SLC5A2 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 91 bp and 86 bp deletion in exon 2.
Images
Key facts
- Cell type
- HEK-293
- Species or organism
- Human
- Tissue
- Kidney
- Form
- Liquid
- Knockout validation
- Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 91 bp and 86 bp deletion in exon 2
Recommended products
SLC5A2 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 91 bp and 86 bp deletion in exon 2.
Key facts
- Cell type
- HEK-293
- Species or organism
- Human
- Tissue
- Kidney
- Form
- Liquid
- Knockout validation
- Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, Homozygous: 91 bp and 86 bp deletion in exon 2
- Concentration
Properties
- Gene name
- SLC5A2
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Sanger Sequencing, Western blot
Cell culture
- Biosafety level
- EU: 2 US: 2
- Viability
- ~ 80%
- Adherent/suspension
- Adherent
- Gender
- Female
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.
- Culture medium
- DMEM (High Glucose) + 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
Although we aim to provide customers with a homozygous clone, feasibility will be dependent on the biology of the protein. Should only heterozygous edits be achieved, you will be notified of the outcome and be asked to confirm whether the cell line is acceptable. All clones will be accompanied with DNA sequencing data, and the mutation description.
Recommended control: Human wild-type HEK-293 cell line (ab277915). 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: DMEM (High Glucose) + 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 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 2x10E4 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 2x10E4 cells/cm2 is recommended.
• Cells should be passaged when they have achieved 80-90% confluence.
• Do not allow the cell density to exceed 7x10E4 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 SLC7A5/LAT1 or LAT1 transporter also known as the large neutral amino acid transporter small subunit 1 plays an important role in facilitating the transport of large neutral amino acids across cell membranes. LAT1 is a heterodimer with a heavy chain known usually as SLC3A2 forming a functional transporter which is around 40-50 kDa in mass. LAT1 is highly expressed in tissues with high metabolic rates like the brain placenta and some cancer cells making it important for nutrient transport in these areas.
- Biological function summary
The LAT1 transporter mediates the uptake of essential amino acids which are critical for protein synthesis and cellular metabolism. It functions as part of the LAT1-SLC3A2 heterodimer complex ensuring effective transport across the blood-brain barrier and into other metabolically active tissues. This activity supports cellular growth and proliferation underlining its importance in rapidly dividing cells such as in cancer.
- Pathways
LAT1 plays a critical role in the mTOR signaling pathway which is central to regulating cell growth and metabolism in response to nutrient availability. LAT1's transport function influences the activation of mTORC1 by ensuring a steady supply of leucine an amino acid that activates this complex. LAT1 is also integral to the cross-pathway interaction with the insulin signaling pathway enhancing nutrient-dependent growth signals in conjunction with other transporters.
- Associated diseases and disorders
Abnormal LAT1 function links closely with cancer and neurological disorders. Its overexpression often marks various tumors where it supports cancer cell survival by sustaining amino acid supply often in conjunction with increased SLC3A2 expression. In neurological contexts LAT1 impairments can contribute to disorders involving neurotransmitter imbalances as it participates in amino acid transport critical for neurotransmitter synthesis.
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2 product images
Sanger Sequencing - Human SLC7A5 knockout HEK-293 cell line (ab288697)
91 bp and 86 bp deletion in exon 2.
Western blot - Human SLC7A5 knockout HEK-293 cell line (ab288697)
False colour image of Western blot: Anti-SLC7A5 antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to SLC7A5. A band was observed at 35 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in SLC7A5 knockout cell line ab288697 (knockout cell lysate ab289590). To generate this image, wild-type and SLC7A5 knockout HEK-293 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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.
Performed under reducing conditions.
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