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.
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