U251 knockout cell line | Exclusive CRISPR-U

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.

CRISPR/Cas9 knockout U251 cell line strategies

Workflow of knockout U251 cell line

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.