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
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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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