IP3R Expressing HEK-293 Cell Lines
Immortalized cell lines (HEK-293), with all three isotypes of the inositol 1,4,5-trisphosphate receptor (IP3R) knocked out, or which express a single IP3R subtype or combinations of two subtypes.
Highlights:
- Available cell lines: IP3R null, IP3R1 null, IP3R2 null, IP3R3 null, IP3R1+, IP3R2+, IP3R3+
- IP3R null HEK-293 created via CRISPR/Cas9 technology, targeting the ITPR1, ITPR2, and ITPR3 genes
- IP3R null cells do not respond to extracellular stimuli which would normally raise intracellular Ca2+
- Useful for studies of IP3R and Ca2+ signaling
Inositol 1,4,5-trisphosphate receptors (IP3R) are a family of 3 intracellular Ca2+ release channels which are responsible for Ca2+ signals which control diverse cellular events including secretion, gene transcription, metabolism and cell fate. Because of their ubiquitous expression of multiple subtypes of the protein, study of individual subtypes or the consequences of their activation in isolation is difficult. Therefore, the Yule lab created a cell line based on the parental HEK 3KO cell in which they have deleted both copies of the three IP3R genes by CRISPR/Cas9 technology and is therefore IP3R null and does not respond to extracellular stimuli which would normally raise intracellular Ca2+. This is the only null IP3R cell line to their knowledge. They have also created HEK cell lines which express the endogenous single IP3R subtype or combinations of two subtypes. These cell lines are useful for studies of IP3R and Ca2+ signaling in general.
From the laboratory of David I. Yule, PhD, University of Rochester.
Immortalized cell lines (HEK-293), with all three isotypes of the inositol 1,4,5-trisphosphate receptor (IP3R) knocked out, or which express a single IP3R subtype or combinations of two subtypes.
Highlights:
- Available cell lines: IP3R null, IP3R1 null, IP3R2 null, IP3R3 null, IP3R1+, IP3R2+, IP3R3+
- IP3R null HEK-293 created via CRISPR/Cas9 technology, targeting the ITPR1, ITPR2, and ITPR3 genes
- IP3R null cells do not respond to extracellular stimuli which would normally raise intracellular Ca2+
- Useful for studies of IP3R and Ca2+ signaling
Inositol 1,4,5-trisphosphate receptors (IP3R) are a family of 3 intracellular Ca2+ release channels which are responsible for Ca2+ signals which control diverse cellular events including secretion, gene transcription, metabolism and cell fate. Because of their ubiquitous expression of multiple subtypes of the protein, study of individual subtypes or the consequences of their activation in isolation is difficult. Therefore, the Yule lab created a cell line based on the parental HEK 3KO cell in which they have deleted both copies of the three IP3R genes by CRISPR/Cas9 technology and is therefore IP3R null and does not respond to extracellular stimuli which would normally raise intracellular Ca2+. This is the only null IP3R cell line to their knowledge. They have also created HEK cell lines which express the endogenous single IP3R subtype or combinations of two subtypes. These cell lines are useful for studies of IP3R and Ca2+ signaling in general.
From the laboratory of David I. Yule, PhD, University of Rochester.
| Catalog Number | Product | DataSheet | Size | AVAILABILITY | Price | Qty |
|---|
| Product Type: | Cell Line |
| Name: | IP3R null: HEK-3KOSingle IP3R isoform expressed: HEKR1, HEKR2, HEKR3Two IP3R isoforms expressed: HEKR2R3, HEKR2R1, HEKR3R1 |
| Cell Type: | HEK-293 *The parental HEK cell line was genotyped to verify its identity prior to generation of the IP3R cell lines |
| Accession ID: | Q14643, Q14571, Q14573, CVCL_HB82, CVCL_HB83, CVCL_HB84, CVCL_HB85, CVCL_HB86, CVCL_HB87, CVCL_HB88 |
| Organism: | Human |
| Source: | Embryonic kidney |
| Morphology: | Fibroblast |
| Biosafety Level: | BSL2 |
| Subculturing: | 1:10; Suggestion: These cells can take a few days to recover when first thawing. It is important to leave the cells alone for the first few days, only supplementing with more media. Typically, the cells bounce back after a few days. |
| Growth Conditions: |
DMEM (High Glucose, 4.5g/L, with L-Glutamine and Pyruvate, 110mg/L), 10% FBS, 5%CO2, 37C. Suggested DMEM & FBS (or equivalent): Gibco, Cat # 11995-065 & Cat # 10437-038, respectively. |
| Cryopreservation: |
Growth Medium + 5% DMSO Note from the provider: The cells may freeze and recover much better when frozen in DMEM + 20% FBS + 5% DMSO. |
| Storage: | Liquid nitrogen |
| Shipped: | Dry ice |
Generation of HEK293 cells with IP3R-null background using CRISPR/Cas9 technology
(A) Confirmation of the absence of all three subtypes of IP3R in HEK-3KO cells. HEK293 cells were transfected with vectors encoding Cas9 endonuclease and sgRNAs targeting corresponding IP3R-encoding alleles. Single clones were propagated, lysed, and processed for immunoblotting with the indicated IP3R subtype-specific antibodies. (B) Ca2+ release from HEK-3KO cells transiently transfected either with vector or cherryR1. Twenty-four hours posttransfection, cells were loaded with Ca2+ indicator Fura-2AM and stimulated with 500 nM trypsin to induce IP3 formation. Ca2+ release was measured as a change in the 340/380 fluorescence ratio. Experiments were repeated five times. Representative traces are shown.
Western Blot of IP3R Expression
Confirmation of expression of the endogenous single IP3R subtype (HEKR1, HEKR2, HEKR3) or combinations of two subtypes (HEKR2R3; HEKR2R1, HEKR3R1).
Adapted from: Alzayady KJ, et al. Sci Signal. 2016 Apr 5;9(422):ra35.
- Alzayady KJ, Wang L, Chandrasekhar R, Wagner LE 2nd, Van Petegem F, Yule DI. Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release. Sci Signal. 2016 Apr 5;9(422):ra35.
- Thillaiappan NB, Chavda AP, Tovey SC, Prole DL, Taylor CW. Ca(2+) signals initiate at immobile IP(3) receptors adjacent to ER-plasma membrane junctions. Nat Commun. 2017 Nov 15;8(1):1505. View Article
- Filadi R, Leal NS, Schreiner B, Rossi A, Dentoni G, Pinho CM, Wiehager B, Cieri D, Calì T, Pizzo P, Ankarcrona M. TOM70 Sustains Cell Bioenergetics by Promoting IP3R3-Mediated ER to Mitochondria Ca(2+) Transfer. Curr Biol. 2018 Feb 5;28(3):369-382.e6. View Article
- Mataragka S, Taylor CW. All three IP(3) receptor subtypes generate Ca(2+) puffs, the universal building blocks of IP(3)-evoked Ca(2+) signals. J Cell Sci. 2018 Aug 10. pii: jcs.220848. View Article
- Atakpa P, Thillaiappan NB, Mataragka S, Prole DL, Taylor CW. IP(3) Receptors Preferentially Associate with ER-Lysosome Contact Sites and Selectively Deliver Ca(2+) to Lysosomes. Cell Rep. 2018 Dec 11. View Article
- Ellefsen KL, Lock JT, Settle B, Karsten CA, Parker I. Applications of FLIKA, a Python-based image processing and analysis platform, for studying local events of cellular calcium signaling. Biochim Biophys Acta Mol Cell Res. 2018 Nov 27. View Article
- Atakpa P, van Marrewijk LM, Apta-Smith M, Chakraborty S, Taylor CW. GPN does not release lysosomal Ca(2+), but evokes ER Ca(2+) release by increasing cytosolic pH independent of cathepsin C. J Cell Sci. 2019 Jan 7. View Article
- Lock JT, Alzayady KJ, Yule DI, Parker I. All three IP(3) receptor isoforms generate Ca(2+) puffs that display similar characteristics. Sci Signal. 2018 Dec 18;11(561). pii: eaau0344. View Article
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