ALKBH5 Knockout UMUC-3 Cell Line
Product classification
Welcome to consult Elem's high-throughput CRISPR service.
Contact us to orderALKBH5 Knockout UMUC-3 Cell Line
Product ID： LM02095091542
Market price
Stock surplus
Antibody custom service consulting
隐藏域元素占位
- 产品描述
- 细胞复苏
- 细胞传代
- 细胞冻存
- 抗体验证结果
-
- Brand: ELEM粒曼
- Commodity name: ALKBH5 Knockout UMUC-3 Cell Line
- Commodity ID: LM02095091542
- Gene Symbol: ALKBH5 ABH5 OFOXD1
- Ensembl ID: ENSG00000091542
- Uniprot ID: Q6P6C2
- 宿主细胞 / 类型: UMUC-3/Human Bladder Transitional Cell Carcinoma
- NCBI Gene ID: 54890
- 规格: 1×10^6 cells/frozen vial
- 生长培养基: RPMI-1640 + 10% FBS + 1% P/S
- 筛选标记: N/A
- 生长特性: Adherent cells, epithelial-like
- 培养条件: Incubator at 37°C with 5% CO2; passage at 1/2 to 1/4 confluence.
- 倍增时间: ~24-36 hours
- 参考换液频率: 2–3 times per week
- 支原体检测结果: Negative
- 敲除效率（Sanger测序）: 100%
- 蛋白质组验证结果: N/A
- 抗体货号: Adding...
- 目标基因介绍: Dioxygenase that demethylates RNA by oxidative demethylation: specifically demethylates N(6)-methyladenosine (m6A) RNA, the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes (PubMed:23177736, PubMed:24489119, PubMed:24616105, PubMed:24778178). Can also demethylate N(6)-methyladenosine in single-stranded DNA (in vitro) (PubMed:24616105). Requires molecular oxygen, alpha-ketoglutarate and iron (PubMed:21264265, PubMed:23177736, PubMed:24489119, PubMed:24616105, PubMed:24778178). Demethylation of m6A mRNA affects mRNA processing and export (PubMed:23177736). Required for the late meiotic and haploid phases of spermatogenesis by mediating m6A demethylation in spermatocytes and round spermatids: m6A demethylation of target transcripts is required for correct splicing and the production of longer 3'-UTR mRNAs in male germ cells (By similarity).
- 细胞开发路径: A stable KO cell line was generated using the CRISPR‑RNP approach; Sanger sequencing confirmed 100% knockout efficiency in the KO cell line.
- 应用: A gene-knockout cell line with high knockout efficiency, particularly well suited for preliminary functional analyses, the development of complex disease models, precision drug screening, and broad‑scale gene discovery studies.
Key words:- ALKBH5 ABH5 OFOXD1
-
01. Preheat the complete culture medium in a 37°C water bath.
02. Thaw the cryovial in a 37°C water bath for 1–2 minutes.
03. Transfer the cryovials into a biosafety cabinet and wipe their surfaces with 70% ethanol.
04. Unscrew the cap of the cryovial and gently transfer the cell suspension into a sterile centrifuge tube containing 9 mL of complete culture medium.
05. Centrifuge at room temperature at 125g for 5–7 minutes, then discard the supernatant.
06. Resuspend the cell pellet in 5 mL of complete culture medium, and transfer the cell suspension to a T25 culture flask.
07. Transfer the cells to a 37°C incubator with 5% CO2 for culture.
08. Recommended passage ratio: 1/2 to 1/4; cells reach confluence in 2–3 days. -
01. When the cell confluence in the culture flask reaches 80%–90% or higher, cells can be passaged.
02. Remove the culture medium, PBS, and trypsin (0.25% Trypsin‑EDTA, Gibco 25200‑056) from the 4°C refrigerator, place them in a 37°C water bath, and once their temperature approaches 37°C, take them out. Spray the exterior of the bottles with 75% ethanol, then transfer them to a biosafety cabinet.03. Remove the culture flask to be passaged from the incubator, spray the exterior of the flask with 75% ethanol, and place it inside a biosafety cabinet.
04. To avoid dispersing the cells, gently rinse the cells along the inner wall of the culture flask with PBS; discard the wash solution, and add 2 mL of PBS to the T25 flask.
05. Add the appropriate volume of trypsin (1.5 mL for a T75 flask, 0.5 mL for a T25 flask), and gently swirl the flask to ensure the enzyme evenly covers the cell‑covered bottom. Adjust the volume as needed based on the specific situation. After approximately 1–2 minutes, when most of the cells have detached, add the corresponding volume of complete culture medium to stop the digestion, then use a 5‑mL pipette to gently pipette up and down until all cells are fully detached.
06. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 300 g for 5 minutes, and discard the supernatant.
07. Transfer 5 mL of complete culture medium to resuspend the cells, adjust the seeding ratio as needed, and replenish the culture flask with complete medium—add up to 13–15 mL for a T75 flask and 5 mL for a T25 flask—then supplement with 1% double antibiotic.
08. After capping the bottle and tightening the lid, gently shake the vial to ensure uniform mixing of the cells, then place it in a 37°C incubator with 5% CO2. -
01. Prepare the cryopreservation solution and pre-cool it in advance.
02. Ensure that the cells to be cryopreserved meet the cryopreservation requirements, and verify the following conditions under a microscope: healthy appearance and morphological characteristics, being in the late logarithmic growth phase, and showing no signs of contamination or senescence.
03. Digest and centrifuge the cells (refer to the subculture protocol for specific steps).
04. Resuspend the cells in cryopreservation medium at 1 mL per tube, gently pipette up and down to ensure uniformity, then aliquot into cryovials.
05. Place the cells in a programmed cooling box and freeze them in a −80°C freezer.
06. Subsequently, the cells are transferred to a liquid nitrogen tank for long-term storage. - In antibody validation
Previous Page
Next Page
Classification： Gene Knockout Cell Line（Cell Line）
Cell Line Information
Gene Symbol
ALKBH5 ABH5 OFOXD1
NCBI Gene ID
54890
Ensembl ID
ENSG00000091542
Uniprot ID
Q6P6C2
Screening marker
N/A
Host cell/type
UMUC-3/Human Bladder Transitional Cell Carcinoma
Specifications
1×10^6 cells/frozen vial
Growth Medium
RPMI-1640 + 10% FBS + 1% P/S
growth characteristics
Adherent cells, epithelial-like
culture condition
Incubator at 37°C with 5% CO2; passage at 1/2 to 1/4 confluence.
doubling time
~24-36 hours
Reference fluid change frequency
2–3 times per week
Mycoplasma test results
Negative
Knock-out validation
Knockout efficiency (Sanger sequencing)
100%
Proteome Validation Results
N/A
Antibody number
Adding...
Antibody validation results
Cell Line Description
Introduction of target gene
Dioxygenase that demethylates RNA by oxidative demethylation: specifically demethylates N(6)-methyladenosine (m6A) RNA, the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes (PubMed:23177736, PubMed:24489119, PubMed:24616105, PubMed:24778178). Can also demethylate N(6)-methyladenosine in single-stranded DNA (in vitro) (PubMed:24616105). Requires molecular oxygen, alpha-ketoglutarate and iron (PubMed:21264265, PubMed:23177736, PubMed:24489119, PubMed:24616105, PubMed:24778178). Demethylation of m6A mRNA affects mRNA processing and export (PubMed:23177736). Required for the late meiotic and haploid phases of spermatogenesis by mediating m6A demethylation in spermatocytes and round spermatids: m6A demethylation of target transcripts is required for correct splicing and the production of longer 3'-UTR mRNAs in male germ cells (By similarity).
Cell development path
A stable KO cell line was generated using the CRISPR‑RNP approach; Sanger sequencing confirmed 100% knockout efficiency in the KO cell line.
Application
A gene-knockout cell line with high knockout efficiency, particularly well suited for preliminary functional analyses, the development of complex disease models, precision drug screening, and broad‑scale gene discovery studies.
Cell Culture Instructions
Cell Resuscitation
01. Preheat the complete culture medium in a 37°C water bath.
02. Thaw the cryovial in a 37°C water bath for 1–2 minutes.
03. Transfer the cryovials into a biosafety cabinet and wipe their surfaces with 70% ethanol.
04. Unscrew the cap of the cryovial and gently transfer the cell suspension into a sterile centrifuge tube containing 9 mL of complete culture medium.
05. Centrifuge at room temperature at 125g for 5–7 minutes, then discard the supernatant.
06. Resuspend the cell pellet in 5 mL of complete culture medium, and transfer the cell suspension to a T25 culture flask.
07. Transfer the cells to a 37°C incubator with 5% CO2 for culture.
08. Recommended passage ratio: 1/2 to 1/4; cells reach confluence in 2–3 days.
cell passage
01. When the cell confluence in the culture flask reaches 80%–90% or higher, cells can be passaged.
02. Remove the culture medium, PBS, and trypsin (0.25% Trypsin‑EDTA, Gibco 25200‑056) from the 4°C refrigerator, place them in a 37°C water bath, and once their temperature approaches 37°C, take them out. Spray the exterior of the bottles with 75% ethanol, then transfer them to a biosafety cabinet.
03. Remove the culture flask to be passaged from the incubator, spray the exterior of the flask with 75% ethanol, and place it inside a biosafety cabinet.
04. To avoid dispersing the cells, gently rinse the cells along the inner wall of the culture flask with PBS; discard the wash solution, and add 2 mL of PBS to the T25 flask.
05. Add the appropriate volume of trypsin (1.5 mL for a T75 flask, 0.5 mL for a T25 flask), and gently swirl the flask to ensure the enzyme evenly covers the cell‑covered bottom. Adjust the volume as needed based on the specific situation. After approximately 1–2 minutes, when most of the cells have detached, add the corresponding volume of complete culture medium to stop the digestion, then use a 5‑mL pipette to gently pipette up and down until all cells are fully detached.
06. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 300 g for 5 minutes, and discard the supernatant.
07. Transfer 5 mL of complete culture medium to resuspend the cells, adjust the seeding ratio as needed, and replenish the culture flask with complete medium—add up to 13–15 mL for a T75 flask and 5 mL for a T25 flask—then supplement with 1% double antibiotic.
08. After capping the bottle and tightening the lid, gently shake the vial to ensure uniform mixing of the cells, then place it in a 37°C incubator with 5% CO2.
cell cryopreservation
01. Prepare the cryopreservation solution and pre-cool it in advance.
02. Ensure that the cells to be cryopreserved meet the cryopreservation requirements, and verify the following conditions under a microscope: healthy appearance and morphological characteristics, being in the late logarithmic growth phase, and showing no signs of contamination or senescence.
03. Digest and centrifuge the cells (refer to the subculture protocol for specific steps).
04. Resuspend the cells in cryopreservation medium at 1 mL per tube, gently pipette up and down to ensure uniformity, then aliquot into cryovials.
05. Place the cells in a programmed cooling box and freeze them in a −80°C freezer.
06. Subsequently, the cells are transferred to a liquid nitrogen tank for long-term storage.
Copyright © Elem Biotechnology (Wuhan) Co., Ltd.
LM