PITRM1 Knockout HCT116 Cell Pool
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Product ID： LM01400107959
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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
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- Brand: ELEM粒曼
- Commodity name: PITRM1 Knockout HCT116 Cell Pool
- Commodity ID: LM01400107959
- Gene Symbol: PITRM1 KIAA1104 MP1 PREP
- NCBI Gene ID: 10531
- Ensembl ID: ENSG00000107959
- Uniprot ID: Q5JRX3
- 宿主细胞 / 类型: HCT116/Human Colon Cancer Cells
- 规格: 1 × 10 ^ 6 cells/frozen storage tube
- 生长培养基: McCoy’s 5A＋10% FBS＋1% P/S
- 筛选标记: N/A
- 生长特性: Adherent cells, epithelial-like
- 培养条件: 37 ℃,5% CO2 incubator, 1/3 to 1/4 passage
- 倍增时间: ~25-48 hours
- 参考换液频率: Change fluid in 2-3 days
- 支原体检测结果: Negative
- 敲除效率（Sanger测序）: >70%
- 蛋白质组验证结果: N/A
- 抗体货号: N/A
- 目标基因介绍: Metalloendopeptidase of the mitochondrial matrix that functions in peptide cleavage and degradation rather than in protein processing (PubMed:10360838, PubMed:16849325, PubMed:19196155, PubMed:24931469). Has an ATP-independent activity (PubMed:16849325). Specifically cleaves peptides in the range of 5 to 65 residues (PubMed:19196155). Shows a preference for cleavage after small polar residues and before basic residues, but without any positional preference (PubMed:10360838, PubMed:19196155, PubMed:24931469). Degrades the transit peptides of mitochondrial proteins after their cleavage (PubMed:19196155). Also degrades other unstructured peptides (PubMed:19196155). It is also able to degrade amyloid-beta protein 40, one of the peptides produced by APP processing, when it accumulates in mitochondrion (PubMed:16849325, PubMed:24931469). It is a highly efficient protease, at least toward amyloid-beta protein 40 (PubMed:24931469). Cleaves that peptide at a specific position and is probably not processive, releasing digested peptides intermediates that can be further cleaved subsequently (PubMed:24931469).
- 细胞开发路径: Stable KO Cell Pool was generated by CRISPR-RNP method; Sanger sequencing results showed that KO Cell Pool knockout efficiency was> 70%
- 应用: The KO Cell Pool with high knockout efficiency is especially suitable for preliminary functional analysis, development of complex disease models, precision drug screening and extensive gene discovery research. KO pool can be directly applied to various types of assays and analyses without the cumbersome process of monoclonal selection, which greatly improves the efficiency of experiments.
Key words:- PITRM1 KIAA1104 MP1 PREP
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01 in 37 DEG C water bath preheat complete medium.
02. Thaw the tubes in a 37°C water bath for 1-2 minutes.
03. Transfer the frozen tubes to a biosafety cabinet and wipe the surface with 70% ethanol.
04. Unscrew the tube cap and gently transfer the cell suspension to a sterile centrifuge tube containing 9mL of complete medium.
05. Centrifuge at 125g for 5-7 minutes at room temperature and discard the supernatant.
06. Resuspend the cell pellet with 5mL of complete medium and transfer the cell suspension to a T25 flask.
07. Transfer the cells to 37°C, 5% CO2 incubator culture.
08. Reference passage ratio: 1/3 to 1/4 passage, full in 2-3 days. -
01. When the confluence of cells in the culture flask is above 80%-90%, the cells can be passaged.
02. Take out the culture medium, PBS, pancreatin (0.25% Trypsin_EDTA Gibco 25200-056), etc. from the refrigerator at 4 ℃, place them in a 37 ℃ water bath when the temperature is close to 37 ℃, take them out, spray 75% alcohol on the surface of the bottle, and place them in a biosafety cabinet.03. Take out the culture bottle to be passaged from the incubator, spray 75% alcohol on the bottle body and place it in the biosafety cabinet.
04. In order to avoid scattering the cells, wash the cells along the upper wall of the culture flask with PBS, wash the cells and discard them. Add 2mL to T25.
05. Add the corresponding volume of pancreatin (T75 plus 1.5mL, T25 plus 0.5mL), and gently shake the bottle body to make the pancreatin spread over the bottom of the cells. The dosage can be appropriately increased or decreased according to the actual situation. When most of the cells were shed after about 1-2min, the digestion was terminated by adding the corresponding volume of complete medium and gently beating with a 5mL pipette until all the cells were shed.
06. Transfer the cell suspension to a 15mL centrifuge tube, centrifuge the suspension 300g for 5min, and discard the supernatant.
07. Transfer 5mL of complete medium to resuspend the cells, adjust the inoculation ratio as required, and supplement the complete medium in the culture flask, add T75 to 13-15mL,T25 to 5mL, and add 1% double antibody.
08. Cover and tighten the bottle cap and gently shake the bottle body to mix the cells evenly and place them in a 37 ℃,5% CO2 incubator. -
01. Prepare the frozen storage liquid and pre-cool it in advance.
02. Ensure that the cells to be frozen meet the cryopreservation requirements and examine the following conditions with a microscope: healthy appearance and morphological characteristics, growth cycle (late logarithmic), no contamination or signs of decline.
03. Digestion and centrifugation of cells (refer to the subculture process for specific steps)
04. Add the frozen storage solution to resuspend the cells according to the amount of 1mL per tube, blow evenly and pack them into the frozen storage tube.
05. Place the cells in a programmed cooling box and freeze in a freezer at -80°C.
06. The cells were subsequently transferred to a liquid nitrogen tank for long-term storage. - Antibody validation in process
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Classification： Gene Knockout Cell Pool（KO Pool）
Cell Line Information
Gene Symbol
PITRM1 KIAA1104 MP1 PREP
NCBI Gene ID
10531
Ensembl ID
ENSG00000107959
Uniprot ID
Q5JRX3
Screening marker
N/A
Host cell/type
HCT116/Human Colon Cancer Cells
Specifications
1 × 10 ^ 6 cells/frozen storage tube
Growth Medium
McCoy’s 5A＋10% FBS＋1% P/S
growth characteristics
Adherent cells, epithelial-like
culture condition
37 ℃,5% CO2 incubator, 1/3 to 1/4 passage
doubling time
~25-48 hours
Reference fluid change frequency
Change fluid in 2-3 days
Mycoplasma test results
Negative
Knock-out validation
Knockout efficiency (Sanger sequencing)
>70%
Proteome Validation Results
N/A
Antibody number
N/A
Antibody validation results
Cell Line Description
Introduction of target gene
Metalloendopeptidase of the mitochondrial matrix that functions in peptide cleavage and degradation rather than in protein processing (PubMed:10360838, PubMed:16849325, PubMed:19196155, PubMed:24931469). Has an ATP-independent activity (PubMed:16849325). Specifically cleaves peptides in the range of 5 to 65 residues (PubMed:19196155). Shows a preference for cleavage after small polar residues and before basic residues, but without any positional preference (PubMed:10360838, PubMed:19196155, PubMed:24931469). Degrades the transit peptides of mitochondrial proteins after their cleavage (PubMed:19196155). Also degrades other unstructured peptides (PubMed:19196155). It is also able to degrade amyloid-beta protein 40, one of the peptides produced by APP processing, when it accumulates in mitochondrion (PubMed:16849325, PubMed:24931469). It is a highly efficient protease, at least toward amyloid-beta protein 40 (PubMed:24931469). Cleaves that peptide at a specific position and is probably not processive, releasing digested peptides intermediates that can be further cleaved subsequently (PubMed:24931469).
Cell development path
Stable KO Cell Pool was generated by CRISPR-RNP method; Sanger sequencing results showed that KO Cell Pool knockout efficiency was> 70%
Application
The KO Cell Pool with high knockout efficiency is especially suitable for preliminary functional analysis, development of complex disease models, precision drug screening and extensive gene discovery research. KO pool can be directly applied to various types of assays and analyses without the cumbersome process of monoclonal selection, which greatly improves the efficiency of experiments.
Cell Culture Instructions
Cell Resuscitation
01 in 37 DEG C water bath preheat complete medium.
02. Thaw the tubes in a 37°C water bath for 1-2 minutes.
03. Transfer the frozen tubes to a biosafety cabinet and wipe the surface with 70% ethanol.
04. Unscrew the tube cap and gently transfer the cell suspension to a sterile centrifuge tube containing 9mL of complete medium.
05. Centrifuge at 125g for 5-7 minutes at room temperature and discard the supernatant.
06. Resuspend the cell pellet with 5mL of complete medium and transfer the cell suspension to a T25 flask.
07. Transfer the cells to 37°C, 5% CO2 incubator culture.
08. Reference passage ratio: 1/3 to 1/4 passage, full in 2-3 days.
cell passage
01. When the confluence of cells in the culture flask is above 80%-90%, the cells can be passaged.
02. Take out the culture medium, PBS, pancreatin (0.25% Trypsin_EDTA Gibco 25200-056), etc. from the refrigerator at 4 ℃, place them in a 37 ℃ water bath when the temperature is close to 37 ℃, take them out, spray 75% alcohol on the surface of the bottle, and place them in a biosafety cabinet.
03. Take out the culture bottle to be passaged from the incubator, spray 75% alcohol on the bottle body and place it in the biosafety cabinet.
04. In order to avoid scattering the cells, wash the cells along the upper wall of the culture flask with PBS, wash the cells and discard them. Add 2mL to T25.
05. Add the corresponding volume of pancreatin (T75 plus 1.5mL, T25 plus 0.5mL), and gently shake the bottle body to make the pancreatin spread over the bottom of the cells. The dosage can be appropriately increased or decreased according to the actual situation. When most of the cells were shed after about 1-2min, the digestion was terminated by adding the corresponding volume of complete medium and gently beating with a 5mL pipette until all the cells were shed.
06. Transfer the cell suspension to a 15mL centrifuge tube, centrifuge the suspension 300g for 5min, and discard the supernatant.
07. Transfer 5mL of complete medium to resuspend the cells, adjust the inoculation ratio as required, and supplement the complete medium in the culture flask, add T75 to 13-15mL,T25 to 5mL, and add 1% double antibody.
08. Cover and tighten the bottle cap and gently shake the bottle body to mix the cells evenly and place them in a 37 ℃,5% CO2 incubator.
cell cryopreservation
01. Prepare the frozen storage liquid and pre-cool it in advance.
02. Ensure that the cells to be frozen meet the cryopreservation requirements and examine the following conditions with a microscope: healthy appearance and morphological characteristics, growth cycle (late logarithmic), no contamination or signs of decline.
03. Digestion and centrifugation of cells (refer to the subculture process for specific steps)
04. Add the frozen storage solution to resuspend the cells according to the amount of 1mL per tube, blow evenly and pack them into the frozen storage tube.
05. Place the cells in a programmed cooling box and freeze in a freezer at -80°C.
06. The cells were subsequently transferred to a liquid nitrogen tank for long-term storage.
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