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.
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.
|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|
|Growth Conditions:||EMEM, 10% FBS, 5% C02, 37C|
|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.|
|Cryopreservation:||Growth Medium + 5% DMSO|
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.
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