Gene-edited HuH-7 Cell Line ---- Powerful Tool for Research in Coronavirus, Drug Metabolism and Cancers

As one of the five internal organs of the human body, the liver is closely related to the body's normal metabolism, detoxification, blood coagulation, and other processes. The liver participates in the body's immunity, it is an indispensable important organ for maintaining the body's life. In the study of liver diseases, a suitable cell model is one of the important tools. However, due to the difficulties in obtaining conventional liver cells, high culture failure, and high cost of culture, the development of liver disease research has been limited. Therefore, liver cell lines with simple culture conditions and stable genetic backgrounds have become a new favorite in liver research. Huh-7 is one of the most common liver cancer line.

 

The Huh-7 cell line was established by Nakabayshi H., and Sato, J. in 1982. It is a cancer cell line derived from a highly differentiated hepatocyte derived from the liver tumor of a 57-year-old Japanese man. Most Huh-7 cells show epithelial-like morphology and have a chromosome number between 55 and 63. Moreover, these cells are highly heterogeneous.

 

Hepatitis virus research: Huh-7 is highly sensitive to the hepatitis C virus (HCV). So far, Huh-7 and its derived cell lines are the only cell lines that can effectively replicate the hepatitis C virus (HCV), so Huh-7 It is often used as a model for studying HCV. It can be used to screen drug candidates against hepatitis C virus and develop new drugs against hepatitis C virus.

 

2. Xenograft cell model: The HuH-7 cells can be used as the CDX model of human hepatocellular carcinoma in a mouse model that enables pre-clinical tumor growth inhibition studies targeting kinase inhibitors (e.g. BZG-4000), FGFR4, anti-EGFRvIII antibodies and other novel anti-tumor growth therapeutics (e.g. sorafenib, silibinin).

3. Drug research: HuH-7 cell line can be applied to study the drug efficacy and metabolism of liver cancer drugs, and to explore the molecular mechanism of drugs.

 

The combination of liver cell lines and CRISPR/Cas9 technology provides new ideas for the research of cancer, drug metabolism and coronavirus!

 

Applying CRISPR/Cas9 to knock out Huh-7's key host factors for viral replication and exploring key genes for coronavirus replication

 

CypA (cyclosporin A binding protein) is an important host factor for replication of many RNA viruses. In addition, some studies showed that the replication of some viruses depends on CypA to varying degrees. These studies used different viruses, cell lines, and experimental designs, making it hard to compare one to another. Scientists have discovered the CypA dependence of three single-stranded sense RNA viruses that can replicate in Huh-7 cells, namely equine arteritis virus (EAV), human coronavirus (HCoV-229E) and Middle East respiratory syndrome coronavirus ( MERS-CoV). They compared the replication of these viruses, which were in the same parent Huh-7 cells or in the CypA gene knockout Huh-7 cells edited by CRISPR/Cas9 technology.

 

The sgRNAs targeting CypA, CypB, CypC, and CypD were transferred into Huh-7 cells by nuclear transfection, and positive cell pools were obtained after screening. Cyp KO Huh-7 cell pool was infected with MERS-CoV (B), HCoV-229E (C), or EAV (D), with 0.01 MOI. The plaque method was to determine virus production at 48h p.i. (B, C) or 32h p.i. (D). Among all four CypKO cell pools, the titer of MERS-CoV and HCoV-229E remained unchanged (B and C). After being infected in the Huh-7 CypA KO cell pool, the EAV virus titer decreased by 2-logs but did not vary in other knockout cell pools. The result proved that CypA did play an important role in the replication of EAV.

 

Since the Huh7 CypA-KO cell pool may have low levels of CypA expression residue, which is still sufficient to support normal levels of MERS-CoV and HCoV-229E replication. In this case, obtaining Huh7 CypA-KO monoclonals would be necessary. Different clones were chosen for target site amplification and sequencing verification, after which CypA-knockout positive clones were selected. Both wild-type Huh-7 cells and CypA-KO Huh-7 clones #1 and #2 were infected by MERS-CoV, HCoV-229E or EAV. In these two CypA-KO cell clones, the inactivation of CypA expression significantly reduced MERS-CoV replication (approximately 300%). Interestingly, in these two clones, there was no effect on HCoV-229E lacking CypA (D). However, for EAV, a ~3-log decrease in virus production (E) was observed, which showed a 10-fold stronger inhibitory effect compared to the previous Huh-7 CypA-KO cell pool.

 

In summary, there was no difference in the replication of MERS-CoV, HCoV-229E and EAV in the CypB-KO, CypC-KO or CypD-KO cell pools, which indicated that the replication of these viruses does not depend on CypB, CypC Or CypD in Huh-7 cells. However, similar to CypA, we cannot rule out the possibility that a very small amount of Cyp is still sufficient to support virus replication effectively. The knock-out of CypA reduced EAV production by about 3-log, while the  titers of MERS-CoV progeny decreased by about 3-fol, Also, HCoV-229E replication remained the same. This study showed that the replication of different single-stranded sense RNA viruses has significant differences in CypA dependence.

 

CRISPR/Cas9-mediated gene knockout and point mutation in Huh-7 cell model facilitate the study of the impact of different genetic differences on drug metabolism

 

In vitro studies of drug metabolism and related gene mutations often use freshly isolated or frozen human/animal liver cells; however, primary liver cells are probably not the best choice because they require liver collection and are expensive. Additionally, primary liver cells are not immortalized , which leads to large differences between different kinds of the cells. In most cases, the cell lines are identical in genome. Therefore, researchers developed a CRISPR/Cas9 modified human hepatocyte cell line so as to continuously study the effects of genetic variation on drug metabolism. To study the effects of CYP3A5 mutations on the metabolism of two enzyme substrates, the sedative or anesthetic midazolam (MDZ) and the immunosuppressant tacrolimus (Tac).

 

About 50% of oral drugs are metabolized by CYP3A4 and CYP3A5, which are the most abundant in the liver have highly variable expression. The loss of function of CYP3A5*3 (rs776746) allele is very common among Caucasians. Tac has a comparatively lower metabolic rate than those with CYP3A5*1 genotype. However, the CYP3A5*1 allele is abundant in African Americans who have rapid metabolism of MDZ, Tac, and other drugs. Therefore, CYP3A5 genotype is vital to determine the appropriate dose of drugs.

 

To present, no commercial liver cell line has been diploid on chromosome 7 and can express CYP3A5*1. Huh-7 cell line can convert substrate MDZ to its metabolite hydroxylated 1-OH-MDZ and 4-OH-MDZ through CYP3A4 activity, though they are not effective in the metabolism of MDZ because of their identity as homozygote to the CYP3A5*3 allele. Therefore, it is necessary to develop a liver cell line that can simulate the rapid metabolic process of drugs that are related to the CYP3A5*1 genotype.

 

The gRNA, Cas9 and ssODN were co-transformed into Huh-7 cells by nuclear transfection method, followed by monoclonal selection. Different clones were selected for target site amplification and sequencing verification, while positive clones with gene knockout were selected. By knocking out or point-mutating the splice junction of the Exon-3B of CYP3A5*3, three CYP3A5*1 cell lines were obtained.

 

Compared with WT  Huh-7, CYP3A5*1/*3sd (heterozygous KO), CYP3A5*1/*1dd (homozygous KO) or CYP3A5*1/*3pm (point mutation) express CYP3A5*1 mRNA, Both CYP3A5 mRNA and protein expression increased. Therefore, through the CRISPR/Cas9 technology, the *3 genotype was successfully transformed into the *1 genotype, thereby activating the expression of CYP3A5 in the Huh-7 cell line. This cell model can accelerate preclinical drug development, save time and money, and more accurately predict drug metabolism, pharmacokinetics, toxicity, and efficacy in different populations or genotypes.

 

Using CRISPR/Cas9 to knockin GFP to the Nanog in Huh-7 cells, revealing the reasons for gender differences in the incidence of hepatocellular carcinoma

 

Hepatocellular carcinoma (HCC) is a common malignant tumor, and its morbidity has obvious gender differences ---- the inclination to men. Studies have shown that the androgen/androgen receptor signal axis is related to the incidence of various hormone-related tumors such as prostate cancer and cervical cancer. As an important place for androgen metabolism, the liver has a high level of androgen in its microenvironment. Hepatocellular carcinoma seems to have a close relationship with the androgen/androgen receptor signal axis.

Cancer Stem Cells (CSCs) are closely related to the occurrence and metastasis of tumors. Their ability to self-renewal and unlimited proliferation is a key factor in cancer development. Studies have revealed that the pluripotency factor Nanog is involved in maintaining the dryness of CSCs. However, it is unclear whether the androgen/androgen receptor signal axis affects the dry maintenance of HCC cells through Nanog-related pathways.

The researchers found that the expression of the androgen receptor is very high in liver cancer tissues and is related to Nanog. Subsequently, the endogenous Nanog of huh7 cells was labeled with GFP by CRISPR/Cas9, which confirmed the co-localization of the androgen receptor and Nanog in HCC cells. The gRNA, Cas9, and Donor were co-transformed into huh-7 cells by nuclear transfection, followed by drug screening and monoclonal cultures selection. Different clones were chosen for target site amplification and sequencing, after which positive clones with homozygous knock-in were selected.

Through subsequent in vitro experiments, the researchers proved that the signal axis can promote the stemness of HCC cells, which would be achieved in a Nanog-dependent manner. By activating the transcription, this effect can be blocked by androgens Or AR degradation enhancer.

 

These studies show that the androgen/androgen receptor signaling axis provides evidence for the inhibition of this axis in HCC therapy by affecting the stemness of tumor cells, which also offers a possible way for the suppression of axons in the treatment of liver cancer.

 

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 (KO, KI, and point mutation) liver cell lines as you desire and satisfying various needs in gene editing.

 

References：

1.       de Wilde A H, Zevenhoven-Dobbe J C, Beugeling C, et al. Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture[J]. Virology, 2018, 517: 148-156.

2.       Dorr C R, Remmel R P, Muthusamy A, et al. CRISPR/Cas9 genetic modification of CYP3A5* 3 in HuH-7 human hepatocyte cell line leads to cell lines with increased midazolam and tacrolimus metabolism[J]. Drug Metabolism and Disposition, 2017, 45(8): 957-965.

3.       Jiang L, Shan J, Shen J, et al. Androgen/androgen receptor axis maintains and promotes cancer cell stemness through direct activation of Nanog transcription in hepatocellular carcinoma[J]. Oncotarget, 2016, 7(24): 36814.

 

CRISPR-U Genome-editing technologies | High-efficiency

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.

Inducible Knockout Cell Lines | 100% Guarantee

1.      Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening

Large-scale genetic screening applying CRISPR/Cas9 technology has become a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biological processes. It is reported that the optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with temporal control.

Researchers have designed multiple drug-inducible sgRNA expression vectors and measured their activities using an EGFP gene disruption assay in 11 human and mouse cell lines. The optimal design allows for tight and inducible control of gene knockout in vitro, and in vivo. Next parallel genome-wide loss-of-function screens were performed using the inducible and constitutive CRISPR-Cas9 systems. In proliferation-based dropout screens, these two approaches have similar performance in discriminating essential and nonessential genes. In a more challenging phenotypic assay that requires cytokine stimulation and cell staining, scientists observed similar sensitivity of the constitutive and drug-induced screening approaches in detecting known hits. Importantly, the minimal leakiness of our inducible CRISPR screening platforms in the absence of chemical inducers in large-scale settings.

 

2.      Conditional gene knockout in human cells with Inducible CRISPR/Cas9

The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based optimized inducible gene knockout method for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single-guide RNAs (sgRNAs) that drive Cas9 activity. To ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease.

3.      Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9

Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are useful tools for elucidating regulatory processes during early development and disease pathogenesis under the human genetic backgrounds. Genetic modification, including gene knockout (KO), further expands the utility of hPSCs in studying gene function in human embryogenesis or human genetic diseases. Thus, precise temporal control of gene KO in hPSCs is often necessary or highly beneficial for elucidating gene functions and molecular pathways that underlie complex human traits. Precise temporal control of gene expression or deletion is critical for elucidating gene function in biological systems. However, the establishment of human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) remains challenging. So scientists explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system and further developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene to speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines with iKO of SOX2, PAX6, OTX2, and AGO2, genes that exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells.

References：

1)     Sun, N., Petiwala, S., Wang, R. et al. Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening. BMC Genomics 20, 225 (2019).

2)     Yuejun Chen, Jingyuan Cao, Man Xiong, Andrew J. Petersen, Yi Dong, Yunlong Tao, Cindy Tzu-Ling Huang, Zhongwei Du, Su-Chun Zhang. Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9. Cell Stem Cell. Volume 17, Issue 2, 2015. Pages. 233-244. ISSN 1934-5909,

3)     Snijders K.E., Cooper J.D., Vallier L., Bertero A. (2019) Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9. In: Luo Y. (eds) CRISPR Gene Editing. Methods in Molecular Biology, vol 1961. Humana Press, New York, NY

CRISPR Stem Cells | High-Efficiency | Ready-to-use | 100% guarantee

Since the gene-editing potential of CRISPR systems was realized in 2013, they have fundamentally transformed our ability to manipulate genomes and been utilized in laboratories across the world for a wide variety of applications. When this gene-editing power is combined with the proliferative potential of stem cells, scientists have leveled up their understanding of cell biology, human genetics and developmental biology, and the future potential of regenerative medicine. 

As a consequence of CRISPR/Cas9 systems, next-generation genome sequencing, and stem cell technologies have matured, the possibilities for their combined use have also enlightened. Here are the latest advances in this rapidly evolving field to showcase the current progress in the intersection of gene-editing (such as gene-knockout, gene-knockin, and pointmutation, etc.) stem cell research.

The ability to modify selectively specific genes provides a powerful tool for characterizing gene functions, performing gene therapy, correcting specific genetic mutations, eradicating diseases, engineering cells and organisms to achieve more innovative functions and obtain transgenic animals as models for disease studies. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has revolutionized genome engineering. The application of CRISPR/Cas9 systems for stem cell research allows researchers to develop new disease models to explore new therapeutic tools and address degenerative diseases. Moreover, there are challenges and future perspectives regarding the use of CRISPR/Cas9 as a helpful technology in the invention of new medical therapies.

 

1.     Using the CRISPR/Cas9 to create stem cells that antagonize IL-1- / TNF-α-mediated inflammation

To create stem cells that antagonize IL-1- and TNF-α-mediated inflammation in an autoregulated manner, researchers applied the genome editing system CRISPR/Cas9. Transgenes encoding a firefly luciferase transcriptional reporter or a cytokine antagonist, either murine IL-1Ra or a chimeric human sTNFR1-murine immunoglobulin G (Bloquel et al., 2004), were targeted to the Ccl2 start codon in murine iPSCs cells (Diekman et al., 2012) using the CRISPR/Cas9 platform.

The results show that genome engineering can be applied successfully to rewire endogenous cell circuits to allow for prescribed input/output relationships between inflammatory mediators and their antagonists. This provides a foundation for cell-based drug delivery or cell-based vaccines via a rapidly responsive, autoregulated system. The customization of intrinsic cellular signaling pathways in stem cells also provides innovative ideas for safer and more effective therapeutic approaches for several kinds of diseases.

 

2.     Using the CRISPR/Cas9 system in genetic modification of pluripotent or multipotent stem cells

Combined with the pluripotency of stem cells, the technology represents a powerful tool to generate various cell types for disease modeling, drug screening, toxicology, and targeted therapies. Generally, the CRISPR/Cas9 system has been applied in genetic modification of pluripotent or multipotent stem cells, after which the cells are differentiated into specific cell types and used for functional analysis or even clinical transplantation. Recent advancement in CRISPR/Cas9 technology has widened the scope of stem cell research and its therapeutic application. This review provides an overview of the current application and the prospect of CRISPR/Cas9 technology, particularly in stem cell research and therapy.

 

3.     combining iPSC and CRISPR/Cas9 technologies for the investigation of the molecular and cellular mechanisms of inherited diseases

CRISPRi permits gene repression at the transcription level, as opposed to RNAi which controls genes at the mRNA level. This allows researchers to repress certain genes within stem cells and decipher their function. Kampmann explains: "For CRISPRi, we target a transcriptional repressor domain (the KRAB domain) to the transcription start site of genes to repress their expression. This knockdown approach is highly effective and lacks the notorious off-target effects of RNAi-based gene knockdown.”Using CRISPR/Cas9 systems(such as gene-knockout, gene-knockin, and point mutation, etc.) to mediate iPSC stem cells is helpful to investigate mechanisms of inherited diseases including immunological, metabolic, hematological, neurodegenerative, and cardiac diseases.

 

Ubigene Biosciences, we provide high-efficiency and 100% guarantee CRISPR services including gene knockout/knock-in and point mutation cell lines. Make Genome Editing Easier is the goal of Ubigene. Our exclusive technologies have been successfully applied in more than 100 types of 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.

 

Ubigene also provides CRISPR-B ™ gene-editing services exclusively for bacteria and fungus, virus packaging such as lentiviruses, adenoviruses, and adeno-associated viruses (AAV), etc.

 

References:

1.     Jonathan M. Brunger, Ananya Zutshi, Vincent P. Willard, Charles A. Gersbach, Farshid Guilak. Genome Engineering of Stem Cells for Autonomously Regulated, Closed-Loop Delivery of Biologic Drugs. Cell. VOLUME 8, ISSUE 5, P1202-1213, MAY 09, 2017.

2.     Jatin Roper, Ömer H. Yilmaz. Breakthrough Moments: Genome Editing and Organoids. Cell Stem Cell, Volume 24, Issue 6, 6 June 2019, Pages 841-842.

3.     Fleischer A., Vallejo-Díez S., Martín-Fernández J.M., Sánchez-Gilabert A., Castresana M., del Pozo A., Esquisabel A., Ávila S., Castrillo J.L., Gaínza E., Pedraz J.L., Viñas M., Bachiller D.

4.     iPSC-Derived Intestinal Organoids from Cystic Fibrosis Patients Acquire CFTR Activity upon TALEN-Mediated Repair of the p.F508del Mutation. Molecular Therapy - Methods and Clinical Development, Volume 17, 2020.

5.     Valenti MT, Serena M, Carbonare LD, Zipeto D. CRISPR/Cas system: An emerging technology in stem cell research. World J Stem Cells. 2019;11(11):937‐956.

6.     Zhang Y, Sastre D, Wang F. CRISPR/Cas9 Genome Editing: A Promising Tool for Therapeutic Applications of Induced Pluripotent Stem Cells. Curr Stem Cell Res Ther. 2018;13(4):243‐251.

7.     Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell, ISSN: 1875-9777, Vol: 13, Issue: 6, Page: 653-8

CRISPR H9C2 cell line | Ubigene CRISPR-UTM | High-Efficiency

The H9C2 line of embryonic rat cardiomyocytes is a subclonal line of the original clonal cell line derived from embryonic BD1X rat heart tissue by Kimes and Brandt (1976). This cell line exhibits many of the properties of skeletal muscle and its parameters are particularly useful in preclinical tests of anticancer drugs, determining their cardiotoxicity, safety, and the possibility of moving to subsequent stages of clinical tests. Therefore H9C2 cell line is commonly used in numerous in vitro studies, some of which involve CRISPR technology to mediates the characteristics of H9C2 cells.

 

H9C2 cells retain some components of the signaling pathway essential for their differentiation into mature cardiac muscle cells. The cell line is used, in particular, for cardiotoxicity analyses of new, mainly anticancer drugs (e.g. doxorubicin), and studies on mechanisms of myocyte damage, and assessment of toxic effects of studied compounds on apoptosis and necrosis in cardiac myocytes. Embryonic H9C2 cardiomyocytes proliferate well in vitro conditions, allowing relatively easy culturing and are suitable to be a CRISPR gene knockout/knockin or over-expression model for in vitro studies of cardiac hypertrophy and supports current work with human cardiomyocyte cell lines for prospective molecular studies in heart development and disease.

 

Applications:

1. Effects of C3G Knockout on Proliferation and Apoptosis in H9C2 Cardiomyocytes

Previous studies found that C3G expression was significantly increased in the myocardium of the non-infarct area around the infarct in rats. Overexpressed C3G can promote cardiomyocyte survival and inhibit cytotoxicity while knocking down C3G can inhibit cardiomyocyte survival and increase cardiomyocyte apoptosis. The CRISPR/Cas9 system built-in knockout C3G recombinant lentivirus was used to infect H9C2 cardiomyocytes to study the speculated effect and mechanism of C3G knockout on the proliferation of H9C2 cardiomyocytes. H9C2 cardiomyocytes were infected with above lentiviruses respectively to investigate the effects of C3G [Crk SH3-domain-binding guanine nucleotide exchange factor] knockout on proliferation and apoptosis in H9C2 cardiomyocytes and their underlying mechanisms.

 

2. Using CRISPR-Cas9 gene-editing technology to knock out Tudor-SN gene of H9c2 cells to inhibit cell cycle arrest and proliferation

CRISPR-Cas9 gene-editing technology was used to knock out the Tudor-SN (Tudor staphylococcal nuclease) gene of rat myocardial H9c2 cells. Researchers observed its effect on H9c2 cell cycle and proliferation. The PX462 plasmid was selected as the vector, and the upstream and downstream sgRNA (single-guided RNA) that can specifically recognize the second exon of Tudor-SN gene in H9c2 cells were designed using software to construct a pair of recombinant plasmids. Subsequently, the pair of plasmids were co-transformed into H9c2 cells, and then positive monoclonal cells were selected for cultivation. Western blotting was used to identify the knockout effect, and the cell cycle and proliferation were detected by flow cytometry and CCK-8=experiment using the successfully knocked out cell lines. Western blotting results showed that Tudor-SN protein was not expressed in positive cells, and the Tudor-SN gene was successfully knocked out. Flow cytometry results showed that Tudor-SN gene knockout cells had a G1 phase arrest. The results of the CCK-8 experiment showed that the proliferation rate of KO cells slowed down. In this experiment, the Tudor-SN gene knockout cell line of H9c2 cells was successfully constructed, and the inhibition of Tudor-SN gene knockout on cell cycle arrest and proliferation was detected, which provided a study for the regulation of Tudor-SN gene on cardiomyocyte function. Convenient tools and research foundation.

 

3. Dock180 knockout inhibits proliferation and promotes apoptosis of rat derived H9C2 cardiomyocytes strain

To investigate the effects of dedicator of cytokinesis 1 (Dock180) knockout on proliferation and apoptosis in rat derived H9C2 cardiomyocytes and their mechanisms, a single guide RNA (sgRNA) targeting rat Dock180 gene was designed and constructed using CRISPR/Cas9 system. A plasmid contained above sgRNA was packaged into lentivirus and selected to knockout Dock180 in the cardiomyocytes. The result showed that Dock180 knockout with CRISPR/Cas9 H9C2 cells can inhibit proliferation and promote apoptosis via p-ERK1/2, Bcl-2, and Bax in H9C2 cardiomyocytes.

 

Ubigene Biosciences, we provide high-efficiency and 100% guarantee CRISPR services including gene knockout/knock-in and point mutation cell lines. Make Genome Editing Easier is the goal of Ubigene. Our exclusive technologies have been successfully applied in more than 100 types of 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.

Ubigene also provides CRISPR-B ™ gene-editing services exclusively for bacteria and fungus, virus packaging such as lentiviruses, adenoviruses, and adeno-associated viruses (AAV), etc.

 

References:

1. GAN Shi-Hu, CUI Xiao-Teng, MA Jin-Zheng, FANG Li-Jiao, LIU Ming-Xia, REN Yuan-Yuan, CAO Xiao-Na, YANG Jie, SU Chao. 2.Using CRISPR-Cas9 gene-editing technology to knock out the Tudor-SN gene of H9c2 cells to inhibit cell cycle arrest and proliferation. Chinese Journal of Biochemistry and Molecular Biology, 2018.07.10.

2. Deng Qin, Liu Cheng, Zhang Jing, Li Gang. The effect of knocking out C3G on the proliferation and apoptosis of H9C2 cardiomyocytes. Chinese Journal of Cell Biology: 1-6[2020-06-09].

3. HU Su-lei, LI Gang, FU Yan-bo, DENG Qin, LIU Cheng. Dock180 knockout inhibits proliferation and promotes apoptosis of rat derived H9C2 cardiomyocytes strain. Basic & Clinical Medicine. 2017, 37(4).

KYSE-150 CRISPR cell line | 100% guarantee | High-efficiency

KYSE-150 cells are poorly differentiated esophageal adenocarcinoma cells that were isolated from the neck esophagus of a 49-year-old Japanese female patient who had received radiotherapy. When researchers discovered this cell line, the patient's cancer tissue had invaded the adjacent tissues. The KYSE-150 cell line is an epithelial cell with an adherent monolayer. It has been reported that cells carry increased oncogenes c-erb-B (8 times) and cyclin D1 (4 times) and that cells can form tumors in nude mice, so KYSE-150 cell line is often used in cancer research involving gene editings, such as CRISPR gene knockout/knock-in and point mutation.

Applications:

1. EZH2 knockout in KYSE-150 cells altered PSMA3-AS1-induced proliferation and migration in esophageal cancer cells

PSMA3-AS1 expression is up-regulated and positively correlated with tumor size and metastasis in esophageal cancer patients. To further explore the biological roles of PSMA3-AS1 in esophageal cancer cells, researchers established stable CRISPR PSMA3-AS1-overexpressing cell lines via lentiviral infection in KYSE150 and KYSE450 cell lines (which have low PSMA3-AS1 expression) and validated the up-regulation of PSMA3-AS1 by RT-qPCR.

CRISPR/Cas9 gene-editing of EZH2 was successfully performed in KYSE150 and KYSE450cells as confirmed by a significant reduction in EZH2 protein expression. CCK-8 and colony formation assays showed that PSMA3-AS1 overexpression did not affect the proliferation of esophageal cancer cells with CRISPR EZH2 knocked out (Figure 6B and 6C). According to wound healing and transwell migration assays, wounding healing and cell migration were not increased in ESCC EZH2-knock out KYSE-150 cells compared to negative control cells.

Clinical-pathological characteristics illustrated that increased expression of PSMA3-AS1 was positively associated with distant metastasis, larger tumor sizes, and a worse prognosis for ESCC patients.

 

2. CRISPR Over-expressed KYSE-150 cells increase chemo-resistance in ESCC

Whole-genome clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR/Cas)-based a lentiviral library is a powerful tool for genome-scale gain-of-function or loss-of-function screening. This system has been proved to be highly effective in identifying drug-resistant genes in vitro. Shalem, Kurata, and Joung have screened out essential genes for drug resistance in melanoma and AML using the CRISPR knockout library. Some researchers attempted to combine the CRISPR library screening strategy with RNA sequencing technology in KYSE-150 Cells to explore the critical genes and potential mechanism for chemo-resistance in ESCC.

To increase the chance of identifying essential genes involved in PTX resistance, an integrated analysis was performed to combine EN-genes in genome-scale CRISPR screening and differentially expressed genes (DE-genes) in KYSE-150 cell line.

Researchers evaluated the potential of CDKN1A, ELAVL2, and TSPAN4 to promote chemo-resistance in ESCC cells KYSE-150. The results showed that overexpression of CDKN1A, ELAVL2, or TSPAN4 could significantly increase the resistance to PTX in both KYSE-180 and KYSE-150 cells. Moreover, overexpressed CDKN1A, ELAVL2, or TSPAN4 could also contribute to DDP-resistance in KYSE-150 cells.

3. CRISPR/Cas9 mediated knockout of DEPTOR in KYSE-510 cells significantly promoted cellular proliferation, migration, and invasion

Researchers found that the expression of DEPTOR negatively regulates the tumorigenic activities of ESCC cell lines. Furthermore, ectopic DEPTOR expression caused significant suppression of the cellular proliferation, migration, and invasion of KYSE150 cells, which has the lowest expression level of DEPTOR. Meanwhile, CRISPR/Cas9 mediated knockout of DEPTOR in KYSE-510 cells significantly promoted cellular proliferation, migration, and invasion. Besides, in vivo assays further revealed that tumor growth was significantly inhibited in xenografts with ectopic DEPTOR expression as compared to untreated KYSE150 cells, and was markedly enhanced in DEPTOR knockout KYSE-510 cells.

In this study, scientists generated stable cell lines that either overexpressing DEPTOR or genetic ablation of endogenous expression of DEPTOR. Since KYSE-150 expresses the lowest endogenous level of DEPTOR among the three cell lines, they stably overexpressed DEPTOR in KYSE-150 cells (pcDNA3.1-DEPTOR). For the same consideration, KYSE-510 cells that express the highest level of DEPTOR were treated with CRISPR/Cas9 system to knockout of DEPTOR (CRISPR-DEPTOR). After the generation of cell lines, pcDNA3.1-DEPTOR displayed a reduced cell proliferation rate as compared to that of KYSE-150 parental cells and empty vector-transfected cells, while CRISPR-DEPTOR cells proliferated significantly faster than control KYSE-510 cells. Furthermore, pcDNA3.1-DEPTOR cells also showed reduced migration. Thus, these results suggested DEPTOR indeed regulates cell proliferation, migration, and invasion in ESCC cells.

 

 

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. Yan-Mei Ji, Xue-Feng Zhou, Jun Zhang, Xiang Zheng, Sheng-Bao Li, Zhi-Qiang Wei, Tao Liu, Dong-Liang Cheng, Ping Liu, Kuncheng Song, Tao Tan, Hua Zhu, Jia-Long Guo. DEPTOR suppresses the progression of esophageal squamous cell carcinoma and predicts poor prognosis. 2016 Mar 22; 7(12): 14188–14198. Published online 2016 Feb 16.

2. Qiu BQ, Lin XH, Ye XD, et al. Long non-coding RNA PSMA3-AS1 promotes malignant phenotypes of esophageal cancer by modulating the miR-101/EZH2 axis as a ceRNA. Aging (Albany NY). 2020;12(2):1843‐1856.

3. Zhao, Wen-Si et al. “Genome-scale CRISPR activation screening identifies a role of ELAVL2-CDKN1A axis in paclitaxel resistance in esophageal squamous cell carcinoma.” American journal of cancer research vol. 9,6 1183-1200. 1 Jun. 2019

U251 knockout cell line | Exclusive CRISPR-U | Ubigene

U251 cell line was derived from a malignant glioblastoma tumor by explant technique, which is commonly used as experimental models in studies about glioblastoma. U-251 MG cell line human has been used to study the mechanism of Pax6 (paired box protein)-associated increase in expression of Dkk3 (Dickkopf 3), a PAX6-knockout U251 cell line via CRISPR/Cas9 is a commonly-used tool for molecular studies of the roles PAX6 have in attenuating glioblastoma tumor progression. Also, the U251 cell line can be used for the extraction of cancer stem cells from the U-251 human glioblastoma cell line, which makes it a popular cell line in the gene-editing filed. Moreover, it is often applied in studies investigating the role of JARID1B (Jumanji AT-rich interactive domain 1B) in the pathogenesis of glioma and the therapeutic efficacy of Irinophore C combined with temozolomide in a glioblastoma tumor model. So U251 cell is a suitable cell line for gene knockout, gene knockdown, and gene knockin, etc.

 

Application:

 

The PAX6-knockout U251 cell line was found to have increased proliferation and colony-forming abilities

The transcription factor PAX6 is expressed in various cancer cell lines, including the U251 cell line. In anaplastic astrocytic glioma, PAX6 expression is inversely related to tumor grade, resulting in low PAX6 expression in Glioblastoma, the highest-grade astrocytic glioma. The U251 cell line is commonly used as experimental models in studies about glioblastoma. So some scientists develop a PAX6 knockout U251 cell line as a tool for molecular studies of the roles PAX6 has in attenuating glioblastoma tumor progression. The CRISPR-Cas9 technique was used to knockout PAX6 in U251 N glioblastoma cells. The guide RNAs were designed to create mutations close to the 5’end of the PAX6 gene, between positions + 25 to + 58 relative to the transcription start site (TSS). Viral transduction of a doxycycline-inducible EGFP-PAX6 expression vector was used to re-introduce (rescue) PAX6 expression in the PAX6 knock out U251 cells. The knockout U251 cells were rigorously characterized by analyzing morphology, proliferation, colony-forming abilities, and responses to oxidative stress and chemotherapeutic agents. The result showed that the knockout U251 cells had increased proliferation and colony-forming abilities compared to WT cells, consistent with clinical observations indicating that PAX6 functions as a tumor-suppressor. For the PAX6 knockout U251 cells, the percentage of cells in the G2/M phase increased compared to PAX6 control cells, indicating that PAX6 keeps U251 N cells in the G1 phase of the cell cycle. Interestingly, PAX6-knockout U251 cells were more resilient to H2O2 induced oxidative stress than wild type cells. The U251 N PAX6-knockout cell lines generated can be used as a tool to study the molecular functions and mechanisms of PAX6 as a tumor suppressor concerning tumor progression and treatment of glioblastoma.

 

 

AEG-1-knockout U251 cells construction and overexpressed AEG-1 gene in U251 cells showed reduced cell migration ability

 

Astrocyte elevated gene-1 (AEG-1) was overexpressed in a diverse array of cancers and played an important role in the development and progression of cancer. Researchers constructed the AEG-1-knockout U251 cell line by CRISPR/Cas9 technology to explore the effect of AEG-1 on the metastasis in U251 Cells. The first step was to synthesize the designed sgRNA targeted to AEG-1, and the sgRNA was cloned into the pX459 plasmid to obtain the AEG-1-pX459 recombinant vector. The recombinant vector was transfected into human glioma U251 cells, and the activity of sgRNA was identified by TA cloning sequencing. Then, the U251 cells transferred with the recombinant vector were screened by puromycin to get the AEG-1-knockout U251 cell line. Western blot assay was used to detect the efficiency of gene knockout. Finally, the migration ability of the AEG-1-knockout cell line was evaluated by the methods of the Transwell and Scratch experiment. The data showed that the AEG-1-pX459 recombinant vector was successfully constructed, and the sgRNA activity was confirmed by TA cloning sequencing, which meant the AEG-1-knockout U251 cell line was successfully established. Western blot assay analysis showed that the knockout efficiency in U125 cells was up to 98%. From the Transwell and Scratch experiment results, the migration ability of the AEG-1-knockout cell line reduced obviously.

 

Knockdown CA-NFATc1 reduced the invasion ability of U251 cell line 

 

Recent studies indicated that the nuclear factor of activated T-cells (NFAT) is a transcription factor that is highly expressed in aggressive cancer cells and tissues, one of which is the U251 cell line. To investigate the role of NFATc1 in U251 cells, researchers established a U251 cell line expressing a constitutively active form of NFATc1 (CA-NFATc1). In this process, small interfering RNA was used to knock down NFATc1 expression in the U251 cells. The result demonstrated that the expression of CA-NFATc1 promoted U251 cancer cell invasion, while siRNA against NFATc1 successfully inhibited the invasion ability of the knockdown U251 cell line. Moreover, the research demonstrated that NFATc1 promoted U251 cell invasion through the induction of cyclooxygenase-2 (COX2) because NFAT transcriptionally regulates the induction of COX-2 induction in U251 cells and binds to the promoter.

 

The effects of knockdown and knockout of CREB in U125 cells 

 

Glioma is a type of tumor that occurs in the brain and accounts for almost 30 % of all brain and central nervous system tumors and 80 % of all malignant brain tumors. To investigate the role of cAMP response element-binding protein (CREB) in the progression of glioma, CREB was then knocked down via siRNA to see if the reduced expression of CREB affects U251 cells proliferation and migration. Moreover, CRISPR/CAS9 mediated knockout of CREB was conducted and the athymic Nude mice model was used to investigate CREB's role in vivo. The result shows that knockdown of CREB via siRNA in glioma cell line U251 significantly inhibited the proliferation and migration of tumor cells. Additionally, knockout of CREB via CRESP/CAS9 inhibited tumor formation of U251 cells in athymic nude mice model. Therefore over-expression of CREB may contribute to the progression of glioma and knockdown of CREB expression may serve as a novel target for therapy.

References:

Wang L, Wang Z, Li J, Zhang W, Ren F, Yue W. NFATc1 activation promotes the invasion of U251 human glioblastoma multiforme cells through COX-2. Int J Mol Med. 2015;35(5):1333‐1340.

Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress. Beate Hegge, Eva Sjøttem, Ingvild MikkolaBMC Cancer. 2018; 18: 496. Published online 2018 May 2.

SHENG Yu-rui, LI Bin, WANG Bin, ZUO Di, MA Lin, REN Xiao-fan, GUO Le, LIU Kun-mei. The Construction of AEG-1-Knockout U251 Cell Line by CRISPR/Cas9 Technology and Study of The Effect of AEG-1 on the Metastasis in U251 Cells[J]. China Biotechnology, 2018, 38(10): 38-47.

Knockdown of CERB expression inhibits proliferation and migration of glioma cells line U251.Zheng KB, Xie J, Li YT, Yuan Y, Wang Y, Li C, Shi YF. Bratislavske Lekarske Listy, 01 Jan 2019, 120(4):309-315.

 

NCI-H1299 KO cell line| High Efficiency | 100% guarantee | Ubigene

NCI-H1299 cell line, also known as H1299 or CRL-5803, is a human non-small cell lung carcinoma (NSCLC) cell line derived from the lymph node. Similar to other immortalized cell lines, H1299 cells can divide infinitely. Also, H1299 cells have a homozygous partial deletion of the TP53 gene, therefore they do not express the tumor suppressor p53 protein that will lead to their proliferative propensity. Moreover, it is reported that these cells can secrete the peptide hormone neuromedin B (NMB), but not gastrin-releasing peptide (GRP). With these interesting characteristics, NCI-H1299 cells become a popular cell line in biological fields that involves gene knockout, gene knock-in, and point mutation.

Lung cancer is the second most prevalent and the most lethal malignancy in both men and women throughout the world. On the other hand, NSCLC accounts for about 85% of lung cancer, which is the leading cause of cancer death in the world. As H1299 cell line was derived from the lymph node, so these cells have been widely used in lung cancer research. Characterizing the accumulated genetic alterations in cancer cells is of importance in both understanding tumor biology and guiding drug design. Besides, it might benefit the patients with a given targeted cancer therapy via gene-customized cancer-related cells, such as gene knockout H1299 cells, etc. As studies showed, NCI-H1299 cell line is a stable cell line with a homozygous knockout of some human genes using CRISPR/Cas9. So NCI-H1299 ko cell line is commonly-used in the treatment of lung cancer.

Application:

Paclitaxel reduced the ability of migration and proliferation of Rsf-1-knockout NCI-H1299

Paclitaxel is a classic drug for the treatment of lung cancer, but it readily induces resistance. Therefore, new ways to overcome drug resistance are urgently needed. Rsf-1 expression is high in lung cancer and increases paclitaxel tolerance through the NF-κB pathway. Many other genes are also involved in drug resistance, such as genes that affect apoptosis and the cell cycle. Chen and colleagues knocked out Rsf-1 in lung cancer cells and xenograft mice using CRISPR-Cas9 technology and tested whether Rsf-1 affects the sensitivity of lung cancer to paclitaxel by regulating the activation of the NF-κB pathway and the expression of its downstream genes. The result indicated that paclitaxel reduced the ability of Rsf-1-knockout NCI-H1299 and NCI-H460 cells to migrate and proliferate, and increased apoptosis. In mice with xenograft tumors derived from Rsf-1-knockout cells, the anti-tumor effect of paclitaxel was enhanced. Therefore, targeting Rsf-1 by CRISPR-Cas9 technology has also become one of the approaches for treating lung cancer.

Knockdown PON2 gene to investigate NCI-H1299 cell line proliferation

Previous research demonstrated that the expression of paraoxonase 2 (PON2), a lactonase/arylesterase with anti-oxidant properties, are markedly enhanced in cancer tissues of NSCLC patients compared with corresponding adjacent non-tumorous tissues. Moreover, increased PON2 expression might contribute to the resistance of NSCLC cells to classical anti-NSCLC therapeutic drugs. However, researchers found that stably reduced PON2 expression by siRNA can reduce the proliferation of NSCLC cells such as NCI-H1299 cells.

To further elucidate the role of PON2 in NSCLC cell proliferation, two gene-editing systems, TALEN and CRISPR/Cas, were applied in the NSCLC cell line NCI-H1299. The result indicated that Cas9 expression was induced by exogenous doxycycline in NSCLC cells in a reversible fashion, providing a platform to investigate how changes in gene expression modulate NSCLC cell proliferation.

References:

1. X. Chen, X. Sun and J. Guan, et al., Rsf-1 Influences the Sensitivity of Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-κB Pathway and Its Downstream Proteins, Cell. Physiol. Biochem., 2017, 44(6), 2322.

2. Antitumor effects of Tubeimoside-1 in NCI-H1299 cells are mediated by microRNA-126-5p-induced inactivation of VEGF-A/VEGFR-2/ERK signaling pathway. Hanbing Shi, Hongxia Bi, Xingyuan Sun, Haiying Dong, Yunfei Jiang, Haijun Mu, Guohua Liu, Weili Kong, Ruizhi Gao, Jiang Su. Mol Med Rep. 2018 Mar; 17(3): 4327–4336.

 

MDA-MB-231 ko cell line | 10x Efficiency | Ubigene

The MDA-MB-231 cell line is a human breast cell line that was established from a pleural effusion of a 51-year-old caucasian female with metastatic mammary adenocarcinoma. This epithelial cell line is one of the most widely used breast cancer cell lines in medical research laboratories, especially those involve CRISPR/Cas9 gene knockout and gene knockdown. 

As a result of lacking ER and PR expression and HER2 amplification, the cell line was initially classed as a “basal” breast cancer cell line. However, it is now recognized as belonging to the claudin-low molecular subtype as it exhibits down-regulation of claudin-3 and claudinin-4, low expression of the Ki-67 proliferation marker, enrichment for markers associated with the epithelial-mesenchymal transition and the expression of features associated with mammary cancer stem cells (CSCs), such as the CD44+CD24-/low phenotype. That’s why many studies had been focusing on the gene-editing MDA-MB-231 cell line.

Application:

1. CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in MDA-MB-231 cells.

Cancer cells require the expression of certain genes that encode proteins necessary for tumor growth. Silencing the expression of these genes or blocking the activity of the proteins can trigger cell death and durable tumor regression. So identifying and characterizing cancer dependencies is a key goal of pre-clinical cancer research. MELK has been implicated as a cancer dependency and putative drug target in multiple cancer types, one of which is the MDA-MB-231 cell line. MELK is overexpressed in breast cancer cells, which is associated with poor patient prognosis. Moreover, knockdown of MELK in MDA-MB-231 cells using RNAi has been reported to block cancer cell proliferation and trigger cell cycle arrest or mitotic catastrophe.

Researchers designed seven guide RNAs (gRNAs) against MELK and cloned each guide RNA into a GFP-expressing vector and transduced the guides into three Cas9-expressing cell lines: the triple-negative breast cancer cell line MDA-MB-231 reported to be addicted to MELK expression. Western blot analysis of MELK protein levels in overexpression MDA-MB-231 cells confirmed that the CRISPR system can effectively ablate MELK expression. 

2. Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells

To explore the functional significance of β1 integrin, researchers established β1-KO MDA-MB-231 cells via a CRISPR/Cas9-based approach. Western blot and FACS analyses were performed to confirm the effective knockout of β1 in the MDA-MB-231 cell line. Surprisingly, the expression of β1 exhibited opposite effects on cell proliferation. These effects were dependent on cell densities and they showed an up-regulation of cell proliferation when cells were cultured under sparse conditions and a down-regulation of cell growth under dense conditions. By comparison with WT cells, the phosphorylation levels of ERK in KO MDA-MB-231 cells were consistently suppressed under sparse culture conditions but consistently up-regulated under dense culture conditions. The phosphorylation levels of EGFR were increased in the KO MDA-MB-231 cells. By contrast, the phosphorylation levels of AKT were decreased in the MDA-MB-231 KO cells. The abilities for both colony and tumor formation were significantly suppressed in the MDA-MB-231 knockout cells, suggesting that β1 plays an important role in cell survival signaling for tumorigenesis. These aberrant phenotypes in the KO cells were rescued in the Res cells. Taken together, these results clearly showed the distinct roles of β1 in cancer cells: the inhibition of cell growth and the promotion of cell survival, which may shed light.

3. Knockdown of BCL2L12 leads to cisplatin resistance in MDA-MB-231 breast cancer cells

Gene BCL2L12 was found to be highly expressed in normal breast tissues and was associated with favorable prognosis in breast cancer patients. Researchers reported that the mRNA levels of BCL2L12 and its transcript variant BCL2L12A could be upregulated upon cisplatin treatment in MDA-MB-231 breast cancer cells. The knockdown of BCL2L12 and BCL2L12A dramatically inhibited cisplatin-induced apoptosis. In contrast, ectopic expressions of each of the proteins promoted cisplatin-induced apoptosis. These results indicated that decreased expressions or loss of BCL2L12 and BCL2L12A may contribute to the cisplatin resistance in breast cancer patients. Furthermore, we found that cisplatin-induced downregulation of β-catenin was partially suppressed in BCL2L12- and BCL2L12A-knocked down MDA-MB-231 cells, which indicated that knockdown of these two proteins may stabilize β-catenin in cisplatin-induced apoptosis. In short, we proposed that BCL2L12 and BCL2L12A may play an important role in cisplatin-induced apoptosis in MDA-MB-231 breast cancer cells.

References:

1. Christopher J Giuliano, Ann Lin, Joan C Smith, Ann C Palladino, Jason M Sheltzer. MELK expression correlates with tumor mitotic activity but is not required for cancer growth. eLife 2018;7:e32838 DOI: 10.7554/eLife.32838.

2. Ann Lin, Christopher J Giuliano, Nicole M Sayles, Jason M Sheltzer. CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials. eLife 2017;6:e24179 DOI: 10.7554/eLife.24179.

3. Yi Hong, Junwu Yang, Weibing Wu, Wenzong Wang, Xiangfei Kong, Yanlin Wang, Xiaojing Yun, Hongliang Zong, Yuanyan Wei, Si Zhang, Jianxing Gu. The knockdown of BCL2L12 leads to cisplatin resistance in MDA-MB-231 breast cancer cells. Volume 1782, Issue 11, November 2008, Pages 649-657.

4. Hou, S., Isaji, T., Hang, Q. et al. Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells. Sci Rep 6, 18430 (2016). https://doi.org/10.1038/srep18430.

 

AGS CRISPR Cell Line | Ubigene | 20x Efficiency

The AGS cell line was derived from fragments of a stomach tissue tumor resected from a 54-year-old caucasian female patient who had received no prior therapy. This cell line is closely related to gastric adenocarcinoma diseases. To date, several models of gastric carcinoma xenografts using different cell lines have been developed. AGS is one of the most common cell lines for the xenograft modeling of gastric cancer; however, there is limited information about the characteristics of this model. So researchers usually apply CRISPR/Cas9 technology to create gene knockout or gene knockin AGS cell lines.

AGS knockout cell line is clonally derived from the AGS cell line which has been transfected with an encoded vector, followed by stable cell selection. This cell line can stably express CRISPR Cas9 nuclease, GFP, and hygromycin resistance gene. In combination with separately transfected sgRNAs, the AGS-Cas9 KO cell line can be used to efficiently generate targeted genomic modifications including gene knockout, gene knockin, gene mutagenesis, gene tagging, etc. It is also an ideal cell line model for sgRNA screening and validation, either individually or in pools. So the AGS knockout cell line is a popular cell model in studies that involve Gastric cancer and diseases.

Application:

Knockout PDEF in AGS cells using CRISPR/Cas9 genome-editing system to study gastric cancer

Gastric cancer is one of the most common malignant tumors worldwide and has the second-highest incidence and mortality rate among malignant tumors in China. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors. Although PDEF plays an important role in tumorigenesis, its biological function in gastric cancer is still unclear. Researchers investigated PDEF expression in the gastric cancer cell line AGS and the normal gastric epithelial cell line GES; The CRISPR/Cas9 genome-editing system was used to knockout PDEF in AGS cells as a model for gastric cancer. were evaluated  Then they used CCK-8, flow cytometry, scratch wound, and transwell assays to evaluate the cell proliferation, apoptosis, migration, and invasion of PDEF-knockout AGS cells respectively. The results illustrated that  Knockout of the PDEF gene significantly inhibited the migration of AGS GC cells, which implied that PDEF plays an important role in the proliferation, migration, and invasion of AGS cells and may serve as a new treatment target in gastric cancer. The PDEF protein in the AGS cells transfected with the recombinant plasmidpX459-sgRNA1 was not expressed at all, within the sensitivity of the western blot assays. Knocked down PDEF expression, have significantly lowered ABS CRISPR cells’ ability of migration, invasiveness, and proliferative capacity.

 

CRISPR/Cas9-mediated LMX1A knockout promoted AGS cell survival and proliferation

LIM homeobox transcription factor 1, alpha (LMX1A) is downregulated in human gastric cancer (GC), functioning as a tumor suppressor. By sequencing analysis of LMX1A mRNA 3’-untranslated region(3’-UTR), it shows that microRNA-9(miR-9) putatively targets human LMX1A. In established gene-edited AGS cells and primary human GC cells, ectopic overexpression of miR-9 by a lentiviral construct decreased LMX1A 3’-UTR activity, causing LMX1A mRNA and protein downregulation. Functional analyses show that miR-9 overexpression enhanced GC cell survival and proliferation. On the contrary, miR-9 inhibition by antagomir-9 lentivirus increased LMX1A 3’-UTR activity to upregulate LMX1A mRNA and protein expression, causing GC cell apoptosis. CRISPR/Cas9-mediated LMX1A knockout promoted AGS cell survival and proliferation. Importantly, miR-9 and antagomiR-9 were ineffective in the function of LMX1A-knockout AGS cells. Therefore, the knockout AGS cell line is helpful in studying the affecting factors of gastric cancer.

 

References:

1. Zhang YQ, Pei JH, Shi SS, et al. CRISPR/Cas9-mediated knockout of the PDEF gene inhibits migration and invasion of human gastric cancer AGS cells. Biomed Pharmacother. 2019;111:76‐85.

2. Xiaohong Zhang, Yanqing Qian, Fan Li, Songhua Bei, Meiyi Li, Li Feng. microRNA-9 selectively targets LMX1A to promote gastric cancer cell progression. Biochemical and Biophysical Research Communications. 2018;405-412.