NRF2 gene knockout for treating cancer
A gene and cancer technology, applied in the field of knockout NRF2 composition, can solve the problem of loss of efficacy
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Embodiment 1
[0130] Example 1: Establishment of NRF2 knockout clone A549 cell line using CRISPR-guided gene editing approach
[0131] method
[0132] cell culture conditions
[0133] Human lung cancer A549 cells were purchased from ATCC (Manassas, VA, USA). A549 is a mature non-small cell lung adenocarcinoma cell line that contains a mutation in the Kelch domain of KEAP1 that results in overexpression of NRF2. It is often used as a standard for discovering new therapeutic agents against cancer. Cells were thawed according to the manufacturer's protocol, and cultured in F-12K medium supplemented with 10% FBS (ATCC, Manassas, VA, USA) and 1% penicillin-streptomycin solution (ATCC, Manassas, VA, USA) ( ATCC, Manassas, VA, USA). Cells were grown and maintained at a concentration of 2x10 3 and 1x10 4 live cells / cm 2 between, and at 37°C and 5% CO 2 incubate. Determine the cell number using a hemocytometer.
[0134] Guide RNA design and construction
[0135] Input the NRF2 gene codi...
Embodiment 2
[0150] Example 2: Increased chemosensitivity in the NRF2 knockout A549 cell line
[0151] method
[0152] MTS cell proliferation assay
[0153] Cell viability was assessed using the CellTiter 96 aqueous non-radioactive cell proliferation assay (Promega, Madison, WI). with 2x10 3 cells / well were inoculated with the A549 cell line and incubated for 24 hours. Then, the cell culture medium was aspirated, the cells were washed with PBS, and then exposed to MTS reagent for 3 hours. After 3 hours of MTS bioreduction by proliferating cells, the absorbance of the formazan product was measured on an Infinite 2000 PRO microplate reader (Tecan, Mannadorf, Switzerland) using a 450 nm filter. Cell viability after drug exposure was assessed using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay. with 2x10 3 cells / well were inoculated with the A549 cell line and incubated for 24 hours. Cells were then treated with cisplatin, carboplatin, or a combination of cisplatin a...
Embodiment 3
[0156] Example 3: Genetically Reengineered A549 Cells Show Slower Growth Rate and Increased Chemosensitivity in a Lung Cancer Xenograft Mouse Model
[0157] method
[0158] Animal experiments and statistical analysis
[0159] The animal experiments presented herein were performed at Washington Biotech Inc., Simpsonville Maryland, under the animal use and care protocols approved by the Animal Care and Use Committee of Washington Biotechnology Inc. (SOP 505, SOP 520, SOP 522, SOP 1610, SOP 1650) AAALAC accredited Animal Welfare Assurance Number A4192-01). Human xenograft models were established using previously reported methods. Kellar et al., Biomed Res. Int. 2015, 1–17 (2015). Female athymic nude mice (Envigo, 5-6 weeks old) were used in this study. Approximately 5x 10 cells suspended in PBS containing 20% Matrigel 6 Cells (wild-type A549 or homozygous knockout (clonal expansion 2-11)) were injected subcutaneously into the right flank of each mouse. Once palpable, tumo...
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