STING1 Knockout RLF Cell Line
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Product ID： LM02195042137
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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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- Commodity name: STING1 Knockout RLF Cell Line
- Commodity ID: LM02195042137
- Gene Symbol: Sting1, Sting, Tmem173
- Ensembl ID: ENSRNOG00000042137
- Uniprot ID: F1M391
- 宿主细胞 / 类型: RLF/rat lung fibroblasts
- NCBI Gene ID: 498840
- 规格: 1×10^6 cells/frozen vial
- 生长培养基: DMEM + 10% FBS + 1% P/S
- 筛选标记: N/A
- 生长特性: Adherent cells, fibroblast-like
- 培养条件: Incubate at 37°C in a 5% CO2 atmosphere, and passage at a ratio of 1/2 to 1/3.
- 倍增时间: ~24-48 hours
- 参考换液频率: 2-3 times/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: Key innate immune signaling adapter that promotes the production of type I interferon (IFN-alpha and IFN-beta) in response to the presence of DNA from bacteria and viruses in the cytosol (PubMed:26669264). Innate immune response is triggered by non-CpG double-stranded DNA from viruses and bacteria delivered to the cytoplasm, which induces production of cyclic dinucleotides that bind and activate STING1: STING1 recognizes and binds cyclic di-GMP (c-di-GMP), a second messenger produced by bacteria, cyclic UMP-AMP (2',3'-cUAMP), and cyclic GMP-AMP (cGAMP), a messenger produced by CGAS in response to DNA in the cytosol. Upon binding to c-di-GMP, cUAMP or cGAMP, STING1 oligomerizes and buds from the endoplasmic reticulum into COPII vesicles, which then form the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). It is then phosphorylated by TBK1 on the pLxIS motif, leading to recruitment and subsequent activation of the transcription factor IRF3 to induce expression of type I interferon and exert a potent antiviral state. Also involved in intercellular immune signaling: cross-activated by 2',3'-cGAMP previously generated in virus-infected cells, triggering type I interferon signaling in macrophages and uninfected neighboring cells to propagate and amplify the antiviral immune response. In addition to promote the production of type I interferon, cGAS-STING signaling also activates the NF-kappa-B signaling: mechanistically, STING1 recruits TRAF6, leading to degradation of the NF-kappa-B inhibitor and subsequent translocation of NF-kappa-B into the nucleus. Activation of NF-kappa-B signaling takes place in the endolysosome and is independent of type I interferon response via direct activation by RNA viruses. Independently of type I interferon production, plays a direct role in autophagy. The ERGIC serves as the membrane source for WIPI2 recruitment and LC3 lipidation, leading to formation of autophagosomes that target cytosolic DNA or DNA viruses for degradation by the lysosome. Promotes autophagy by acting as a proton channel that directs proton efflux from the Golgi to facilitate MAP1LC3B/LC3B lipidation. The autophagy- and interferon-inducing activities can be uncoupled and autophagy induction is independent of TBK1 phosphorylation (By similarity). Autophagy is also triggered upon infection by bacteria: following c-di-GMP-binding, which is produced by live Gram-positive bacteria, promotes reticulophagy (By similarity). The proton channel activity also regulates lysosome biogenesis by activating transcription factors TFEB and TFE3, driving the expression of lysosome-related genes: activated STING1 in post-Golgi vesicles induces proton efflux and lipidation of GABARAP, sequestering the FLCN-FNIP complex and blocking mTORC1-dependent inhibition of TFEB and TFE3 (By similarity). May be involved in transduction of apoptotic signals via its association with the major histocompatibility complex class II (MHC-II) (By similarity).
- 细胞开发路径: A stable KO cell line was generated using the CRISPR-RNP method; Sanger sequencing confirmed 100% knockout efficiency in the KO cell line.
- 应用: Gene knockout cell lines (KO Cell Lines) with high knockout efficiency are particularly well suited for preliminary functional analyses, the development of complex disease models, precision drug screening, and broad gene discovery studies.
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01. Pre-warm 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 cryovial to a biosafety cabinet and wipe its surface 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 125g for 5–7 minutes at room temperature, 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: Passage at a ratio of 1/2 to 1/3; cells will confluence in 2–3 days. -
01. Cell passage can be performed when the cell confluence in the culture flask reaches 80%–90% or higher.
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 remove them when their temperature approaches 37°C. After spraying the surfaces of the bottles with 75% ethanol, transfer them to a biosafety cabinet.03. Remove the culture flask to be passaged from the incubator, spray the flask with 75% ethanol, and place it in a biosafety cabinet.
04. To avoid detaching the cells, rinse them with PBS along the inner wall of the culture flask; discard the wash solution, and add 2 mL of PBS to a T25 flask.
05. Add the appropriate volume of trypsin (1.5 mL for T75, 0.5 mL for T25), and gently swirl the flask so the enzyme evenly coats the bottom. Adjust the volume as needed. After about 1–2 minutes, when most cells have detached, add an appropriate volume of complete culture medium to neutralize the trypsin, 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 300g for 5 min, and discard the supernatant.
07. Resuspend the cells in 5 mL of complete medium, adjust the seeding ratio as needed, and replenish the complete medium in the culture flasks—add up to 13–15 mL for T75 flasks and 5 mL for T25 flasks—and supplement with 1% double antibiotic.
08. After capping the bottle and tightening the cap, gently shake the bottle to mix the cells evenly, then place it in a 37°C, 5% CO2 incubator. -
01. Prepare the cryopreservation solution and pre-cool it in advance.
02. Ensure that the cells to be cryopreserved meet the cryopreservation requirements; examine the following conditions under a microscope: healthy appearance and morphological characteristics, appropriate growth phase (late logarithmic phase), and absence of contamination or signs of decline.
03. Digest and centrifuge the cells (refer to the subculture protocol for specific steps).
04. Resuspend the cells in cryopreservation solution at 1 mL per tube, mix thoroughly by pipetting, and aliquot into cryovials.
05. Place the cells in a programmed cooling box and freeze them in a -80°C freezer.
06. The cells are subsequently transferred to a liquid nitrogen tank for long-term storage. - In antibody validation
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Classification： Gene Knockout Cell Line（Cell Line）
Cell Line Information
Gene Symbol
Sting1, Sting, Tmem173
NCBI Gene ID
498840
Ensembl ID
ENSRNOG00000042137
Uniprot ID
F1M391
Screening marker
N/A
Host cell/type
RLF/rat lung fibroblasts
Specifications
1×10^6 cells/frozen vial
Growth Medium
DMEM + 10% FBS + 1% P/S
growth characteristics
Adherent cells, fibroblast-like
culture condition
Incubate at 37°C in a 5% CO2 atmosphere, and passage at a ratio of 1/2 to 1/3.
doubling time
~24-48 hours
Reference fluid change frequency
2-3 times/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
Key innate immune signaling adapter that promotes the production of type I interferon (IFN-alpha and IFN-beta) in response to the presence of DNA from bacteria and viruses in the cytosol (PubMed:26669264). Innate immune response is triggered by non-CpG double-stranded DNA from viruses and bacteria delivered to the cytoplasm, which induces production of cyclic dinucleotides that bind and activate STING1: STING1 recognizes and binds cyclic di-GMP (c-di-GMP), a second messenger produced by bacteria, cyclic UMP-AMP (2',3'-cUAMP), and cyclic GMP-AMP (cGAMP), a messenger produced by CGAS in response to DNA in the cytosol. Upon binding to c-di-GMP, cUAMP or cGAMP, STING1 oligomerizes and buds from the endoplasmic reticulum into COPII vesicles, which then form the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). It is then phosphorylated by TBK1 on the pLxIS motif, leading to recruitment and subsequent activation of the transcription factor IRF3 to induce expression of type I interferon and exert a potent antiviral state. Also involved in intercellular immune signaling: cross-activated by 2',3'-cGAMP previously generated in virus-infected cells, triggering type I interferon signaling in macrophages and uninfected neighboring cells to propagate and amplify the antiviral immune response. In addition to promote the production of type I interferon, cGAS-STING signaling also activates the NF-kappa-B signaling: mechanistically, STING1 recruits TRAF6, leading to degradation of the NF-kappa-B inhibitor and subsequent translocation of NF-kappa-B into the nucleus. Activation of NF-kappa-B signaling takes place in the endolysosome and is independent of type I interferon response via direct activation by RNA viruses. Independently of type I interferon production, plays a direct role in autophagy. The ERGIC serves as the membrane source for WIPI2 recruitment and LC3 lipidation, leading to formation of autophagosomes that target cytosolic DNA or DNA viruses for degradation by the lysosome. Promotes autophagy by acting as a proton channel that directs proton efflux from the Golgi to facilitate MAP1LC3B/LC3B lipidation. The autophagy- and interferon-inducing activities can be uncoupled and autophagy induction is independent of TBK1 phosphorylation (By similarity). Autophagy is also triggered upon infection by bacteria: following c-di-GMP-binding, which is produced by live Gram-positive bacteria, promotes reticulophagy (By similarity). The proton channel activity also regulates lysosome biogenesis by activating transcription factors TFEB and TFE3, driving the expression of lysosome-related genes: activated STING1 in post-Golgi vesicles induces proton efflux and lipidation of GABARAP, sequestering the FLCN-FNIP complex and blocking mTORC1-dependent inhibition of TFEB and TFE3 (By similarity). May be involved in transduction of apoptotic signals via its association with the major histocompatibility complex class II (MHC-II) (By similarity).
Cell development path
A stable KO cell line was generated using the CRISPR-RNP method; Sanger sequencing confirmed 100% knockout efficiency in the KO cell line.
Application
Gene knockout cell lines (KO Cell Lines) with high knockout efficiency are particularly well suited for preliminary functional analyses, the development of complex disease models, precision drug screening, and broad gene discovery studies.
Cell Culture Instructions
Cell Resuscitation
01. Pre-warm 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 cryovial to a biosafety cabinet and wipe its surface 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 125g for 5–7 minutes at room temperature, 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: Passage at a ratio of 1/2 to 1/3; cells will confluence in 2–3 days.
cell passage
01. Cell passage can be performed when the cell confluence in the culture flask reaches 80%–90% or higher.
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 remove them when their temperature approaches 37°C. After spraying the surfaces of the bottles with 75% ethanol, transfer them to a biosafety cabinet.
03. Remove the culture flask to be passaged from the incubator, spray the flask with 75% ethanol, and place it in a biosafety cabinet.
04. To avoid detaching the cells, rinse them with PBS along the inner wall of the culture flask; discard the wash solution, and add 2 mL of PBS to a T25 flask.
05. Add the appropriate volume of trypsin (1.5 mL for T75, 0.5 mL for T25), and gently swirl the flask so the enzyme evenly coats the bottom. Adjust the volume as needed. After about 1–2 minutes, when most cells have detached, add an appropriate volume of complete culture medium to neutralize the trypsin, 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 300g for 5 min, and discard the supernatant.
07. Resuspend the cells in 5 mL of complete medium, adjust the seeding ratio as needed, and replenish the complete medium in the culture flasks—add up to 13–15 mL for T75 flasks and 5 mL for T25 flasks—and supplement with 1% double antibiotic.
08. After capping the bottle and tightening the cap, gently shake the bottle to mix the cells evenly, then place it in a 37°C, 5% CO2 incubator.
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; examine the following conditions under a microscope: healthy appearance and morphological characteristics, appropriate growth phase (late logarithmic phase), and absence of contamination or signs of decline.
03. Digest and centrifuge the cells (refer to the subculture protocol for specific steps).
04. Resuspend the cells in cryopreservation solution at 1 mL per tube, mix thoroughly by pipetting, and aliquot into cryovials.
05. Place the cells in a programmed cooling box and freeze them in a -80°C freezer.
06. The cells are subsequently transferred to a liquid nitrogen tank for long-term storage.
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