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Cell Lines For Screening Odorant And Aroma Receptors

An odorant, cell-based technology for use in the field of assays for the identification of odorants and/or aroma compounds or such modulators, capable of solving the problems of inefficient, cumbersome molecular biology methods

Inactive Publication Date: 2018-04-13
FIRMENICH SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing techniques for developing cell lines stably expressing the endogenous RTP1 gene involve inefficient and cumbersome molecular biology methods of inserting DNA into cultured cells that do not otherwise express the gene

Method used

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  • Cell Lines For Screening Odorant And Aroma Receptors
  • Cell Lines For Screening Odorant And Aroma Receptors
  • Cell Lines For Screening Odorant And Aroma Receptors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Genome editing strategies induce constitutive activation of the endogenous RTP1 gene in HEK293T cells.

[0140] A strategy to develop enhanced expression of functional odorant receptors in a heterologous expression system is described. Using a newly available genome editing technology called CRISPR / Cas9, the silenced (inactive) endogenous RTP1 gene in normal HEK293T cells was specifically targeted by introducing a constitutive promoter (CMV) upstream of its coding sequence Activated constitutively. figure 1 ) The RTP1 gene is located on chromosome 3, showing the DNA sequence around its start site. The Cas9 endonuclease is guided by a 20 base pair (bp) guide RNA (gRNA) homologous to the target. Upon delivery to cells (GeneArt CRISPR Nuclease (CD4 Enriched) Vector Kit, cat#A21175), the guide RNA molecule and Cas9 protein form an active complex that induces the desired double-strand DNA break (DSB) upstream of the coding sequence . Boxes indicate the RTP1 gene on chrom...

Embodiment 2

[0142] Selection of modified cell lines endogenously expressing the RTP1 gene.

[0143] Several control steps help characterize the modification of the cell line and its integrity. image 3 ) shows a schematic representation of wild-type and recombinant alleles. Gray lines indicate relative amplicon positions (not to scale) of PCR and RT-PCR experimental results for DNA genotyping and RNA expression controls, respectively. Figure 4 ) Genomic DNA from puromycin-resistant cell lines was extracted and PCR was performed to differentiate non-recombinant wild-type (WT) from modified cell lines (Mod.). PCR1 amplified the 2.0 kb band only in wild-type HEK293T cells, but not in the modified cell line. The modified line should produce a 4.0 kb band with PCR 1, but this is not possible due to the length and complexity of the genome structure. Genotyping of the modified cell line failed to yield a 2.0 kb band, indicating homozygous integration of the CMV promoter. Correct integration...

Embodiment 3

[0145] Characterization of RTP1 protein expression.

[0146] RTP1 protein expression in selected modified cell lines was determined by Western blot analysis using RTP1-specific antibodies. The long (RTP1L) and short (RTP1S) protein forms can be derived from the endogenous RTP1 gene. The genome modification strategy described here involves introducing the CMV promoter upstream of the RTP1L start codon to avoid any modification of the endogenous coding sequence; RTP1L is thus expected to be expressed. However, the results showed that the modified cell lines were heavily biased towards the expression of RTP1S, suggesting that the endogenous RTP1 gene preferentially expresses a short version without further genome editing. The latter is the preferred form as it is known to better promote cell surface expression of odorant receptors. Figure 6 ) shows a Western blot of RTP1 protein. Arrows indicate the expected protein sizes for RTP1S - 25 kDa, RTP1L - 28 kDa and the control pro...

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Abstract

Provided herein is a cell line with improved odorant receptor function comprising an activated endogenous RTP1 gene, which further expresses an RTP1 protein. Further provided herein is a method for specifically activating an endogenous RTP1 gene in a eukaryotic cell using a CRISPR / Cas9 derived technique. Also provided herein is a method for identifying compounds with desired effects such as perfume or aroma modulators in said cell line.

Description

technical field [0001] The technical field relates to odorants and aroma receptors, and assays useful for the identification of odorants and / or aroma compounds or such modulators. This assay is more specifically directed to engineered cell lines that exhibit improved odorant receptor activity. Background technique [0002] Odors are initially encoded in the peripheral olfactory system (i.e., the nose) through interactions between volatile flavor and aroma compounds and odorant receptor (OR) proteins residing on the membranes of olfactory receptor neurons in the olfactory epithelial tissue middle. Such interactions occur in an odor-specific combinatorial manner, where any single OR can be activated by multiple odorants, and conversely, most odorants are able to activate several different ORs. For a given odorant / aroma compound or mixture, these receptor interactions generate neurophysiological signals in the brain and ultimately conscious odor perception. The approximately...

Claims

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Application Information

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IPC IPC(8): C12N5/10C07K14/47G01N33/68
CPCC07K14/47C12N5/0636G01N33/68G01N2500/10G01N2333/705C12N2510/02C12N9/22C12N5/10C12N15/85C12N2310/20C07K14/4705C07K14/705C12N15/11C12N15/86C12N2800/80C12N2830/00G01N33/502
Inventor H·Y·郑P·菲斯特M·罗杰斯
Owner FIRMENICH SA
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