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Method of identifying production using gene expression

A product and reporter gene technology, applied in the field of product identification, can solve problems such as ligand quantity and characteristic limitations

Inactive Publication Date: 2002-05-22
ROHM & HAAS CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Some of the disadvantages of these existing detection systems include the need for high concentrations of ligand in the labeled product and limitations in the number and identity of ligands that can be used in this detection method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1: EcR gene switch

[0110] One embodiment of the ecdysone gene switch is to prepare an acceptor plasmid (the above-mentioned first nucleotide molecule) and a reporter plasmid (the above-mentioned second nucleotide molecule).

[0111] The recipient plasmid pIE1VP16CfEcRCDEF contains the CfEcR CDEF domain fused to the VP16 activation domain and whose expression is under the control of the baculovirus IE1 promoter. The construction of this plasmid has two steps. To construct the vector pIE1VP16, the IE1 promoter region of AcMNPV (described in Ayres et al., supra) was amplified using primers with NdeI and BgllII restriction enzyme sites. This amplified product was cloned into a plasmid vector containing the VP16 activation domain (described in Pellett et al., supra), and a multiple cloning site followed by the SV40 polyA signal. The CfEcR CDEF domain was amplified using primers appended with BamHI and XbaI (described in Kothapalli et al., supra). This amplified ...

Embodiment 2

[0115] Add 1.0166g of ligand markers, methoxyfenoxide (N'-tert-butyl-N'-(3,5-dimethylbenzoyl)-3-methoxy-2-methylbenzoyl) to a 100ml volumetric flask hydrazide). Solution A was obtained by diluting the solution to a volume of 100 ml with absolute ethanol. 1 ml of solution A contains approximately 10 mg of ligand label.

[0116] Add 10ml (±0.04ml) of the above solution A into a 100ml volumetric flask to prepare solution B. The obtained solution was diluted to a volume of 100 ml with absolute ethanol. 1 ml of the obtained solution B contained about 1 mg of the ligand label.

[0117] Add 10ml (±0.04ml) of the above solution B into a 100ml volumetric flask to prepare solution C. The obtained solution was diluted to a volume of 100 ml with absolute ethanol. 1 ml of the obtained solution C contained about 0.1 mg of the ligand label.

[0118] sample#

[0119]After the samples in Table 1 were diluted 1:100 in acetonitrile / water (1 / 1), each gasoline and vodka sample was ...

Embodiment 3

[0122] The insect cell line BRL-AG2 is derived from the boll weevil (Anthomus grandis) (see Stiles et al., In Vitro Cell Dev. Biol., 28A:355-363 (1992)). Another insect cell line, L57, was generated by modifying the orangutan Drosophila Kc cell line to silence the expression of ecdysone receptors. Each insect cell line was transfected with the recipient and reporter plasmids described in Example 1 above.

[0123] In this insect cell-based ligand marker assay, 200,000 of the transfected BRL-AG2 cells or L57 cells described above were distributed in each well of a 48-well plate. To these cells are added 0.5 [mu]l of a negative control such as BMSO, or the labeled product of Example 2 (vodka or gasoline). These cells were maintained at 25[deg.] C. for 48 hours in medium containing the ligand marker solution of Example 2 or a positive control (ie methoxyfenoxide in ethanol) or a negative control (ethanol only). These cells were harvested and resuspended in reporter lysis buffer ...

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PUM

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Abstract

A method for identifying a product involves the steps of: (1) associating with the product a marker ligand; and (2) detecting the marker ligand in the product at a later point in time as a means of identifying the product by contacting the product with a detector composition. The detector composition comprises one or more first nucleotide sequences encoding one or more natural or synthetic ligand-dependent transcription factors, wherein said factors comprise at least one ligand binding domain, at least one DNA binding domain and at least one transactivation domain; and a second nucleotide sequence encoding a reporter gene under the regulatory control of a receptor response element or a modified or synthetic response element. The detector composition, a cell line containing the first and second nucleotide sequences, kits using them and products marked with specific marker ligands are useful in this method.

Description

field of invention [0001] The present invention relates generally to the field of product identification; more specifically, to the use of biotechnology systems in marking products for identification. Background of the invention [0002] The interaction between various ligands and their intracellular binding receptors has been exploited in a variety of fields where receptor-induced triggering of a promoter enables a gene encoding a protein of interest to be activated on that promoter. expression under regulation. These inducible expression systems allow the protein of interest to be produced in the cell at an appropriate time point. One such receptor system is the insect steroid hormone receptor system disclosed in US Patent 5,514,578. [0003] These systems are used in the screening of drugs or new compounds. For example, International Patent Application WO 92 / 27356, published June 3, 1999, relates to a method for identifying modulators of receptor function by combining ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12N15/09C12Q1/00C12Q1/02C12Q1/66C12Q1/68G01N33/566
CPCC12Q1/6897C12Q1/00
Inventor B·温斯坦L·H·凯勒S·R·帕里
Owner ROHM & HAAS CO
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