ISG15 Knockout raw264.7 Cell Line
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Product ID： LM02042035692
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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
-
- Commodity name: ISG15 Knockout raw264.7 Cell Line
- Commodity ID: LM02042035692
- Gene Symbol: Isg15, G1p2, Ucrp
- Ensembl ID: ENSMUSG00000035692
- Uniprot ID: Q64339
- 宿主细胞 / 类型: RAW 264.7/mouse monocyte-macrophage leukemia cells
- NCBI Gene ID: 100038882
- 规格: 1×10^6 cells per cryovial
- 生长培养基: DMEM + 10% FBS + 1% P/S
- 筛选标记: N/A
- 生长特性: Adherent cells, irregularly shaped
- 培养条件: Incubator at 37°C with 5% CO2; passage at a ratio of 1/6 to 1/8.
- 倍增时间: ~12-30 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: Protein-level validation has been completed.
- 抗体货号: Adding...
- 目标基因介绍: Ubiquitin-like protein which plays a key role in the innate immune response to viral infection either via its conjugation to a target protein (ISGylation) or via its action as a free or unconjugated protein. ISGylation involves a cascade of enzymatic reactions involving E1, E2, and E3 enzymes which catalyze the conjugation of ISG15 to a lysine residue in the target protein. Its target proteins include SERPINA3G/SPI2A, JAK1, MAPK3/ERK1, PLCG1, TRIM25, STAT5A, MAPK1/ERK2 and globin. Isgylation of the viral sensor IFIH1/MDA5 promotes IFIH1/MDA5 oligomerization and triggers activation of innate immunity against a range of viruses, including coronaviruses, flaviviruses and picornaviruses. Can also isgylate: RIGI which inhibits its function in antiviral signaling response, IRF3 which inhibits its ubiquitination and degradation as well as EIF4E2 which enhances its cap structure-binding activity and translation-inhibition activity. Exhibits antiviral activity towards both DNA and RNA viruses, including influenza A and B virus, sindbis virus (SV) and herpes simplex type-1 (HHV-1). Plays a significant role in the control of neonatal Chikungunya virus (CHIKV) infection by acting as a putative immunomodulator of pro-inflammatory cytokines. Protects mice against the consequences of Chikungunya virus infection by down-regulating the pathogenic cytokine response, often denoted as the cytokine storm. Plays a role in erythroid differentiation. The secreted form of ISG15 can: induce natural killer cell proliferation, act as a chemotactic factor for neutrophils and act as a IFN-gamma-inducing cytokine playing an essential role in antimycobacterial immunity. The secreted form acts through the integrin ITGAL/ITGB2 receptor to initiate activation of SRC family tyrosine kinases including LYN, HCK and FGR which leads to secretion of IFNG and IL10; the interaction is mediated by ITGAL (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:- Isg15
- G1p2
- Ucrp
-
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: 1/6 to 1/8; cells reach confluence in 2–3 days. -
01. When the cells in the culture flask reach a confluence of 80%–90% or higher, cell passage can be performed.
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 conditions. 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 solution.
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. - Western blot (WB) analysis was performed on lysates from both wild-type and knockout cells using a specific monoclonal antibody; the target protein band was detectable in WT cells but absent or markedly reduced in KO cells.
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Classification： Gene-knockout cell line (protein levels validated)
Cell Line Information
Gene Symbol
Isg15, G1p2, Ucrp
NCBI Gene ID
100038882
Ensembl ID
ENSMUSG00000035692
Uniprot ID
Q64339
Screening marker
N/A
Host cell/type
RAW 264.7/mouse monocyte-macrophage leukemia cells
Specifications
1×10^6 cells per cryovial
Growth Medium
DMEM + 10% FBS + 1% P/S
growth characteristics
Adherent cells, irregularly shaped
culture condition
Incubator at 37°C with 5% CO2; passage at a ratio of 1/6 to 1/8.
doubling time
~12-30 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
Protein-level validation has been completed.
Antibody number
Adding...
Antibody validation results
Cell Line Description
Introduction of target gene
Ubiquitin-like protein which plays a key role in the innate immune response to viral infection either via its conjugation to a target protein (ISGylation) or via its action as a free or unconjugated protein. ISGylation involves a cascade of enzymatic reactions involving E1, E2, and E3 enzymes which catalyze the conjugation of ISG15 to a lysine residue in the target protein. Its target proteins include SERPINA3G/SPI2A, JAK1, MAPK3/ERK1, PLCG1, TRIM25, STAT5A, MAPK1/ERK2 and globin. Isgylation of the viral sensor IFIH1/MDA5 promotes IFIH1/MDA5 oligomerization and triggers activation of innate immunity against a range of viruses, including coronaviruses, flaviviruses and picornaviruses. Can also isgylate: RIGI which inhibits its function in antiviral signaling response, IRF3 which inhibits its ubiquitination and degradation as well as EIF4E2 which enhances its cap structure-binding activity and translation-inhibition activity. Exhibits antiviral activity towards both DNA and RNA viruses, including influenza A and B virus, sindbis virus (SV) and herpes simplex type-1 (HHV-1). Plays a significant role in the control of neonatal Chikungunya virus (CHIKV) infection by acting as a putative immunomodulator of pro-inflammatory cytokines. Protects mice against the consequences of Chikungunya virus infection by down-regulating the pathogenic cytokine response, often denoted as the cytokine storm. Plays a role in erythroid differentiation. The secreted form of ISG15 can: induce natural killer cell proliferation, act as a chemotactic factor for neutrophils and act as a IFN-gamma-inducing cytokine playing an essential role in antimycobacterial immunity. The secreted form acts through the integrin ITGAL/ITGB2 receptor to initiate activation of SRC family tyrosine kinases including LYN, HCK and FGR which leads to secretion of IFNG and IL10; the interaction is mediated by ITGAL (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: 1/6 to 1/8; cells reach confluence in 2–3 days.
cell passage
01. When the cells in the culture flask reach a confluence of 80%–90% or higher, cell passage can be performed.
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 conditions. 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 solution.
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.
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