DGCR8 KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided.
Images
Key facts
- Cell type
- A549
- Species or organism
- Human
- Tissue
- Lung
- Form
- Liquid
- Knockout validation
- Next Generation Sequencing, Sanger Sequencing, Western blot
Alternative names
C22orf12, D16H22S788E, D16Wis2, DGCR8 microprocessor complex subunit, DGCR8_HUMAN, DGCRK 6, DiGeorge syndrome critical region 8, DiGeorge syndrome critical region gene 8, Gy1, Microprocessor complex subunit DGCR8, pasha
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DGCR8 KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided.
Key facts
- Cell type
- A549
- Species or organism
- Human
- Tissue
- Lung
- Form
- Liquid
- Knockout validation
- Next Generation Sequencing, Sanger Sequencing, Western blot
- Disease
- Carcinoma
- Concentration
Properties
- Gene name
- DGCR8
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Next Generation Sequencing, Sanger Sequencing, Western blot
Quality control
- STR analysis
- CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
- EU: 1 US: 1
- Adherent/suspension
- Adherent
- Gender
- Male
Handling procedures
- Initial handling guidelines
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.- Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not allow the cell density to exceed 7x104 cells/cm2.
- Culture medium
- F-12K + 10% FBS
- Cryopreservation medium
- Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Storage
- Shipped at conditions
- Dry Ice
- Appropriate short-term storage conditions
- -196°C
- Appropriate long-term storage conditions
- -196°C
Notes
Recommended control: Human wild-type A549 cell line (ab288558). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: F-12K + 10% FBS
Initial handling guidelines:
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x103-1x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.
Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 6x104 cells/cm2 is recommended.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
- Do not allow the cell density to exceed 7x104 cells/cm2.
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
The DGCR8 protein also known as Pasha in some organisms serves an important role in the microRNA (miRNA) processing machinery. It has a molecular mass of approximately 90 kDa and shows expression ubiquitously but with higher levels in tissues with active cell division like the brain and lungs. DGCR8 acts as a partner in a complex with Drosha and together they initiate the microRNA maturation process by cleaving primary miRNA transcripts into precursor miRNA in the nucleus.
- Biological function summary
DGCR8 partners with Drosha to form the Microprocessor complex an important player in miRNA biogenesis. This complex identifies and accurately processes primary transcripts into precursor miRNAs a step essential for proper gene regulation. DGCR8's role is critical for maintaining the normal cellular function enabling the precise control of miRNA production which in turn modulates gene expression post-transcriptionally. Aberrations in its function affect the regulation of genes involved in cell cycle differentiation and development.
- Pathways
DGCR8 holds a significant influence on the RNA interference (RNAi) pathway interacting closely with Drosha to regulate miRNA synthesis. This pathway is fundamental for the regulation of genetic networks involved in cellular development and homeostasis. In the context of the miRNA processing pathway DGCR8 links to proteins like Dicer which further processes precursor miRNAs in the cytoplasm. Proper function of these pathways ensures the balanced expression of mRNAn important for healthy cellular activities.
- Associated diseases and disorders
DGCR8 misregulation is associated with the development of DiGeorge syndrome and certain cancers. Alterations in its function can lead to compromised miRNA maturation impacting genes that control cell growth and survival. In DiGeorge syndrome haploinsufficiency of DGCR8 may contribute to the associated developmental anomalies. In cancer altered DGCR8 expression can disrupt miRNA profiles interacting further with proteins such as Argonaute to influence the dysregulation of gene expression profiles commonly observed in tumorigenesis.
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3 product images
Western blot - Human DGCR8 knockout A549 cell line (ab287368)
Western blot: Anti-DGCR8 antibody [EPR18757] (Anti-DGCR8 antibody [EPR18757] ab191875) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-DGCR8 antibody [EPR18757] ab191875 was shown to bind specifically to DGCR8. A band was observed at 90-100 kDa in wild-type A549 cell lysates with no signal observed at this size in DGCR8 knockout cell line. To generate this image, wild-type and DGCR8 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-DGCR8 antibody [EPR18757] (Anti-DGCR8 antibody [EPR18757] ab191875) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: DGCR8 knockout A549 cell lysate at 20 µg
Lane 3: HeLa cell lysate at 20 µg
Lane 4: PC-3 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Next Generation Sequencing - Human DGCR8 knockout A549 cell line (ab287368)
160 bp deletion after Ser372 of the WT protein.
Sanger Sequencing - Human DGCR8 knockout A549 cell line (ab287368)
160bp deletion after Ser373 of the WT protein.
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