAs was assessed by a mitochondrial tetrazolium assay. The results suggested that the CRISPR/Cas9 system could efficiently destroy HBV expressing templates (genotypes A-D) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV cccDNA in chronic HBV infection patients.

4. The CRISPR/Cas9 genome editing methodology as a weapon against human viruses

Curing chronic HBV infection will require the specific eradication of the persistent HBV cccDNA from infected cells. Scientists designed eight gRNAs against HBV and showed that the CRISPR/Cas9 system significantly reduced the production of HBV core and HBsAg proteins in the HuH-7 cells transfected with an HBV-expression vector. Further, this system could cleave intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo in a mouse model resulting in a reduction in serum HBsAg level. Thus, the CRISPR/Cas9 system could disrupt the HBV templates both in vitro and in vivo and may have the potential in eradicating persistent HBV infection. 

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.

Vero knockout cell line | Ubigene | 20x Efficiency

Introduction of Vero cell line:

 

Vero cells are derived from the kidney of a normal adult African green monkey on March 27 in the 1960s, and are one of the more commonly used mammalian continuous cell lines in microbiology and molecular and cell biology research, including studies that involve gene-editing such as gene knockout/knock-in that require CRISPR/Cas9 technology. At its 93rd passage, the cell line was brought to the National Institute of Allergy and Infectious Diseases at the National Institutes of Health in the United States and was provided to the American Type Culture Collection (ATCC) in 1966. 

 

Vero cell line, which is also anchorage-dependent cell line, has been used extensively in virology studies but has also been used in many other applications, including the propagation and study of intracellular bacteria and parasites, and assessment of the effects of chemicals, toxins, and other substances on mammalian cells at the molecular level. Therefore gene-edited Vero cells, such as Vero knockout cell lines, are getting more and more popular in studies in these fields. Besides, Vero cells have been licensed in the United States for production of both live (rotavirus, smallpox) and inactivated (poliovirus) viral vaccines, and throughout the world, Vero cells have been used for the production of several other viruses, including Rabies virus, Reovirus, and Japanese encephalitis virus. These studies are mostly related to Vero CRISPR/cas9 mammalian cells, Vero knockdown cell line, virus packaging, and Vero point mutation cell line. Vero cells can also be used as host cells for eukaryotic parasites such as Trypanosoma. 

Vero cells grew well during subculture and found clear cell membrane boundaries and good cytoplasmic transparency. The morphology of Vero cells is relatively complete, and the rate of cell proliferation is relatively fast. Vero cells can be adherent after 24 hours of cell culture after being transferred to flasks. Cells can reach a relatively stationary phase after three days of culture. Cells can grow into a monolayer on the fifth day of culture and cells can be cultured until the twelfth day. It was found that the growth was dense, and the cells began to age until the fourteenth day. The cell growth rate after spinning was slower than before spinning, but the monolayer cells lasted longer than before spinning. No mycoplasma growth and contamination were found during mycoplasma inspection. The results of cell type analysis showed that there were no obvious abnormalities in the karyotype of Vero cells, and there was no significant change in chromosome number.

 

Applications of Vero cell line:

 

Vero cells can be used for a variety of research purposes. Vero cells are widely used in the study of molecular mechanisms of viral infections, the production of vaccines, and recombinant proteins. Vero cells were found to be highly sensitive to many types of viruses shortly after they were established, including simian vacuole virus, measles virus, rubella virus, arthropod-borne virus, and adenovirus. It was later found to be susceptible to bacterial toxins, including diphtheria toxin, heat-labile enterotoxins, and Shiga-like toxins. Therefore, Vero cells are suitable for being a gene-customizing model such as CRISPR knockout cell that involves virus infection.

 

1. Enhancing viral vaccine production using engineered knockout Vero cell lines

 

The global adoption of vaccines to combat disease is hampered by the high cost of vaccine manufacturing. The work described herein follows two previous publications that report a strategy to enhance poliovirus and rotavirus vaccine production through genetic modification of the Vero cell lines used in large-scale vaccine manufacturing. CRISPR/Cas9 gene-editing tools were used to knockout Vero target genes previously shown to play a role in polio- and rotavirus production. Subsequently, small-scale models of current industrial manufacturing systems were developed and adopted to assess the increases in polio- and rotavirus output by multiple stable knockout cell lines. Unlike previous studies, the Vero knockout cell lines failed to achieve the desired target yield increases. These findings suggest that additional research will be required before implementing the genetically engineered Vero cell lines in the manufacturing process for polio- and rotavirus vaccines to be able to supply vaccines at reduced prices.

2. Gene-edited Vero cells as rotavirus vaccine substrates

 

Rotavirus (RV) is the main cause of severe gastroenteritis worldwide and can lead to a large number of gastroenteritis-related illnesses in children under 5 years of age. Oral-attenuated live attenuated RV vaccine can prevent disease, but the high manufacturing cost and maintenance of cold chain identified a subset of viral cell host genes by siRNA. When knocking down RV replication, these antiviral host genes are used alone CRISPR -Cas9 deleted. The results showed that Vero cells with EMX2 gene deletion had higher RV replication and antigen production than other tested Vero cell-substrate components, which provided the possibility of improving the production of RV vaccines.

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.

Hela KO cell line | CRISPR-U | Ubigene

Introduction of HeLa cells

HeLa cells, like other cell lines, are termed as an immortal cell line used in scientific research, which can divide an unlimited number of times in a laboratory cell culture plate as long as being they are maintained and sustained in a suitable environment. It is commonly applied in gene-editing research, where HeLa cells become CRISPR/Cas9 KO cell line.

HeLa cells are the oldest and most commonly used human cell line, as well as the most frequently-used CRISPR cell line. The line was originated from cervical cancer cells of a 31-year-old African-American woman in 1951. The cells from this cell line were observed that they grew very fast and doubled every 20-24 hours, unlike other specimens that died out previously. This cell line was found to be remarkably durable and prolific, which causes it to be used widely in scientific studies, such as the HeLa knockout cell line.

Application of HeLa knockout cells

Since HeLa cells were the first human cells to be successfully cloned in 1953, they have continually been used for research into cancer, AIDS, the effects of radiation and toxic substances, gene mapping, and countless other scientific pursuits. For example, HeLa cells were used by Jonas Salk to test the first polio vaccine in the 1950s which made HeLa cells highly desirable for polio vaccine testing since results could be easily obtained. Moreover, HeLa cells have also been instrumental in the development of Human papillomavirus (HPV) vaccines.

In 1965, Henry Harris and John Watkins created the first human-animal hybrid by fusing HeLa cells with mouse embryo cells. This enabled advancements in mapping genes to specific chromosomes, which would eventually lead to the Human Genome Project, involving gene knockout and gene knockin by CRISPR technologies.

1) Establishment of SQSTM1/p62 gene knockout Hela cells by CRISPR/Cas9

To explore the efficiency of CRISPR / Cas9 gene-editing directed against p62 double sgRNA, researchers have successfully used CRISPR / Cas9 gene-editing technology to knock out the p62 gene. The cell dilution method and puromycin screening were also applied to separately confirm the success rate of double sgRNA and single sgRNA guided gene editing. Western blot analysis showed that double sgRNA-directed p62 knockout efficiency was higher than single sgRNA-directed knockout. Target-sequence sequencing analysis suggested that the p62-encoding gene had large deletion mutations.  The result of stable knock-out Hela cell line after H₂O₂ treatment showed that p62 gene knock-out can significantly inhibit the early apoptosis induced by H₂O₂ in Hela cells. Therefore, the p62 knockout Hela cell line has been successfully established. The double sgRNA-guided CRISPR / Cas9 gene-editing system may be a more effective editing tool.

2) Construction of stable Cdc25C knockout HeLa cell strains using CRISPR/Cas9 gene-editing system

The CRISPR / Cas9 gene-editing system was used to knock out the cell division cycle 25 homolog C (Cdc25C) gene in human cervical cancer cell HeLa to construct a Cdc25C gene stable knockout cell line. The method was based on CRISPR / Cas9 target design rules, design small guide RNA (sgRNA) upstream and downstream that specifically recognize the relevant sequence of the first exon of the Cdc25C gene, construct a eukaryotic recombinant expression plasmid. After sequencing and identification, the recombinant plasmid is transfected to In HeLa cells, puromycin (puromycin) resistance screening was used to stably knock out the Cdc25C gene cell line, and then Western blotting was used to identify the Cdc25C knockout effect. Finally, flow cytometry was used to detect the effect of gene knockout on the cell cycle. Results The cell lines with stable knock-out of the Cdc25C gene were screened, and Cdc25C knock-out significantly affected the G2 / M phase process. Conclusion The endogenous Cdc25C gene knock-out cell line was successfully obtained using CRISPR / Cas9 technology to study the function of Cdc25C in the cell cycle process And the occurrence of related cancers has laid the foundation.

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.

HEK 293 knockout cell line | 20x Efficiency | Ubigene

HEK 293 cell line:

HEK 293 cell lines, which are also called HEK 293, HEK-293, 293 cells, are human embryonic kidney 293 cells that were generated in 1973.  Just as the name implies, they are a typical cell line which is originated from the embryonic kidney cells in the human body. At first, the cells were legally extracted from a healthy aborted fetus. For many years, HEK 293 cell lines were assumed to be generated by the transformation of a fibroblastic or epithelial cell. Although these substances are abundant in kidneys, the original adenovirus transformation was quite inefficient. Therefore, researchers started to wonder if the cell that produced the HEK 293 line would be unusual. A series of experiments were conducted to analyze the genomes and transcriptomes of the cell line afterward. The results showed that the HEK 293 pattern was similar to that of adrenal cells with several neuronal properties. As a consequence, HEK 293 cells are supposed to be used as an in vitro model of adrenal cells instead of the typical kidney cells.

In terms of cell structure, HEK 293 cells are hypotriploid and contain only 1/3 of the number of chromosomes of a haploid human gamete. Additionally, these embryonic kidney cells are grown in tissue culture. So far, HEK 293 cells have been commonly used in cell research for many years. Their growth is comparatively reliable and they are more likely to do transfection.

HEK 293 cells can directly grow and direly in culture. They are usually used as hosts in gene expression. Moreover, this cell line is widely used due to its transferability through various techniques such as calcium phosphate method that can achieve almost 100% efficiency.

The application of HEK 293 cell line:

HEK 293 cell line can be used in various research. For instance, it can be used in studying the effects of drugs on sodium channels, indubitable RNA interference system, and nuclear export signals in proteins, etc.

To be more specific, HEK 293 cells are applied in the propagation of adenovirus vectors. It is an effective way to use viruses to evolve target genes and transfer them into the cells. However, viruses also pose risks as a result of their character as pathogens. Therefore, in order to propagate this kind of virus vectors, a cell line that can express some missing genes is required. Since HEK 293 cells contain many adenovirus genes, it is perfect to use them to spread adenovirus vectors in which these genes(es. E1, E2) have been deleted.

HEK293 cells with CRISPR/Cas mediated quadruple gene knockout improved protein and virus production

CRISPR/Cas9 recognizes the target sequence with gRNA, and guide Cas9 endonuclease to cut the upstream of PAM, resulting in the double-strand (DSB) break of the target site DNA. To repair the DSB, the cell uses its own DNA repair mechanism to add, delete or replace pieces of DNA sequences via Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ).

Researchers used CRISPR / Cas technology to knock out four pro-apoptotic genes (Caspase3, Caspase6, Caspase7, and AIF1) and successfully produced anti-apoptotic cell lines in HEK293 cells. Apoptosis plays an important role in the pathophysiology. However, from a biopharmaceutical point of view, active apoptosis of host cells during viral packaging or protein expression is undesirable because it reduces the efficiency of virus or protein production. Compared with wild type cells, the edited cell line showed higher expression levels of pro-apoptotic proteins, and the packaging efficiency of viruses carrying these proteins was also much higher. This gene-editing cells not only produced anti-apoptotic cell lines that can be used to produce apoptosis-inducing proteins or viruses that express these proteins but also provided methods for constructing other anti-apoptotic cell lines.

HEK293-based human expression system stably expressing recombinant erythropoietin

HEK293 cells are a particularly attractive expression system for recombinant protein production because they not only have the ability to generate human glycosylation profiles and have established regulatory track records but also have multiple advantages. First, it is carboxylated at glutamate and sulfated at tyrosine. Secondly, it is easy to operate, and can quickly produce recombinant proteins through transient gene expression. Third, it can be used for stable recombinant protein production. Some researchers used the CRISPR / Cas9 system to generate the GLUL-KO HEK293 cell line, targeted sequencing of the GLUL genome site, produced HEK293 cells that produced EPO, and found a mechanism for the stable expression of recombinant erythropoietin in the human body.

Reference:

1. hin, C.L., Goh, J.B., Srinivasan, H. et al. A human expression system based on HEK293 for the stable production of recombinant erythropoietin. Sci Rep 9, 16768 (2019).

2. Z. Dong, Z. Hu, Q. Qin, F. Dong, L. Huang, J. Long, P. Chen, C. Lu, and M. Pan, CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm, Insect Molecular Biology, 28, 1, (112-122), (2018).

CHO ko cell line | High Efficiency | Ubigene

CHO cells are also called Chinese hamster ovary cells, which are an epithelial cell line that originated from the ovary of the Chinese hamster. Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins. This cell line is usually used in biological and medical research. Also, it is the most widely used mammalian host in terms of the industrial production of therapeutic proteins. 

 

 

Construction of a gene knockout CHO cell line using gene targeting method

 

Since CHO cell line is a major host for therapeutic antibody production, constructing productive CHO cell lines is important. There are two major transfection methods that are commonly used in building characterized CHO cell lines, one is random integration and the other one is gene targeting. The former one, which causes variation in antibody productivity, would usually affect transgene expression levels.

 

However, in gene-targeting methods, exogenous genes are inserted into a specific chromosomal region. This method is based on homologous recombination using sequences targeting a specific genomic region of the host cell. Researchers used CRISPR/Cas9 system as a gene-targeting method, which induces double-strand breaks(DSBs) via guide RNA and Cas9, which increases the efficiency of homologous recombination. Therefore, the CRISPR/Cas9 vector system can efficiently insert exogenous genes into CHO cell lines. 

 

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells

 

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. Currently, there are two main CHO expression systems that have been widely used, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection.

 

To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of the glutamine-dependent growth of all GS-knockout cell lines.

 

Characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

 

With a GS-knockout CHO cell line12 and promoter engineering, the GS-based system can be effective in cell line generation even in the absence of MSX1. Using two different host cell lines (CHO-K1 and GS-knockout CHO (GS KO), mAb producing rCHO cell clones were generated by a single round of selection at various MSX concentrations.

 

GS-knockout CHO cell lines with an improved selection stringency. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

A schematic diagram of the process for mAb producing clone generation and long-term culture for testing the production stability.

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.

https://www.ubigene.us/service/

Reference:

Fan L, Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye CC, Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

 

Aga, M., Yamano, N., Kumamoto, T. et al. Construction of a gene knockout CHO cell line using a simple gene targeting method. BMC Proc 9, P2 (2015). 

 

Noh, S.M., Shin, S. & Lee, G.M. Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies. Sci Rep 8, 5361 (2018).

 

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.