Posted by on July 5, 2020 7:14 am
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this is the knockout KYSE-150 cell line successfully cheats-source well Bio

KYSE-150 cells are poorly differentiated esophageal adenocarcinoma cells, from a receiving radiation therapy of the 49-year-old Japanese female patient with the neck of the esophagus is separated out. When the researchers found that the cell line, the patient’s cancer tissue has invaded the adjacent tissue. KYSE-150 cell line having adhesion to a monolayer of epithelial cells. As reported, the cell carrying the oncogene c-erb-B 8-fold and cyclin D1(4 times), and cells in Nude mice formed tumors, and therefore the KYSE-150 cell line often used to refer to a gene-edit the cancer research, for example, the CRISPR gene knockout/knock-in and point mutations.

applications:

1. In KYSE-150 cells in the knockout of EZH2 change of the PSMA3-AS1-induced esophageal cancer cell proliferation and migration

in esophageal cancer patients, PSMA3-AS1 expression is upregulated and associated with tumor size and metastasis were positively correlated. In order to further explore the PSMA3-AS1 in esophageal cancer cells in the biological effects, researchers through a slow virus infection in KYSE150 and KYSE450 cell lines(PSMA3-AS1 expression is low in the establishment of a stable CRISPR PSMA3-AS1 overexpression cell lines, and verified its upregulation by RT-qPCR detection PSMA3-AS1.

EZH2 CRISPR / Cas9 gene editing has been in KYSE150 and KYSE450 cells successfully carried out, and by the EZH2 protein expression is significantly reduced confirmed. CCK-8 and colony formation analysis showed that the knockout CRISPR EZH2 after PSMA3-AS1 overexpression does not affect the esophageal cancer cell proliferation figure 6B and 6C River. According to the wound healing and transwell migration analysis, and the negative control cells as compared to the ESCC EZH2 knockout KYSE-150 cells in wound healing and cell migration did not increase.

clinical pathological features showed that the PSMA3-AS1 expression was increased with distant metastases, larger tumor size and ESCC patients with poor prognosis showed a positive correlation.

2. CRISPR overexpressed KYSE-150 cells increased ESCC in chemical resistance

based on the full genome clusters of regularly spaced short palindromic repeats/based on CRISPR lentiviral library(CRISPR / Cas is used for genome-scale gain of function or loss of function screening of the powerful tool. The system has been demonstrated in vitro identification of drug resistant genes is very effective. Shalem, the Kurata and Joung the use of CRISPR gene knockout library screening a melanoma and AML resistance of the basic gene. Some researchers tried to the CRISPR library screening strategies and RNA sequencing technologies combined with in KYSE-150 cells, to explore the ESCC of the key genes and chemical resistance of the underlying mechanisms.

in order to increase the identification involved in the PTX resistance of the necessary genes of the opportunity to conduct a comprehensive analysis to the genome-scale CRISPR screening of the EN gene and KYSE-150 cell line differences in gene expression of DE genes combined together.

researchers assessed CDKN1A, AND ELAVL2 and TSPAN4 enhanced ESCC cells KYSE-150 chemical resistance potential. The results showed that CDKN1A, AND ELAVL2 or TSPAN4 over-expression can significantly increase the KYSE-180 KYSE-150 cells to PTX resistance. In addition, over-expression of CDKN1A, the ELAVL2 or TSPAN4 may also contribute to the KYSE-150 cells to DDP resistance.

3. CRISPR / Cas9-mediated KYSE-510 cells DEPTOR knockdown significantly promoted cell proliferation, migration and invasion

the researchers found that DEPTOR expression negative regulation of ESCC cell lines of tumorigenic activity. In addition, ectopic DEPTOR expression caused by KYSE150 cells proliferation, migration and invasion was significantly suppressed, and KYSE150 cells DEPTOR expression level to a minimum. At the same time, CRISPR / Cas9-mediated KYSE-510 cells DEPTOR knockdown significantly promoted cell proliferation, migration and invasion. In addition, in vivo tests further show that with untreatedThe KYSE150 cells compared to ectopic DEPTOR expression in the xenograft tumor growth was significantly inhibited, whereas in the DEPTOR gene knockout of KYSE-510 cells, the tumor growth was significantly enhanced.

in this study, scientists generated the stable cell lines, these cell lines either over-expression of DEPTOR, either by genetic elimination of endogenous DEPTOR expression. Due to the KYSE-150 in the three cell lines Express low endogenous DEPTOR, so they are in KYSE-150 cells, pcDNA3. 1-DEPTOR in a stable manner over-expression of DEPTOR in. For the same considerations, by the CRISPR/Cas9 system processing Express the highest level of DEPTOR of KYSE-510 cells to knockout DEPTOR(CRISPR-DEPTOR in. Cell lines generated with the KYSE-150 parental cells and empty vector transfected cells compared with pcDNA3. 1-DEPTOR exhibit reduced cell proliferation rate, and CRISPR-DEPTOR cell proliferation is significantly faster than the control KYSE-510 cells. In addition, pcDNA3. 1-DEPTOR cells also showed reduced migration.

gene knock in addition to the cutting efficiencynot only can let a person have to generate human glycosylation profile and to establish the regulation of the recording capacity, and has a variety of advantages, is a particularly attractive recombinant protein expression system. First of all, it is in the glutamic acid on the carboxyl group in tyrosine on the sulfation. Second, the operation is simple, by transient gene expression can rapidly produce recombinant proteins. Third, can be used for stable recombinant protein production. Some researchers use gene knock-cutting efficiency system to produce gene edited cell lines, for GLUL genomic loci for targeted sequencing, to produce produce produce EPO stably transfected cell lines, and found that recombinant erythropoietin in the human body within the stable expression mechanisms.

source wells bio according to research needs, the combination of target genes for gene stably transfected knockout programme design.

scenario 1: a small fragment of the gene knockout program, the gRNA located in exon 2 at both ends of the intron, the knock except for the exon encoding the nucleotide number of non-3 ratio, the knock can be caused by shift code.

Scenario 2: frameshift gene knockout program, the gRNA located in the exon, the missing base number is for the non-3 multiples, knockout can occur frameshift mutation.

Scenario 3: a large fragment of the gene knockout program, the entire gene coding sequence knockout, to achieve a large fragment of the knockout effect.

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 rid malignant phenotypes of esophageal cancer by modulating the miR-101/EZH2 axis as a ceRNA. Aging (Albany NY). 2020;12(2):18431856.

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

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Published at Sun, 05 Jul 2020 11:13:18 +0000