LEPR Knockout HAP1 Cell pool

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LM01010116678

Product ID： LM01010116678

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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: LEPR Knockout HAP1 Cell pool
- Commodity ID: LM01010116678
- Gene Symbol: LEPR DB OBR
- Ensembl ID: ENSG00000116678
- Uniprot ID: P48357
- 宿主细胞 / 类型: HAP1/Chronic myeloid leukemia
- NCBI Gene ID: 3953
- 规格: 1×10^6 cells/ cryovials
- 生长培养基: IMDM + 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, passage 1/3 to 1/5
- 倍增时间: ~16 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）: >70%
- 蛋白质组验证结果: Completed protein level verification
- 抗体货号: Adding
- 目标基因介绍: [Isoform A]: May transport LEP across the blood-brain barrier. Binds LEP and mediates LEP endocytosis. Does not induce phosphorylation of and activate STAT3.||[Isoform E]: Antagonizes Isoform A and isoform B-mediated LEP binding and endocytosis.||Receptor for hormone LEP/leptin (Probable) (PubMed:22405007). On ligand binding, mediates LEP central and peripheral effects through the activation of different signaling pathways such as JAK2/STAT3 and MAPK cascade/FOS. In the hypothalamus, LEP acts as an appetite-regulating factor that induces a decrease in food intake and an increase in energy consumption by inducing anorexinogenic factors and suppressing orexigenic neuropeptides, also regulates bone mass and secretion of hypothalamo-pituitary-adrenal hormones (By similarity) (PubMed:9537324). In the periphery, increases basal metabolism, influences reproductive function, regulates pancreatic beta-cell function and insulin secretion, is pro-angiogenic and affects innate and adaptive immunity (PubMed:25060689, PubMed:12504075, PubMed:8805376). Control of energy homeostasis and melanocortin production (stimulation of POMC and full repression of AgRP transcription) is mediated by STAT3 signaling, whereas distinct signals regulate NPY and the control of fertility, growth and glucose homeostasis. Involved in the regulation of counter-regulatory response to hypoglycemia by inhibiting neurons of the parabrachial nucleus. Has a specific effect on T lymphocyte responses, differentially regulating the proliferation of naive and memory T-cells. Leptin increases Th1 and suppresses Th2 cytokine production (By similarity).
- 细胞开发路径: Stable KO cell pool generated using CRISPR-RNP method; Sanger sequencing results show KO cell pool knockout efficiency >70%
- 应用: Gene knockout cell pools (KO Cell Pool) with high knockout efficiency are particularly suitable for preliminary functional analysis, the development of complex disease models, precision drug screening, and extensive gene discovery research. KO pools can be directly applied to various types of assays and analyses without the need for cumbersome single-clone selection processes, significantly improving experimental efficiency.
Key words:
- LEPR DB OBR
01. Prewarm the complete medium in a 37℃ water bath.
02. Thaw the cryovial in a 37℃ water bath for 1-2 minutes.
03. Transfer the cryovial to a biological safety cabinet and wipe the surface with 70% ethanol.
04. Unscrew the cap of the cryovial 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 culture flask.
07. Transfer the cells to a 37℃, 5% CO2 incubator for culture.
08. Reference passage ratio: passage 1/3 to 1/5, confluent in 2-3 days.
01. When the cell confluence in the culture bottle reaches 80%-90%, cell passage can be performed.
02. Take out the culture medium, PBS, trypsin (0.25% Trypsin_EDTA Gibco 25200-056), etc., from the 4℃ refrigerator and 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, and place it in a biosafety cabinet.
04. To avoid cell dispersion, rinse the cells with PBS along the upper wall of the culture bottle. After washing the cells, discard the solution. Add 2mL to T25.
05. Add the corresponding volume of trypsin (T75 add 1.5mL, T25 add 0.5mL), and gently shake the bottle to spread the trypsin evenly over the bottom of the cells. The amount can be appropriately increased or decreased according to the actual situation. After about 1-2 minutes, when most cells detach, add the corresponding volume of complete culture medium to terminate digestion, and gently blow with a 5mL pipette until all cells detach.
06. Transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 300g for 5min, and discard the supernatant.
07. Resuspend the cells with 5mL of complete culture medium, adjust the inoculation ratio as needed, and add complete culture medium to the culture bottle, T75 to 13-15mL, T25 to 5mL, and add 1% double antibody.
08. After tightening the bottle cap, gently shake the bottle to mix the cells evenly, and place it in a 37℃, 5% CO2 incubator.
01. Prepare freezing solution and pre-chill it.
02. Ensure the cells to be frozen meet the freezing requirements. Check the following status under a microscope: healthy appearance and morphological characteristics, growth cycle (late logarithmic phase), no signs of contamination or decline.
03. Digest and centrifuge the cells (refer to the subculture procedure for specific steps).
04. Add freezing solution to resuspend the cells at 1 mL per tube. After mixing well, dispense them into cryovials.
05. Place the cells in a programmable freezing container and freeze them in a -80℃ freezer.
06. Subsequently, transfer the cells to a liquid nitrogen tank for long-term storage.
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