Claims
- 1. A ligand dependent transactivation system for gender-sorting in a test animal, comprising;
a) a first DNA construct comprising a nucleic acid molecule encoding a receptor having specific binding affinity for said ligand operably linked to a promoter; b) a second DNA construct comprising a promoter containing a plurality of ligand specific response elements, said promoter being operably linked to a reporter gene; c) a third DNA construct comprising a nucleic acid sequence encoding an enzyme for cleaving a naturally occurring biological conjugate, said biological conjugate comprising said ligand, said nucleic acid sequence encoding said enzyme being operably linked to a promoter sequence; and d) a host cell comprising said first, second and third DNA constructs, expression of said reporter gene in said host cell being dependent upon cleavage of said ligand from a biological conjugate present in a biological sample isolated from said test animal, reporter gene expression being indicative of the presence of a ligand which indicates the sex of said test animal.
- 2. A system as claimed in claim 1, wherein said first DNA construct encodes a receptor selected from the group of sex related nuclear receptors consisting of estrogen receptor alpha, estrogen receptor beta, androgen receptor, progesterone receptor, glucocorticoid receptor, adrenocorticoid receptor, thyroid hormone receptor alpha and beta, retinoic acid receptor alpha, beta and gamma, retinoic acid X receptor alpha, beta and gamma, peroxysome proliferator activating receptor alpha, delta and gamma, vitamin D receptor, dioxin receptor, and liver X receptor.
- 3. A system as claimed in claim 1 wherein said test animal is selected from the group consisting of chickens and turkeys.
- 4. A system as claimed in claim 1, wherein said third construct encodes an enzyme selected from the group consisting of beta-glucuronidase, alpha-glucuronidase, sulfatases, SUMO specific proteases, SUMO specific hydrolases, and cytochrome P450 enzymes.
- 5. A system as claimed in claim 1, wherein said biological conjugate is selected from the group of conjugates consisting of estrone-sulfate, estradiol-17 beta-sulfate, estrone glucuronide, and estradiol 17-beta-glucuronide.
- 6. A system as claimed in claim 1, wherein said cleavage of said biological conjugate releases a ligand selected from the group consisting of 17-beta-estradiol and estrone.
- 7. A system as claimed in claim 1, wherein at least one of said promoters in said DNA constructs of steps a), b) and c) is an inducible promoter selected from the group consisting of CUP1, HSP70, HSP26, HSP104, SSA4, galactose-inducible promoters, GAL1, and GAL10.
- 8. A system as claimed in claim 1, wherein at least one of said promoters in said DNA constructs of steps a), b) and c), is a constitutive promoter selected from the group consisting of ADH1, GPD, and CUP1.
- 9. A system as claimed in claim 1, wherein said first, second and third DNA constructs are expression vectors, wherein each of said expression vectors comprises sequences which enable replication in both prokaryotes and eukaryotes.
- 10. A system as claimed in claim 1, wherein said host cell is selected from the group consisting of a yeast cell, an insect cell, a mammalian cell, and a bacterial cell.
- 11. A system as claimed in claim 10, wherein said yeast is Saccharomyces cerevisiae.
- 12. A system as claimed in claim 1, wherein said reporter gene is selected from the group consisting of β-galactosidase, alkaline phosphatase, green fluorescent protein, red fluorescent protein, chloramphenicol acetyltransferase, and surface molecules recognized by immunospecific antibodies.
- 13. A system as claimed in claim 1, wherein said biological sample is selected from the group consisting of allantoic fluid, blood, urinates, saliva, culture media of test animal tissue and extract from test animal tissue.
- 14. A system as claimed in claim 1, wherein the construct of step b) comprises a plurality of response elements selected from the group consisting of estrogen response elements, androgen response elements and nuclear receptor response elements.
- 15. A method for detecting the presence of sex determinative ligands which transactivate nuclear receptors in a ligand-dependent manner in a biological sample, comprising:
a) providing a host cell containing a first DNA construct having a nucleic acid molecule encoding a receptor having binding affinity for a sex determinative ligand operably linked to a first promoter; a second DNA construct comprising a second promoter containing a plurality of sex determinative ligand response elements, said second promoter being operably linked to a reporter gene and a third DNA construct comprising a nucleic acid sequence encoding an enzyme for cleaving a naturally occurring biological conjugate, said biological conjugate comprising said sex determinative ligand, said nucleic acid sequence encoding said enzyme being operably linked to a third promoter sequence; b) contacting said host cell with a biological sample suspected of containing said sex determinative ligand; and c) assessing levels of expression of said reporter gene in said host cell, said expression being dependent upon cleavage of said sex determinative ligand from a biological conjugate if present, in said biological sample.
- 16. A method as claimed in claim 15, wherein said first DNA construct encodes a receptor selected from estrogen receptor alpha, estrogen receptor beta, androgen receptor, progesterone receptor, glucocorticoid receptor, adrenocorticoid receptor, thyroid hormone receptor alpha and beta, retinoic acid receptor alpha, beta and gamma, retinoic acid X receptor alpha, beta and gamma, peroxysome proliferator activating receptor alpha, delta and gamma, vitamin D receptor, dioxin receptor, and liver X receptor isolated from different organisms.
- 17. A method as claimed in claim 15, wherein said third DNA construct encodes an enzyme selected from the group consisting of beta-glucuronidase, alpha-glucuronidase, and sulfatases.
- 18. A method as claimed in claim 15, wherein said biological conjugate is selected from the group of conjugates consisting of estrone-sulfate, estradiol-17 beta-sulfate, estrone glucuronide, and estradiol 17-beta-glucuronide.
- 19. A method as claimed in claim 15, wherein said cleavage of said biological conjugate releases a ligand selected from the group consisting of 17-beta-estradiol and estrone.
- 20. A method as claimed in claim 15, wherein at least one of said promoters in said DNA constructs of steps a), b) and c) is an inducible promoter selected from the group consisting of CUP1, HSP70, HSP26, HSP104, SSA4, galactose-inducible promoters, GAL1 and GAL10.
- 21. A method as claimed in claim 15, wherein at least one of said promoters in said DNA constructs of steps a), b) and c), is a constitutive promoter selected from the group consisting of ADH1, GPD, and CUP1.
- 22. A method as claimed in claim 15, wherein said first, second and third DNA constructs are expression vectors, wherein each of said expression vectors comprises sequences which enable replication in both prokaryotes and eukaryotes.
- 23. A method as claimed in claim 15, wherein said host cell is selected from the group consisting of a yeast cell, an insect cell, a mammalian cell, and a bacterial cell.
- 24. A method as claimed in claim 23, wherein said yeast is Saccharomyces cerevisiae.
- 25. A method as claimed in claim 15, wherein said reporter gene is selected from the group consisting of β-galactosidase, alkaline phosphatase, green fluorescent protein, red fluorescent protein, chloramphenicol acetyltransferase, and surface molecules recognized by immunospecific antibodies.
- 26. A method as claimed in claim 15, wherein said biological sample is selected from the group consisting of allantoic fluid, blood, urinates, saliva, culture media of test animal tissue, and extract from test animal tissue.
- 27. A method as claimed in claim 15, wherein said second promoter comprises a plurality of response elements selected from the group consisting of estrogen response elements, androgen response elements, and nuclear receptor response elements.
- 28. A method as claimed in claim 15, wherein the activity of said reporter is determined using a method selected from the group consisting of determination of enzymatic activity using enzymatic substrates, detection of protein encoded by said reporter gene using an antibody immunologically specific for said protein.
- 29. The method as claimed in claim 28, wherein said reporter gene encodes a fluorescent reporter protein.
- 30. A kit comprising the test system of claim 1.
- 31. An estrogen dependent transactivation system for gender-sorting in avian species, comprising;
a) a first DNA construct comprising a nucleic acid molecule encoding an estrogen receptor operably linked to a promoter; b) a second DNA construct comprising a promoter containing a plurality of estrogen response elements, said promoter being operably linked to a reporter gene; c) a third DNA construct comprising a nucleic acid sequence encoding a glucuronidase enzyme for cleaving a naturally occurring estrogen-glucuronide conjugate, said estrogen-glucuronide conjugate comprising estrogen, said nucleic sequence encoding glucuronidase being operably linked to a promoter sequence; d) a yeast cell comprising said first, second and third DNA constructs, expression of said reporter gene in said yeast cell being dependent upon release of estradiol from said glucuronide conjugate present in an allantoic sample of an animal, reporter gene expression levels being correlated with the presence of a ligand which indicates a sex of said animal; e) media for yeast cell growth and enhancement of glucuronidase expression, secretion and activity; and f) a substrate and protocol to assess said reporter gene expression.
- 32. A method for detecting the presence of estrogen [which transactivates nuclear receptors] in a ligand-dependent manner in a biological sample, comprising:
a) providing a yeast cell comprising a first DNA construct having a nucleic acid molecule encoding an estrogen receptor operably linked to a first promoter; a second DNA construct comprising a second promoter containing a plurality of estrogen response elements, said promoter being operably linked to a reporter gene, and a third DNA construct comprising a nucleic acid sequence encoding a glucuronidase enzyme for cleaving naturally occurring estrogen-glucuronide conjugates, said nucleic acid sequence encoding a glucuronidase enzyme being operably linked to a promoter sequence; b) contacting said yeast cell with a biological sample of an animal suspected of containing estrogen; and c) assessing levels of expression of said reporter gene in said yeast cell, expression of said reporter gene being dependent upon release of estradiol from said glucuronide conjugate present in said biological sample, reporter gene expression levels being correlated with the presence of a ligand which indicates a sex of said animal.
- 33. The method of claim 32, wherein said biological sample is selected from the group consisting of allantoic fluid, blood, urinates, saliva, culture media of test animal tissue, and extract from test animal tissue.
Parent Case Info
[0001] This application claims priority to U.S. Provisional Application No. 60/286,010, filed Apr. 23, 2001, the entire disclosure of which is incorporated by reference herein.
PCT Information
Filing Document |
Filing Date |
Country |
Kind |
PCT/US02/12590 |
4/23/2002 |
WO |
|
Provisional Applications (1)
|
Number |
Date |
Country |
|
60286010 |
Apr 2001 |
US |