Transgenic animals for analyzing CYP3A4 cytochrome P450 gene regulation

Abstract

The invention relates to the generation of non-human transgenic animals comprising a reporter construct for producing a detectable amount of a reporter molecule operably linked to a transcriptional regulatory nucleic acid molecule from the human CYP3A4 gene located between the initiation of transcription site of the gene and a position located 13,000 nucleotides upstream from the site. The invention also relates to the use of these animals for determining the effect of a compound, particularly, but not exclusively, a xenobiotic or steroid, on the regulation of expression of the CYP3A4 gene in a human.

1 . A non-human mammal comprising:
 a regulatory nucleic acid molecule which is capable of regulating transcription of the human CYP3A4 gene and which comprises a nucleotide sequence that is identical to a sequence of the human CYP3A4 gene located between the initiation of transcription site of the gene and a position located at least 13,000 nucleotides upstream from the site; and   a reporter nucleic acid molecule for producing a detectable amount of a reporter molecule for indicating regulation of transcription of the reporter nucleic acid molecule by the regulatory nucleic acid molecule;   wherein the reporter and regulatory nucleic acid molecules are arranged to permit the regulatory nucleic acid molecule to regulate transcription of the reporter nucleic acid molecule.   
     
     
         2 . A non human mammal comprising:
 a regulatory nucleic acid molecule comprising a nucleotide sequence that is identical to the nucleotide sequence of the human CYP3A4 gene that extends about 13,000 nucleotides upstream from the initiation of transcription site of the gene; and   a reporter nucleic acid molecule for producing a detectable amount of a reporter molecule for indicating regulation of transcription of the reporter nucleic acid molecule by the regulatory nucleic acid molecule;   wherein the reporter and regulatory nucleic acid molecules are arranged to permit the regulatory nucleic acid molecule to regulate transcription of the reporter nucleic acid molecule.   
     
     
         3 . A mammal according to  claim 2  wherein the regulatory nucleic acid molecule comprises the sequence shown in SEQ ID NO:1. 
     
     
         4 . A non-human mammal comprising:
 a regulatory nucleic acid molecule comprising a nucleotide sequence that is identical to the sequence of the human CYP3A4 gene that extends about 8,000 nucleotides upstream from a position about 3,000 nucleotides upstream from the initiation of transcription site of the gene; and   a reporter nucleic acid molecule for producing a detectable amount of a reporter molecule for indicating regulation of transcription of the reporter nucleic acid molecule by the regulatory nucleic acid molecule;   wherein the reporter and regulatory nucleic acid molecules are arranged to permit the regulatory nucleic acid molecule to regulate transcription of the reporter nucleic acid molecule.   
     
     
         5 . A mammal according to  claim 4  wherein the regulatory nucleic acid molecule comprises the sequence shown SEQ ID NO:2. 
     
     
         6 . A non-human mammal comprising:
 a regulatory nucleic acid molecule which is capable of regulating transcription of the human CYP3A4 gene and which comprises a nucleotide sequence that is identical to the sequence of the human CYP3A4 gene that extends about 600 nucleotides upstream from a position about 7,200 nucleotides upstream of the initiation of transcription site of the gene; and   a reporter nucleic acid molecule for producing a detectable amount of a reporter molecule for indicating regulation of transcription of the reporter nucleic acid molecule by the regulatory nucleic acid molecule;   wherein the reporter and regulatory nucleic acid molecules are arranged to permit the regulatory nucleic acid molecule to regulate transcription of the reporter nucleic acid molecule.   
     
     
         7 . A mammal according to  claim 6  wherein the regulatory nucleic acid molecule comprises the sequence shown in SEQ ID NO:3. 
     
     
         8 . A non-human mammal comprising:
 a regulatory nucleic acid molecule which is capable of regulating transcription of the human CYP3A4 gene and which comprises a nucleotide sequence of a fragment of the human CYP3A4 gene consisting of from nucleotide positions −7836 to −7207 contiguous with −362 to +53; and   a reporter nucleic acid molecule for producing a detectable amount of a reporter molecule for indicating regulation of transcription of the reporter nucleic acid molecule by the regulatory nucleic acid molecule;   wherein the reporter and regulatory nucleic acid molecules are arranged to permit the regulatory nucleic acid molecule to regulate transcription of the reporter nucleic acid molecule.   
     
     
         9 . A mammal according to  claim 3 , further comprising at least one human transcription factor for regulating transcription of a human CYP3A4 gene. 
     
     
         10 . A mammal according to  claim 9  wherein the transcription factor is a nuclear receptor. 
     
     
         11 . A mammal according to  claim 10  wherein the nuclear receptor is a heterodimer of the human pregnane X receptor and human 9-cis retinoic acid receptor or a heterodimer of human constitutive androstane receptor-β and human 9-cis retinoic acid receptor. 
     
     
         12 . A mammal according to  claim 3  wherein the reporter nucleic acid molecule is capable of producing a reporter molecule selected from the group of reporter molecules consisting of firefly luciferase, β-galactosidase, alkaline phosphatase, green fluorescent protein or chloramphenicol acetyl transferase. 
     
     
         13 . A mammal according to  claim 3  wherein the mammal is a mouse. 
     
     
         14 . A tissue of a mammal according to  claim 3 . 
     
     
         15 . A tissue according to  claim 14  wherein the tissue is an embryo. 
     
     
         16 . A method of determining whether a compound is capable of effecting the transcription of a human CYP3A4 gene the method comprising the following steps:
 administering the compound to a non human mammal according to any one of the preceding claims; and   determining whether the reporter molecule is produced by the reporter nucleic acid molecule in the mammal.   
     
     
         17 . A method according to  claim 16  wherein the production of the reporter molecule indicates that the binding compound is capable of effecting the transcription of the human CYP3A4 gene.