# Patent

## Title
Compositions and methods for gender sorting

## Abstract

A yeast based transactivation assay for gender sorting is disclosed.

## Background
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.


FIELD OF THE INVENTION
The present invention relates to a novel method for detection of target molecules, such as hormones, using a yeast-based transactivation system. More particularly, the invention relates to methods for accurate and efficient gender sorting in mammalian and avian species.
BACKGROUND OF THE INVENTION
Various publications and patent documents are referenced in this application to describe the state of the art to which the invention pertains. Complete citations for these references are found at the end of the specification. Each of these publications or patents is incorporated by reference herein.
Sex separation of chicks at hatch is an important aspect of the poultry industry. The US poultry industry produces 8.9 billion broilers, 360 million broiler breeders, 360 million turkeys and 1.6 billion layers annually. These annual production numbers equate to roughly 160 million birds hatched each day which need to be sexed. Varied segments of the poultry industry must sex segregate offspring for different reasons. All turkeys are sex segregated due to the large differences in the growth rate, market age, management practices and nutritional requirements between male and female birds. In the layer or table egg industry, chicks must be sexed at hatch as only the females, or birds which lay eggs, are kept while all male chicks are discarded. The broiler breeder and turkey breeder industries produce both male and female lines that are eventually crossed to produce a hybrid commercial bird. The males or off-sex chicks produced from the female lines are discarded while the female chicks or off-sex chicks from the male lines are discarded. Disposal of unwanted chicks creates animal welfare and waste disposal issues for the industry. The meat type chicken or broiler industry prefer to sex segregate males from females to gain feed efficiency, improve uniformity at the processing plant and reduce production costs. The broiler industry often refrains from sex separate rearing due to the high costs of sexing and the need for trained sexers. A higher percentage of the broiler industry would sex segregate if a rapid, inexpensive, automated method were available.
Current sexing methods used in the poultry industry involve manual procedures that require highly trained professionals with specialized skills. Sexing methods involve either the Japanese method of vent sexing or feather sexing of chicks that carry a slow feathering gene (1, 2, 3). The manual sexing methods have several drawbacks. These include: 1) scheduling of manual labor; 2) increased processing time before chicks can be placed in the field with access to food and water; and 3) stress and damage to the chicks or poults. Manual sexing remains one of the few manual processing steps practiced in the highly automated poultry industry.
The Japanese or vent sexing method originated in the 1920's when the Japanese discovered that within the first or second fold of a chick's vent there are distinctive characteristics such as spots, lines and folds that reveal its sex (4, 5). Vent sexing is considered an art and generally practiced only by Asian individuals with excellent manual dexterity and Zen-like concentration (6). Vent sexers attend school for six months and then undergo a trainee program for two to five years until they are considered a qualified sexer based on speed and accuracy. Qualified sexers can vent sex 2,000 chicks per hour at 98% accuracy but only 5 to 10% of individuals attending vent sexing school attain qualified sexer status (6). The rapid rate at which a vent sexer must pick up, sex and sort baby chicks can result in a 1.4 to 1.8% increase in cull rate and 0.5 to 1.0% increase in early chick mortality due to chick damage during handling (7). Vent sexers are contract workers who travel from hatchery to hatchery working long tedious hours. Travel of these contract workers from hatchery to hatchery poses a bio-security threat for the poultry industry.
Feather sexing of chickens inbred for the K gene for slow feathering is the other commonly used method for sex segregation at hatch (3). Day-old chicks carrying the K gene can be sex segregated by examining the relative length of the primary and covert feathers of the wing, with the females carrying genes for fast feathering and the males carrying genes for slow feathering. Feather sexing is less expensive than vent sexing and does not require as skilled a labor force. Feather sexing is not applicable to turkeys, the majority of broiler breeds, some layer breeds, and also carries disadvantages. The k gene for slow feathering is closely linked to an endogenous virus (8), which can cause immunological tolerance to lymphoid leukosis (9) and is therefore a disadvantage in breeding stock. It is noteworthy that slow feathering males usually do not feather well in the brooder house especially during hot weather which often results in slower growth and increased cannibalism and that females carrying the K gene exhibit reduced egg production. Some poultry breeds exhibit sex specific color differences in feathers, however these breeds are not as commercially viable as other breeds. Color sexing can result in a higher rate of sexing mistakes than feather sexing.
Methods to sex segregate eggs before hatching have been previously attempted without success. A sex-linked gene, Sa1 for imperfect albinism was reported by Hutt and Cole (10) to have the potential to sex chicks at one day of age on the basis of eye color. The albino female chicks have pink eyes while the normal males have black eyes. The difference in eye color can also be used to accurately sex segregate embryos by candling the eggs on Day 10 of incubation. Candling or light illumination of the intact egg reveals the dark eye of the male chicken embryo but not the pink eye of the albino female. Use of the Sa1 gene to sex segregate poultry was not pursued as the gene carries deleterious effects in turkeys and negative production effects in chickens.
Development of a quick, accurate, inexpensive automated method to sex segregate eggs before hatching would significantly increase profitability of the global broiler industry. Automated sexing gives rise to several benefits. These include 1) better feed and processing efficiencies; 2) reduced incubation space requirements; 3) greater selection of breeds without constraints of selection for a slow feathering gene; 4) reduced liability and reliability concerns associated with eliminating manual labor; 5) speedier hatchery processing; and 6) reduced animal welfare concerns associated with discarding male chicks in the layer egg industry.
In light of all the foregoing, the development of a quick, inexpensive, automated method to sex segregate eggs before hatching is highly desirable.
SUMMARY OF THE INVENTION
The present invention provides a method and kit for detecting target molecules in a sample. In a preferred embodiment, methods and kits for determining the sex of avian species while in the egg are disclosed. The cell-based sensor has additional applications in detection of glucuronidated steroid hormones, metabolites and xenobiotics. The ultra sensitive method of the invention may also be used to advantage to gender sort other animal species, and to assess patient populations for their susceptibility to drugs and predisposition to disease.
In an exemplary embodiment of the invention, a biological sample containing a sex-indicating hormone is isolated from the egg. Genetically engineered host cells comprising a sex hormone-dependent transactivation system are then contacted with the biological fluid. The engineered cells respond to the level of sex hormone by activating a reporter gene. Female eggs, which contain a significant amount of 17 β estradiol, a hormone that is absent from male embryos, give a positive response in cell-based sensor assays of the invention and are sorted as females.
In another embodiment of the invention, a ligand dependent transactivation system for gender-sorting in a test animal is provided, wherein the system comprises 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. In another embodiment of the invention, the 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.
In yet another embodiment, a method for detecting the presence of sex determinative ligands which transactivate nuclear receptors in a ligand-dependent manner in a biological sample are provided, wherein the method comprises: 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. In a particular embodiment, the 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.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the difference of estradiol levels in pg/ml in the allantoic fluid of male and female broiler 17-day old embryos.
FIGS. 2A–2C are schematic diagrams of the estrogen-dependent transactivation system of the present invention.
FIG. 3A is a schematic diagram of the LacZ-reporter gene with estrogen response elements (ERES). The yeast reporter plasmid, YRpE2, contains two copies of an ERE (Lyttle et al., 1992) located upstream of the iso-1-cytochrome c (CYC1) promoter which is coupled to the E. coli beta-galactosidase gene (lacZ). This yeast-E. coli multicopy shuttle plasmid contains URA3 as a yeast transformation marker.
FIG. 3B is a schematic diagram of the LacZ-reporter gene with EREs. The yeast reporter plasmid, YRpE2SSA4-lacZ, contains two copies of an ERE (Lyttle et al., 1992) located upstream of the yeast heat shock inducible (SSA4, Genbank Accession number: J05637) promoter which is coupled to the E. coli beta-galactosidase gene (lacZ). This yeast-E. coli multicopy shuttle plasmid contains URA3 as a yeast transformation marker.
FIG. 4 (YEpE12-ERα) is a schematic diagram of the yeast expression plasmid utilized in the practice of the present invention (Graumann et al., 1996). A yeast expression plasmid is shown which encodes full-length human estrogen receptor alpha. The coding sequence is inserted into the yeast expression vector to produce a ubiquitin (UBI)-fusion protein under the control of a CUP1 promoter. TRP1 is the tryptophan selectable marker and 2 μm is for replicating yeast DNA.
FIG. 5 (pRS425-GPD-Ub-GUS) shows the yeast expression plasmids for expressing the E. coli GUS (beta-glucuronidase gene; Jefferson et al, 1986). The N-terminal coding sequence of the GUS gene is fused in frame with the C-terminal of the human ubiquitin gene. The recombinant protein is under GPD promoter. LEU2 is the leucine (LEU2) selectable marker and 2 μm facilitates replication in yeast.
FIGS. 6A and 6B show a pair of graphs showing that the gender sorting system of the invention efficiently sorts male and female turkeys as well as layer chickens.
FIG. 7 is a graph showing a transactivation assay using resorufin at concentration 0.00025 mg/mL and allantoic fluids (20 ul allantoic/80 ul of yeast. Samples 768–817). Incubation time was 4 h, followed by addition and incubation with substrates for an additional 10 min at 37° C.
FIG. 8 is a graph showing a comparison of time/response in two transactivation assays in response to estrogen (concentration 5 nM) using YRpE2 (CYC1 promoter based) and YRpE2SSA4-lacZ (heat shock promoter based) reporter vector at an ONPG (substrate) concentration of 0.2%. The incubation times with hormone were varied as shown prior to addition of substrates and subsequent substrate incubation time (15 minutes) at 30° C.
FIG. 9 shows the nucleic acid sequence of the CYC1-based estrogen inducible reporter YRpE2 (SEQ ID NO: 7). It comprises the E. coli replication origin and bacterial selection marker for Ampicillin resistance, the yeast 2 micron S. cerevisiae replicative origin, yeast selection marker URA3, and the E. coli beta-galactosidase gene which is operably linked to the CYC1 core promoter. The 2× estrogen response element (2×ERE) unit is operably linked to the CYC1 promoter in the upstream region.
FIG. 10 shows the nucleic acid sequence of the SSA4-based estrogen inducible reporter YRpE2SSA4-lacZ (SEQ ID NO: 8). It comprises the E. coli replication origin and bacterial selection marker for Ampicillin resistance, the yeast 2 micron S. cerevisiae replicative origin, the yeast selection marker URA3, and the E. coli beta-galactosidase gene which is operably linked to the SSA4 core promoter. The 2×ERE unit is operably linked to the SSA4 promoter in the upstream region.
FIG. 11 shows the nucleic acid sequence of the yeast expression vector for human estrogen receptor alpha—YEpE12-ERα (SEQ ID NO: 9). It comprises the E. coli replication origin and bacterial selection marker for Ampicillin resistance, the yeast 2 micron S. cerevisiae replicative origin, yeast selection marker TRP1, and a human estrogen receptor fused in frame with human ubiquitin which is operably linked to the yeast CUP1 promoter.
FIG. 12 shows the nucleic acid sequence of the yeast expression vector for the E. coli glucuronidase gene—pRS425-GPD-Ub-GUS (SEQ ID NO: 10). It comprises the E. coli replication origin and bacterial selection marker for Ampicillin resistance, the yeast 2 micron S. cerevisiae replicative origin, the yeast selection marker LEU2, and the E. coli beta-glucuronidase gene which is fused in frame with human ubiquitin which is operably linked to the yeast GPD promoter.



DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, compositions, methods and kits are provided for the accurate and efficient detection of biological target molecules. A yeast-based biosensor is disclosed to identify and characterize target molecules isolated from biological samples. In a preferred aspect of the invention, a gender sorting method is provided which enables pre-hatch sorting of female embryos from male embryos.
I. Definitions
The following definitions are provided to facilitate an understanding of the present invention.
As used herein, a “reporter gene” encodes a molecule whose expression may be assayed; such genes include, without limitation, β-galactosidase (LacZ), alkaline phosphatase, amino acid biosynthetic genes, e.g., the yeast LEU2, HIS3, or LYS2 genes, nucleic acid biosynthetic genes, e.g. URA3 or ADE2 genes, the chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (GFP), red fluorescent protein such as DsRd or any surface antigen gene for which specific antibodies are available. Additionally “reporter gene” may encompass any gene of interest whose expression may be detected. The term “reporters” may be used broadly to include systems where expression is detected based on ligand binding induced alterations in receptor structure. Such alterations in receptor structure may be assessed using a variety of methods well known to those of skill in the art, and include without limitation, assessing degradation, recycling, and molecular association rates of ligand bound receptor based on gain or loss of a detectable label. Such labels may be radioactive, fluorescent or chemiluminescent.
A “promoter” is a DNA sequence located proximal to the start of transcription at the 5′ end of an operably linked transcribed sequence. The promoter may contain one or more regulatory elements or modules that act together in coordinating and regulating transcription of the operably linked gene. An inducible promoter is a promoter that responds to the presence of different biochemical stimuli. Such promoters include, but are not limited, to the CUP1 promoter, heat shock promoters, galactose-inducible promoters, glycolytic promoters such as alcohol dehydrogenase (ADH) glyceraldehyde phosphate dehydrogenase (GPD) and the like.
“Operably linked” describes two macromolecular elements arranged such that modulating the activity of the first element induces an effect on the second element. In this manner, modulation of the activity of a promoter element may be used to alter and/or regulate the expression of an operably-linked coding sequence. For example, the transcription of a coding sequence that is operably linked to a promoter element is induced by factors that “activate” the promoter's activity; transcription of a coding sequence that is operably-linked to a promoter element is inhibited by factors that “repress” the promoter's activity. Thus, a promoter region is operably-linked to the coding sequence of a protein if transcription of such coding sequence activity is influenced by the activity of the promoter.
“Fusion construct” refers generally to recombinant genes which encode fusion proteins. Such fusion constructs may include operably linked nucleic acids isolated from two different genes.
A “fusion protein” is a hybrid protein, i.e., a protein that has been constructed to contain domains from at least two different proteins. An exemplary fusion protein, as described herein is a hybrid protein which possesses (a) a transcriptional regulatory domain from a transcriptional regulatory protein, or (b) a DNA binding domain from a DNA binding protein linked to a heterologous protein to be assayed for interaction. The structure of the fusion protein is such that the transcriptional regulatory domain and the DNA binding domain are arranged in a manner that allows both domains to be biologically active. The protein that is the source of the transcriptional regulatory domain is different from the protein that is the source of the DNA binding domain. In other words, the two domains are heterologous to each other.
The transcriptional regulatory domain of the fusion protein may either activate or repress transcription of target genes, depending on the native biological activity of the domain.
The term “fusion protein gene” refers to a DNA sequence that encodes a fusion protein. A fusion protein gene may further provide transcriptional and translational regulatory elements for the transcriptional and translational control thereof.
“Expression” is the process by which the information encoded within a gene is revealed. If the gene encodes a protein, expression involves both transcription of the DNA into mRNA, the processing of mRNA (if necessary) into a mature mRNA product, and translation of the mature mRNA into protein.
A nucleic acid molecule, such as a DNA or gene is said to be “capable of expressing” a polypeptide if the molecule contains the coding sequences for the polypeptide operably linked to expression control sequences which, in the appropriate host environment, facilitate transcription, processing and translation of the encoded genetic information into a protein product.
The phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given reference sequence. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
As used herein, a “cloning vector” is any entity that is capable of delivering a nucleic acid sequence into a host cell for cloning purposes. Examples of cloning vectors include plasmids or phage genomes. A plasmid which replicates autonomously in a host cell is especially preferred. Alternatively, a nucleic acid molecule which stably integrates into the host cell's chromosomal DNA and is inherited by daughter cells may be employed. Optionally, such vectors include a number of endonuclease recognition sites to facilitate manipulation of the sequence in a controlled and targeted fashion. Cloning vectors of the invention may also comprise sequences conferring resistance to selection agents, often referred to herein as selectable marker genes. For example, “a marker gene” may be a gene which confers resistance to a specific antibiotic on a host cell.
As used herein, an “expression vector” is a vehicle or vector similar to the cloning vector but is especially designed to provide an environment that facilitates expression of the cloned gene product after transformation into the host. Such vectors contain regulatory elements for expression in prokaryotic and/or eukaryotic hosts as well as sequences conferring selection properties of cells containing the expression vector. Optionally, enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites may be included.
A “host” refers to any organism or cell line that is the recipient of a cloning or expression vector. In preferred embodiments, the host of the invention is a yeast cell or a cultured animal cell such as a mammalian or insect cell. Especially preferred is the yeast host Saccharomyces cerevisiae. 
A “transformed cell” is any cell into which (or into an ancestor of which) exogenous DNA has been introduced by means of recombinant DNA techniques.
“Ligands” are small compounds such as chemical molecules or small peptides that are able to bind to the target proteins (for example, the receptor heterodimers described herein). By interaction with a target protein, ligands change conformation of the protein and thereafter activate or inactivate the protein.
“Response elements” are specific DNA sequences located in promoters of inducible genes; such inducers may include chemicals, hormones, metals such as zinc, cadmium or copper, heat shock and transcription factors. Nuclear receptors in the form of homodimers, heterodimers or monomers bind specifically to DNA response elements to activate or repress transcription of the targeted genes in the presence or the absence of ligands for the nuclear receptors.
II. Methods and Compositions for Gender Sorting
Cost effective methods for sex segregation of avian eggs require a sex specific target which can be detected at 98% accuracy, at a rate of 10,000 to 20,000 eggs per hour, and can be offered to the poultry industry at a cost of less than 5 cents per egg sexed. Potential sex specific targets include: 1) differences in physiologic parameters such as heart; 11, 12); 2) presence of the W specific chromosome in females (13, 14, 15); 3) presence of anatomical differences such as gonads; and 4) chemical differences which may be present in the blood or extra-embryonic fluids of the avian embryo.
Methods that may be potentially useful for gender sorting are set forth in Table 1.







TABLE 1







Comparison of LifeSensors Gender Sorting


Sensors with Other Technologies
















Background
Ease of


Technology
Speed
Sensitivity
Cost
Interference
Automation





LifeSensor
yes
Yes
yes
no
yes


Biosensors
no
No
yes
no
yes


Electronic
yes
No
yes
yes
yes


Nose


NIR
yes
No
yes
yes
yes


Spectrometry


Ion Mobility
yes
No
yes
yes
yes


Spectroscopy


Fluorescent
yes
Yes
yes
yes
no


Polarization


Antibody
yes
Yes
no
no
yes


Based


Technologies


Photoacoustics
yes
No
yes
no
yes


Imprint
yes
No
yes
no
yes


Polymers


Mass
no
Yes
no
no
no


Spectrometry


Laser Induced
yes
No
yes
yes
yes


Fluorescent


Spectrometry









Numerous analytical techniques to detect estradiol or other sex specific compounds were evaluated. As can be seen from Table 1 only the LifeSensors technology described herein meets the desired criteria of cost, sensitivity, speed, accuracy, safety and ease of application to a commercial hatchery environment.
Sex specific chemicals present in blood or extra-embryonic fluids provide optimal analytical targets for meeting the above criteria. Suitable chemical based targets are male or female-specific hormones. It is well established that male and female embryos exhibit significant differences in hormone levels in sera and in male or female gonadal tissue culture systems (16, 17, 18). The data provided in these studies suggested that differences in the estrogen levels of male and female embryos would be greater than differences in the testosterone levels.
Differences in sex hormone levels are also evident in the allantoic fluid. In 1983, Gill and coworkers reported the presence of estrone sulfate, estradiol-17β sulfate, estradiol-17α sulfate, estrone glucuronide, estradiol-17α glucuronide and estradiol-β glucuronide in pg/ml quantities in the allantoic fluid of female embryos but not in the allantoic fluid of male embryos. No estrogens were present in the amniotic fluid of either sex (19). The amniotic fluid is primarily maternally derived and acts as a nutrient source and a cushion from mechanical and thermal insults.
The present invention is directed to methods for detecting the presence of sex specific estrogen levels in the chorioallantoic fluid of avian eggs using a yeast-based genetic system. The use of chorioallantoic fluid is preferred over the use of either blood or amniotic fluid as the allantoic fluid is proximal to the outside of the egg and is not in direct contact with the fluid surrounding the embryo. The allantois is separated from the eggshell only by the inner and outer shell membranes and the chorioallantoic membrane. Although the allantoic sac containing the allantoic fluid encompasses the entire periphery of the embryonated egg, the allantoic fluid accumulates at the top and upper sides of the egg directly underneath the membranes overlying the air space. The accumulation at the top of the egg is due to gravity and displacement by the dense embryo and yolk sac.
The allantoic fluid is recognized as an excretory medium for the nitrogenous metabolites of the embryo (20). The allantoic fluid begins to form around Day 5 of incubation. It attains a maximum volume of 6.1 mls on Day 13 of incubation, wanes in volume as incubation continues due to moisture loss and fluid resorption but is still present in significant volumes (1 to 2 mls) on Day 18 of incubation (20). The accumulation of solids in the allantoic fluid is continuous throughout the developmental period. The presence of significant amounts of allantoic fluid, which is readily accessible, between Days 13 and 18 of incubation render the allantois a feasible target for detection of a sex specific chemical.
It should also be noted that the estrogen present in allantoic fluids is not an active form, as it exists in the form of conjugates with either glucuronides or sulfates. While immunoassay is available for detecting conjugated estrogen levels, the method is not extremely sensitive nor, more importantly, cost-effective.
The presence of estrogens in the allantoic fluid of Day 17 embryos has been further investigated in efforts to assess the reliability of estrogens as a sex specific chemical marker. The estradiol levels in the allantoic fluid of male and female Day 17 broiler chicken embryos were measured using a commercially available RIA kit (Diagnostic Products, Los Angeles, Calif.).
The estradiol level in the allantoic fluid of male embryos was either non-detectable or less than 42 pg/ml. The estradiol level in the allantoic fluid of female embryos was between 184 and 830 pg/ml, at least four times higher than the levels detected in male embryos. These data indicate that estrogen conjugates can be used to segregate female from male embryos.
The presence of testosterone in the allantoic fluid has also been investigated as androgens may interfere with analysis of estradiol due to similarity in molecular structures. Studies have shown that testosterone levels were undetectable in the allantoic fluid of Day 17 broiler embryos using a commercial RIA kit (Diagnostic Products, Los Angeles, Calif.).
Studies were also conducted to assess the optimal period of embryonic incubation for estradiol detection using the analytical methods described herein. Estradiol was initially detected by our methods on Day 13 of incubation and coincided with reports by Gill and coworkers (19). As shown herein, estrogen levels peaked at Day 17 of incubation.
In accordance with the present invention, a yeast-based genetic system and methods of use, thereof are provided to facilitate gender sorting in poultry. The methods provided herein enable the rapid and efficient sorting of eggs based on the presence of sex hormones in allantoic fluid.
II. Preparation of Nucleic Acid Molecules Encoding the Proteins of the Invention and Uses Thereof in Assay Methods and Kits
A. Nucleic Acid Molecules
Nucleic acid molecules encoding the expression vectors of the invention may be prepared by two general methods: (1) They may be synthesized from appropriate chemical starting materials, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.
The availability of nucleotide sequence information, for the estrogen receptor, as well as for secretory signals from alpha-mating factor or yeast SUC2 gene facilitates synthesis of DNA constructs containing such sequences. Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule encoding a construct of the present invention, must be synthesized in stages due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, a 3 kilobase double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be ligated such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct the entire 3 kilobase double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector. In alternative embodiments of the invention, the human estrogen receptor α (referred to herein as “estrogen receptor”), reporter gene and E. coli gene for expression of glucuronidase can be substituted with similar genes from other biological sources. Suitable candidate genes for such substitution include, without limitation, estrogen receptor β which also has high affinity for the hormone and does mediate transactivation in eukaryotic cells in response to 17β estradiol. In addition, one can replace human estrogen receptor with a homologous receptor molecule from mouse or other higher eukaryotic estrogen receptors. The E. coli beta-glucuronidase gene can be replaced with a snail gene that expresses glucuronidase, or any other glucuronidases that can cleave the 17β estradiol glucuronides. For example, the Lactobacillus gasseri gusA gene with a pH optima of 5 also provides a suitable candidate GUS gene (Russell and Klaenhammer, 2001). Since the pH of extracellular yeast media is acidic, this enzyme is well suited for use in the gender sorting sensor as an extra step to adjust the pH can be omitted.
Estrogen conjugates are cleaved by β-glucuronidases to produce 17β estradiol. The pure estrogen molecule binds to the resident estrogen receptor to transactivate a reporter gene. In accordance with the present invention methods for assessing estrogen levels in allantoic, or amniotic fluid or any other biological or chemical estrogens and their ability to transactivate the reporter genes described herein are provided. The constructs containing the estrogen receptor encoding sequences described herein may be substituted either with mutated (modified or altered receptor) or a receptor from another species.
Similarly, the glucuronidase enzyme of the invention may be derived from different species. The E. coli β-glucuronidase has been used in the studies described herein. However, β-glucuronidases from rat or human may have altered properties that may make them more robust and efficient than the E. coli glucuronidase. The use of glucuronidase from Lactobacillus gusA (GenBank Accession number: AF305888) is also encompassed within the scope of the present invention. In addition to estrogen glucuronides, biological fluids may contain other forms of modified estrogens such as estrogen sulfates. Under these circumstances appropriate de-conjugating enzymes such as sulfatases can be employed to convert the estrogen conjugate into 17 β estradiol. Thus, in this situation, glucuronidases can be replaced with sulfatases.
Nucleic acid sequences encoding the components of the expression plasmids of the invention may be isolated from appropriate biological sources using methods known in the art. For example, RNA isolated from a mammalian or insect cell may be used as a suitable starting material for the generation of cDNA molecules encoding the different receptor proteins.
In accordance with the present invention, nucleic acids having the appropriate level of sequence homology with the protein coding region of the DNA molecules of the present invention may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, using a hybridization solution comprising, for example, 5×SSC, 5× Denhardt's reagent, 1.0% SDS, 100 mg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37–42° C. for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes at room temperature in 2×SSC and 0.1% SDS; (3) 30 minutes-l hour at 37° C. in 1×SSC and 1% SDS; (4) 2 hours at 42–65° C. in 1×SSC and 1% SDS, changing the solution every 30 minutes.
One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is as follows (21):

Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/#bp in duplex

As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1–1.5° C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42° C. Such a sequence would be considered substantially homologous to the sequences of the present invention.
Nucleic acids encoding the fusion proteins of the invention may be maintained as DNA in any convenient cloning vector. In one embodiment, clones are maintained in plasmid cloning/expression vectors, such as pRS plasmids series (27), or YEpE12 derivative plasmids (26, 28). pBluescript plasmids (Stratagene, La Jolla, Calif.) or recombinant baculovirus transfer vector plasmids, such as pFastBac vectors (Gibco-BRL, Gaithersburg, Md.) that are propagated in insect and E. coli host cells, may also be employed.
The nucleic acids of the invention may also be used as starting materials for the generation of sequence variants or truncation mutants of the nucleic acids of the invention using any number of synthetic and molecular biologic procedures well known in the art including, but not limited to, truncation at available restriction sites and site-directed mutagenesis techniques. Particular mutations may give rise to receptor proteins with altered characteristics such as increased or decreased ligand binding activity.
B. Fusion Proteins
In one embodiment of the invention, the estrogen receptors of the invention are expressed in yeast as ubiquitin fusion proteins. Ubiquitin fusion enhances but is not necessary for protein expression in yeast. After translation of recombinant proteins, the 76 amino acids of ubiquitin sequence in the N-terminus are cleaved by the host ubiquitin pathway and native proteins are released. It is widely known that the presence of the ubiquitin sequence improves the expression of proteins of interest in yeast and E. coli (26, 29).
Attachment of SUMO, a 101 amino acid protein, to the N-terminus of GUS also enhances the expression and extracellular secretion of the protein into the media. SUMO is a homologue of ubiquitin that is conjugated to other proteins via the epsilon amino groups of lysine (Yeh et al 2000, Mahajan et al 1997). The Smt3 gene in yeast encodes for SUMO (Li and Hochstrasser 2000). Similar to the case with ubiquitin conjugation and de-conjugation to other proteins, SUMO can be cleaved from the fusion protein by SUMO specific proteases (Li and Hochstrasser 2000, Kim et al 2000). Therefore, SUMO-fusion proteins can also be expressed and cleaved in eukaryotic and prokaryotic cells. Since SUMO protein or SUMO enzymes are absent in prokaryotes, sumolated proteins expressed in E. coli are not cleaved in the absence of SUMO specific hydrolase or protease. In eukaryotic cells, such as yeast, a SUMO-GUS fusion is cleaved by cellular SUMO specific hydrolases (Suzuki et al 1999, Kim et al 2000, Li and Hochstrasser 2000).
The glucuronidase enzymes of the invention are expressed in yeast after attachment of secretory signals and as ubiquitin fusion protein(s) as well. The attachment of the secretory signals in the N-terminal region of glucuronidase or ubiquitin facilitates extracellular secretion of the enzyme following translation. A wide variety of secretion signal sequences are available, including but not limited to, the N-terminus of alpha factor or SUC2. FIG. 5 shows an exemplary plasmid that was robust in secreting E. coli GUS in yeast. Similar plasmids may be generated containing glucuronidase genes from other species, under different secretory signals using different promoters and different hosts to produce active GUS that can cleave the estrogen-glucuronides conjugates.
In a preferred embodiment of the invention, nucleic acid sequences encoding the N-terminus of glucuronidase were operably linked at the C-terminus to a ubiquitin gene. The presence of the ubiquitin moiety at the N-terminus of glucuronidase facilitated and enhanced secretion of the enzyme. See Table 8.
In an alternative embodiment of the invention, the fusion proteins may be prepared using in vitro expression methods known in the art. In this fashion, glucuronidase may be supplied exogenously to the transactivating system of the invention. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such as pSP64 or pSP65 for in vitro RNA synthesis, followed by cell-free translation of the RNA in a suitable cell-free translation system, such as extracts of wheat germ, rabbit reticulocytes or HeLa cells. In vitro transcription and translation systems are commercially available (e.g., Promega Biotech, Madison, Wis.; Gibco-BRL, Gaithersburg, Md.).
C. Assay Methods and Kits
In yet another embodiment of the invention, assays are provided wherein intact cells expressing the proteins of the invention are contacted with biological samples containing molecules suspected of affecting the intracellular activity of the estrogen receptor. After a suitable time period, the effects of such agents on estrogen dependent transactivation activity are measured. Such activity may be quantitated in any number of ways. For example, such cell systems may utilize a reporter system in which the production of the reporter signal is dependent on estrogen dependent transactivation. Numerous reporters may serve equally well in this application including but not limited to, beta-galactosidase, alkaline phosphatase, fluorescent green protein and the like. Furthermore, the methods of the invention may be practiced in bacterial, fungal, insect, avian, mammalian or plant cells. However, yeast-based cell systems are preferred due to low cost and the feasibility of growing yeast cells in plastic devices.
Assays for screening estrogen-containing biological fluid samples are also provided. Assays involving the cell-based systems of the invention may be formatted in any number of configurations. Particularly useful for evaluating large numbers of agents and materials are high throughput screening formats. Traditionally such assays were typically formatted in 96 well plates. However, 384, 864 and 1536 well plates or a custom designed plate may be used in such high throughput assay systems. These systems are often automated using robotics technologies to allow manipulation and processing of large numbers of samples.
According to another aspect of the invention, antibodies immunologically specific for various proteins may be used in the gender sorting assays of the present invention. Such antibodies may be monoclonal or polyclonal. Additionally, antibody fragments having binding affinity for various proteins may also be used to advantage in the gender sorting assays. Such antibody fragments comprise Fab, Fab′, F(ab′)2, F(v) and Sfv generated recombinantly. Such antibodies or fragments thereof may be used to advantage in the methods and kits of the present invention as described below.
In another aspect, the invention includes kits to facilitate the use of the compositions and methods disclosed herein. Exemplary kits include the expression plasmids and yeast strains of the invention, and/or variants thereof. Also provided are beta-glucoronidase enzymes or sulfatase enzymes or plasmids encoding the same. Protocols for use of the compositions of the invention for the particular application and the necessary reagents to carry out the application are also provided. Such reagents may include, but not be limited to, buffers, solvents, media and solutions, substrates and cofactors, vectors and host cells, and detection or reporter reagents. Accessory items may include vials, vessels, reaction chambers and instruction sheets.
The following protocols are provided to facilitate construction of the expression plasmids for use in the methods and kits of the present invention.
Yeast Media, Strains and Plasmids
Standard yeast media were prepared as described (22). YPD composed of Yeast Extract (20 grams per liter), Peptone (20 g/liter), Dextrose (20 g/Liter) is preferred for use in the invention as most yeast strains grow in this media. Yeast selective media (complete-drop out) used to maintain plasmids composed of yeast nitrogen base 1.7 g/L, ammonium sulfate 5 g/L, dextrose 20 g/L, different amino acids and other supplements were added depending on the requirements of the particular yeast strain. For example if the yeast plasmid contains LEU2 marker, the leucine is dropped out from the media in order to select the plasmid.
Yeast strains suitable for use in the present invention include the yeast strain Y4727, snq2::Δ pdr5::kanMX: Matα his3-Δ200 leu2-Δ0 lys2-Δ0 met5-Δ0 trp1-A63 ura3-Δ0 snq2::Δ pdr5::kanMX (original strain Y4747 was a gift from Dr. Jeff Boeke) BJ1991 Mat α prb1-1122 pep4-3 leu2 trp1 ura3-52 gal2 (a gift from Dr. Beth Jones), and RS188: MATa leu2-3,112 trp1-1 ura3-1 ade2-1 his3-11,15 can1-100,
Construction of the β-Glucuronidase Expression Plasmids
A series of yeast expression plasmids have been constructed, all of which are yeast-E. coli multicopy shuttle plasmids, containing LEU2 as a yeast transformation marker. See Table 8. FIG. 5 depicts an exemplary plasmid of the invention that is suitable for GUS production in our assay—plasmid pRS425-GPDp-UB-GUS is comprised of plasmid pRS425 (23) E. coli GUS from reference 24, human ubiquitin (amplified from plasmid YEpE12; Graumann et al., 1996) and S. cerevisiae GPD promoter (plasmid pRS423GPDp was described in Mumberg et al., 1995). See FIG. 4 herein. However, as described herein, additional plasmids for Gus expression were constructed and tested. Those plasmids include pRS425-ADH1p-GUS, pRS425-ADH1p-α-factor-GUS1, pRS425-GPDp-Ub-GUS1, pRS425-GPDp-Ub-α-factor-GUS1, pRS425-GPDp-α-factor-Ub(pro)-GUS1, pRS425-GPDp-α-factor-Ub(met)-GUS1 and pRS425-CUP1p-Ub-GUS1. pRS425-GUS1 was produced by cloning the XhoI-SacI fragment (containing E. coli β-Glucuronidase from plasmid pGUS1 (24) into the XhoI-SacI sites of plasmid pRS425 (23). The next construction involved addition of a promoter, and giving rise to plasmid pRS425-ADH1p-GUS. An XhoI-HindIII fragment containing the ADH1 promoter was inserted into the XhoI-HindIII sites of the plasmid pRS425-GUS. The ADH1 promoter XhoI-HindIII fragment was cloned using polymerase chain reaction (PCR), followed by amplification of the ADH1 promoter from the plasmid pGRIP1 (25). The following primers were used to amplify the full length ADH1 promoter: ADH1-XhoI: 5′-gctcgagagcacagatgcttcgttg-3′ (SEQ ID NO: 1) and ADH1-HindIII: 5′-gcaaagcttggagttgattgtatgc-3′ (SEQ ID NO: 2). The underlining indicates the nucleotide sequence of the XhoI and HindIII restriction sites. PCR of the DNA fragment involved amplification in 30 cycles (96° C.—30 sec., 54° C.—1 min. and 72° C.—3 min.) using high replication fidelity Deep Vent Polymerase (New England Biolabs). The PCR product was then digested with XhoI and HindIII, and subsequently cloned into the XhoI-HindIII sites of pRS425-GUS. pRS425-ADH1p-α-factor-GUS, comprises a secretory signal fused in frame with the N-terminus open reading frame of GUS. Nucleic acid sequences encoding the alpha factor signal from plasmid pPIC9 (Invitrogen) were amplified by PCR using the primers alpha-HindII (forward): 5′-acc aag ctt att cga agg atc caa acg atg ag-3′ (SEQ ID NO: 3) and alpha-HindIII (reverse): 5′-gcc aag ctt gga gcc tct ctt ttc tcg aga g-3′ (SEQ ID NO: 4). Again the underlining indicates the nucleotide sequence of the restriction HindIII site. The PCR product was digested with Hind III and subsequently cloned into the Hind III site of pRS425-ADH1p-GUS, in frame with the open reading frame of GUS. Similarly the plasmids pRS425-GPDp-Ub-GUS1, pRS425-GPDp-Ub-α-factor-GUS1, pRS425-GPDp-α-factor-Ub(pro)-GUS1, pRS425-GPDp-α-factor-Ub(met)-GUS1 were constructed using plasmids pRS425-GPDp as an original vector. The ubiquitin coding DNA fragment was amplified from YEpE12 (Graumann et al., 1996). The plasmid pRS425-CUP1p-Ub-GUS1 was constructed by replacing the GPDp-Ub fragment from plamsid pRS425-GPDp-Ub-GUS1 with CUP1p-Ub fragment derived from plasmid pRS425-CUP1-Ub-CfUSP (Tran et al., 2001).
Yeast Transformation
Briefly, yeast cells were cultured in 5 mL of YPD liquid medium overnight at 30° C. The cell culture was diluted 1:20 in YPD media and further grown until cell density reached an O.D of 0.6. at OD600. The cells were pelleted by centrifugation and washed with 10 mL of 10 mM Tris-Hcl pH 8.00, 1 mM EDTA (TE) containing 0.1M lithium acetate. Pelleted cells were resuspended in 10 mL of TE plus 0.1M lithium acetate and incubated at 30° C. for 1 hour. Cells were collected and resuspended in 0.5 mL of TE plus 0.1M lithium acetate. Aliquots of cells (50 μL), were incubated for 15 minutes at 30° C. with 1–2 μg plasmid DNA in the presence of 5 μg of denatured salmon sperm DNA as DNA carrier. In the case of co-transformation, the indicated plasmids were added simultaneously. 50% polyethyleneglycol of molecular weight 4000, diluted in TE, was added to a final concentration of 35%. DMSO was added to the transformation solution simultaneously to a final concentration 3%. The samples were mixed well by vortexing and incubated for an additional 30 minutes at 30° C. The cell mixture was then heated for 15 minutes at 42° C. Cells were collected and plated onto appropriate yeast selective media. Transformants of 4 plasmids were selected on complete media without tryptophan, uracil, leucine or histidine.
A. Accurate and Efficient Estrogen Transactivation as assessed by β-Galactosidase Activity Assays using o-nitrophenyl-beta-D-galactopyranoside (ONPG) as a substrate.
Yeast cells containing yeast expression plasmids for human estrogen receptor and estrogen-inducible reporter gene were grown overnight in selective liquid media at 30° C. and were diluted in pre-warmed liquid selective media to 0.1 at OD600 (ODculture). See Lyttle et al., 1992. 100 μl of the cell culture was spiked into each well of a 96-well microtiter plate. Ligand (2 μl diluted in DMSO) was added to each well to produce a final concentration of 2% DMSO. As a control, 2 μl of DMSO were also added to additional test wells. The final concentration of 17-β estradiol ranged from 100 pM to 10 nM.
The cells were incubated in the presence of the ligand in a shaker at 30° C. After 4 hours of incubation, 100 μl of 2×“Z” Sarcosine-ONPG buffer (120 mM Na2HPO4, 80 mM NaH2PO4, 20 mM KCl, 2 mM MgSO4, 100 mM beta-mercaptoethanol (Sigma), pH 7.0, 0.4% lauroyl sarcosine (Sigma), 4 mg/mL ONPG (Diagnostic Chemicals limited) was added to each well and the plate was further incubated at 37° C. The 2×“Z” Sarcosine-ONPG buffer was freshly prepared or stored at −20° C. prior to use. After incubation at 37° C. for 1 hour, the reaction was stopped by adding 100 μL of quenching solution 0.5 M Na2CO3 and the OD405 (ODreaction) was determined. Beta-galactosidase activity was measured in a micro plate reader (Biotek) at a wave length of 405–450 nanometers. The relative activity of β-galactosidase was calculated as follows:

(1000×ODreaction)/(tincubation×ODculture).











“Z” Buffer



















 16.1 g
Na2HPO4 7H2O
(60 mM)



 5.5 g
NaH2PO4 H2O
(40 mM)



 0.75 g
KCl
(10 mM)



0.246 g
MgSO4 7H2O
(1 mM)



 2.7 ml
beta-mercaptoethanol
(50 mM)










B. Accurate and Efficient Gender Sorting of Allantoic fluids, using Exogenously supplied GUS-ONPG as β-Galactosidase substrate.
The present assay is similar to the estrogen-induced transactivation assay described above. However, in this assay, estrogen-conjugates present in allantoic fluids from 17-day incubated eggs are tested. Unless present at very high concentrations (approximately 10 micromolar (μM)) estrogen conjugates themselves do not induce receptor transactivation because of the low affinity of the estrogen receptor for such conjugates. This concentration greatly exceeds that present in allantoic fluids which is approximately 200 pM. Thus, the present assay consists of two steps: a) release of estrogen from conjugated forms present in allantoic fluids by Glucuronidase (from snail juice, Sigma, cat. #G7017); and b) assessing the released hormone for its ability to stimulate production of the reporter gene in the yeast based transactivation assay.
Initially, allantoic fluids are exposed to beta-glucuronidase. 75 ul of each allantoic fluid sample was placed into a microcentrifuge tube. To each tube 40 ul of 0.2 M Acetate Buffer pH 4.6 was added. 20 ul of beta-glucuronidase from snail (Sigma cat. #G7017) was added and the entire reaction mixed well. The reactions were incubated at 37° C. for 1 hour. Beta-glucuronidase will cleave the estrogen-conjugates in allantoic fluids thereby releasing free estrogen. In order to inactivate the GUS enzymes, the reactions were heated at 95° C. for 5 minutes. The GUS heat inactivation step is required when snail GUS is used as the snail GUS contains other enzymes that also cleave ONPG. Of note, when purified E. coli GUS was used (Sigma #G-7396), the GUS inactivation step was not required because E. coli GUS does not interfere with ONPG cleavage. The released estrogen was utilized in a transactivation assay in yeast. Yeast cells containing yeast expression plasmids for human estrogen receptor (YEpE12) and an estrogen-inducible reporter gene (YRpE2) were grown overnight in selective liquid media at 30° C. and diluted in pre-warmed liquid selective media to a density of 0.1 at OD600 (ODculture). 100 μl of cell culture was spiked into each well of a 96-well microtiter plate. Four to eight μl of the GUS-treated allantoic fluids were added to the yeast cell suspension. The cells were incubated in the presence of the ligand in a shaker at 30° C. After 4 hours of incubation, 100 μl of 2×“Z” Sarcosine-ONPG buffer (120 mM Na2HPO4, 80 mM NaH2PO4, 20 mM KCl, 2 mM MgSO4, 100 mM beta-mercaptoethanol (Sigma), pH 7.0, 0.4% lauroyl sarcosine (Sigma), 4 mg/mL ONPG (Diagnostic Chemicals limited) was added to each well and the plate was further incubated at 37° C. The 2×“Z” Sarcosine-ONPG buffer was freshly prepared or stored at −20° C. prior to use. After incubation at 37° C. for 1 hour, the reaction was stopped by adding 100 μL of quenching solution 0.5 M Na2CO3 and the OD405 (ODreaction) 25 determined. Beta-galactosidase activity was measured in a micro plate reader (Biotek) at a wave length of 405–450 nanometers (nm) as described above.
Preferably, the estrogen source for analysis is obtained from the allantoic fluid of the egg that is treated with beta-glucuronidase enzyme. β-glucuronidase may be exogenously supplied or endogenously produced from yeast cells harboring a Gus expression plasmid.
C. Accurate and Efficient Gender Sorting using Estrogen-conjugate containing Allantoic Fluids, Endogenous GUS-ONPG as β-Galactosidase substrate.
Yeast cells BJ1991 containing yeast expression plasmids for human estrogen receptor (YEpE12; See Graumann et al., 1996) and estrogen-inducible reporter gene (YRpE2) and a vector for GUS expression (pRS425-GPDp-Ub-GUS1) were able to grow in standard synthetic minimum medium (20 g glucose, 5 g ammonium sulfate, and 1.7 g yeast nitrogen base per liter). Notably, the optimal pH for E. coli GUS activity is 6.8 while yeast standard synthetic medium has a pH 5.2. Yeast cells were grown overnight in special selective liquid media (SSLM, standard liquid media mix with 10×NEB4 buffer in 9:1 ratio, pH 6.4) at 30° C. and were diluted in pre-warmed SSLM to 0.2 at OD600 (ODculture). 80 μl. of the cell culture was spiked to each well of a 96-well microtiter plate. 20 ul of allantoic fluid from 17-day incubated eggs were added to the wells. As controls, 20 μl of H2O or 17-β estradiol (1 nM) were added to additional test wells. The cells were incubated in the presence of the ligand in a shaker at 30° C. After 4 hours of incubation, 100 μl of 2×“Z” Sarcosine-ONPG buffer [120 mM Na2HPO4, 80 mM NaH2PO4, 20 mM KCl, 2 mM MgSO4, 100 mM beta-mercaptoethanol (Sigma), pH 7.0, 0.4% lauroyl sarcosine (Sigma), 4 mg/mL ONPG (Diagnostic Chemicals limited] was added to each well and the plate was further incubated at 37° C. for 1 hour. The 2×“Z” Sarcosine-ONPG buffer was freshly prepared or stored at −20° C. prior to use. After incubation at 37° C. for 1 hour, the reaction was stopped by adding 100 μL of quenching solution 0.5 M Na2CO3 and the OD405 (ODreaction) determined. Beta-galactosidase activity was measured in a micro plate reader (Biotek) at a wave length of 405–450 nanometers as previously described.
















NEB4 (10 × Buffer)
500 mM Potassium acetate




200 mM Tris-Acetate




100 mM magnesium acetate




pH 7.










For some applications, it may be advantageous to use MP medium, which can be packed as a dry powder, as a substitute for SSLM (standard liquid medium mix with 10×NEB4 buffer in 9:1 ratio, pH 6.4). MP is combination of standard synthetic medium with potassium acetate at a final concentration of 50 mM. MP medium has a similar buffering capacity to that of SSLM medium, but it may be prepared and shipped as a dry powder.
D. Accurate and Efficient Gender Sorting of Allantoic Fluids, using Exogenously supplied E. coli GUS added directly into the assay media, YRpE2SSA4-lacz as a reporter and ONPG as the β-Galactosidase substrate.
For applications in which the gender sorting assays of the present invention are used in large scale operations, the following modifications may be used to advantage. These modifications render the gender sorting assays even more robust and, therefore, amenable to high throughput processes. Accordingly, an additional reporter has been constructed which utilizes the rapid response, inducible heat shock promoter SSA4. The present inventors have also discovered that the use of MP medium, which can be packed as a dry powder, eliminates the need for supplementary beta-mercaptoethanol in the “Z” buffer (see below). The modified assay is similar to the estrogen-induced transactivation assay described above. In this assay, however, estrogen-conjugates present in allantoic fluids from Day 17 incubated eggs were tested.
Briefly, yeast strain BJ1991 comprising yeast expression plasmids expressing the human estrogen receptor (YEpE12) and an estrogen-inducible reporter gene (YRpE2SSA4-lacZ; FIG. 3B) were grown overnight in MP selective liquid media at 30° C., after which the overnight cultures were diluted in MP liquid selective media to an OD600 of 0.2. Purified E. coli GUS (Sigma #G-7396) was added to the diluted yeast cell culture to a final concentration 1 unit per 100 μl. Eighty microliters of the yeast cell culture was spiked to each well of a 96-well microtiter plate. To each well, 20 μl of allantoic fluid from Day 17 incubated eggs were added. The yeast cell/allantoic fluid mixture was incubated at 30° C. for 1 hour, after which 100 μl of 2×“Z” Sarcosine-ONPG buffer without beta-mercaptoethanol (120 mM Na2HPO4, 80 mM NaH2PO4, 20 mM KCl, 2 mM MgSO4, pH 7.0, 0.4% lauroyl sarcosine, 4 mg/mL ONPG) was added to each well and the plate was further incubated at 37° C. The 2×“Z” Sarcosine-ONPG buffer was freshly prepared or stored at −20° C. prior to use. After incubation at 30° C. for 30 minutes reaction colors were determined using a CCD camera to measure the number of pixels in each wells.
EXAMPLE I
17 β-estradiol Dependent Transactivation in Yeast Cells Expressing Estrogen Receptor
In accordance with the present invention, compositions and methods are provided for pre-hatch gender sorting in avian eggs. It has previously been demonstrated that the concentration levels of estrogens in the conjugated form is higher in female allantoic fluids as compared to male allantoic fluids (19) and FIG. 1. Thus, this fluid provides ideal starting material for gender sorting of chicken eggs which is based on estrogen concentration levels.
Using several classic methods (radioimmunoassays, etc), one can determine the concentration of estrogens in different biological samples. However, these methods are cumbersome, expensive and not readily adaptable to high throughput formats. The present method described and claimed herein provides robustness, cost-effectiveness and is 100% accurate, thereby satisfying industrial requirements.
As shown in Table 2, the transactivation system of the invention responds only to a specific ligand for the estrogen receptor, 17-β estradiol. Other ligands (Table 2) have very little transactivating activity. Both estrone sulfate and estrone glucuronide have very little to no effect on estrogen transactivation. Furthermore, the yeast transactivation assay is 100-fold more sensitive to 17-β estradiol when compared to mammalian cell assays and no transactivation was observed with the indicated concentration of estrogen glucuronides. The data in the following Table 2 demonstrate that the human estrogen receptor-driven yeast sensor is extremely sensitive and selective for 17-β Estradiol.







TABLE 2







Specificity and selectivity of estrogen sensor. Cross-activity was


determined at concentration of 50% maximum potency.










Compound
Percent Cross Reactivity*














17-β Estradiol
100



Estrone
8



16-Hydroxyestrone
4.0



Estriol
0.4



Estrone Sulfate
<0.25



Estrone Glucuronide
<0.25



2-Methoxyestrone
<0.25



4-Hydroxytamoxifen
<0.25



Androstenediol
0.08



Testosterone
<0.05



Dihydrotestosterone
<0.05



Mesterolone
<0.05



Dehydroepiandrosterone
<0.05



Androstenedione
<0.05



Androsterone
<0.05



Progesterone
<0.025



17-Hydroxyprogestreone
<0.025



Danasol
<0.025



Corticosterone
<0.025



Hydrocortisone
<0.025










EXAMPLE II
Treatment of Estrogen Conjugates from Allantoic Fluids with Exogenous GUS Followed by Yeast Transactivation Assay
A two step treatment of allantoic fluids with exogenous GUS [snail juice GUS (Sigma #G-7017) or bacterial Gus (Sigma #G-7896)] and subsequent use of a yeast estrogen sensor to estimate the level 17β-estradiol released from the allantoic estrogen conjugates is described. A schematic diagram of the assay is shown in FIGS. 2A–2C. The yeast strain used in the present example has been transformed with a yeast expression vector for human estrogen receptor and an estrogen-inducible reporter gene. As described above, in the absence of GUS treatment, allantoic fluids do not induce transactivation of the estrogen biosensor. Accordingly, allantoic fluids were treated with Glucuronidase isolated from snail juice (Sigma cat#G-7017) or bacterial GUS (Sigma Cat #G 7896). Treatment of allantoic fluids with snail GUS has been described above. Treatment of allantoic fluids with bacterial GUS is described hereinbelow.
GUS Treatment
Removal of glucuronides was performed in a total reaction volume of 100 μL, containing 10 μL of NEB 3 buffer 10× (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothretol, pH 7.9) or NEB4 buffer (50 mM potassium acetate 20 mM Tris-Acetate, 10 mM magnesium acetate, pH 7), 50 μL of allantoic fluids, and 10 units of bacterial GUS enzyme. The final concentration of the estrogen-glucuronide conjugates was 1 μM. The reaction mixture was incubated at 37° C. for 1 hour; and stopped by heating at 95° C. for 5 minutes to inactivate the enzyme. After treatment with GUS enzyme, the 17β-estradiol is released from the conjugate. The reporter assays are then performed as follows:

    1. 4 to 8 μL of each GUS treated allantoic fluid reaction mix was spiked into the wells of a 96 well microplate.
    2. To each well 100 μL of yeast estrogen sensor cell suspension was added, incubated at 30° C. as described above in Example I.
    3. The activity of the reporter gene (beta-galactosidase) was measured by adding 100 μl of 2×“Z”-ONPG as described above.


Of note, purified glucuronidase isolated from E. coli (Sigma #G-7896), does not interfere with ONPG, and the heating step described in the previous example is required only when using snail juice GUS. Purified glucuronidase from E. coli was able to cleave estrogen conjugates with equal efficiency. However, the optimal pH of the reactions catalyzed by snail juice Gus and E. coli derived Gus differ significantly (pH 5.2 for snail juice and pH 6.8 for bacterial GUS). The data presented in Table 3 shows a comparison of conventional dissection of bird embryos for sex determination and the yeast based transactivation system of the invention using Gus treated estrogen conjugates isolated from allantoic fluids. As shown in Table 3, there is 100% agreement in sex determination using the two methods.







TABLE 3







Ability to gender sort allantoic fluid from


Day 17 Hyaline layer embryos











Optical



Gender
Density














M
.328



M
.332



M
.338



F
1.204



F
2.261



M
.352



F
2.664



F
2.424



F
2.300



F
2.057










The experimental protocol described above employs only 4 μL of allantoic fluid. The data shown in table 3 are based on the determination of estrogen levels after allantoic fluids from hyaline layer embryos were treated with snail β-glucuronidase and subsequently tested in β-galactosidase reporter assays as described above. Among 10 allantoic fluid samples tested, four of the allantoic fluids were from male embryos while six of the allantoic fluid samples were collected from female embryos. As shown herein, that female allantoic fluid samples showed activity equivalent to 4–8 nM estradiol in the undiluted allantoic fluid after β-glucuronidase treatment. The signal elicited by the allantoic fluid samples collected from male embryos was equal to that of the buffer control.
Sex Specific Signal Readily Determined Regardless of Egg Size or Flock Age from which Chicken Eggs are Derived.
Allantoic fluid samples were collected from eggs of a young flock, a flock in peak production and an old flock. The eggs from the young flock were small in size while the eggs from the old flock were large. The yeast-based estrogen sensor described above was used to determine the level of 17-β estradiol. The sex of the embryos was identified at 100% accuracy regardless of flock age. See Table 4.







TABLE 4







Effect of broiler flock age on ability to


gender sort allantoic fluid











Flock Age


Sex by
Optical
(Weeks in


ID
Density
Lay)












F
2.869
2


F
1.992
2


M
.384
2


F
2.676
2


F
4.000
2


M
0.460
24


F
2.845
24


M
.385
24


F
2.374
24


M
.441
24


M
.395
34


M
.341
34


F
2.319
34


F
2.405
34


M
.399
34









Thus, the data show that using yeast as a host, male and female eggs can be differentiated based on the level of estrogen receptor transactivation observed in the LifeSensorsTM assay. The robustness of the estrogen sensor was tested under different conditions, such as incubation time of eggs, chick species, contamination of blood in the allantoic fluids and the like. All of the experiments demonstrated that the yeast-based estrogen sensor is extremely robust and was successful in determination of the chicken gender 100% of the time.
The ability of the estrogen sensor was also evaluated to determine the sex of different chicken species. See Table 5. Five allantoic fluid samples from each of three different broiler breeds were tested for analysis. Arbor Acres×Classic Yield (AA×CY), Hubbard×Petersen (H×P), and Cobb×Cobb (C×C) strains were the three different breeds evaluated. The assay clearly distinguished between Day 17 male and female embryos regardless of breed. Optical densities less than 0.650 were considered male and optical densities greater than 0.650 were considered female.







TABLE 5







Effect of broiler breed and gender sorting of


allantoic fluid by Estrogen Sensor Assay.









Sample

Optical


Sex
Breed
Density












M
AA X CY
.469


M
AA X CY
.437


F
AA X CY
1.519


F
AA X CY
1.430


M
AA X CY
.492


M
H X P
.368


M
H X P
.444


F
H X P
2.869


M
H X P
.389


F
H X P
1.642


F
C X C
2.187


M
C X C
.649


F
C X C
2.613


M
C X C
.471


M
C X C
.458









Sex Specific Signal Present in the Allantoic Fluid Samples of Turkey can Also be Detected by Yeast-Based Estrogen Sensor.

Ten allantoic fluid samples from Day 22 Nicholas Turkey embryos were analyzed using classical estrogen analysis by radioimmunoassays. The assay-clearly delineated the sex of the turkey embryos. See FIG. 6. Yeast-based estrogen sensor was subsequently used to gender sort. See Table 6. Samples with optical densities less than 0.700 are male and samples with optical densities greater than 0.700 are females. Turkey embryos had a higher average optical density than the chicken samples tested.







TABLE 6







Ability to gender sort allantoic fluid from


Day 22 Nicholas Turkey embryos.











Optical



Sex
Density














M
.354



F
2.891



F
2.866



F
4.000



M
.419



M
.445



M
.453



F
4.000



F
4.000



F
4.000










Yeast-Based Estrogen Sensor can Differentiate Sex at Different Embryonic Stages.

In order to assess whether the methods of the invention were effective at differentiating sex at different embryonic stages, allantoic fluid samples were obtained from embryos at different stages and assessed for estrogen transactivating activity. See Table 7







TABLE 7







Effect of embryo age on ability to


gender sort allantoic fluids.











Embryo


LS ID
SEX ID
Age












.438
M
13


.566
F
13


.853
F
13


.342
M
13


.305
M
13


.338
M
15


.340
M
15


.376
M
15


.389
M
15


.368
M
15


.531
M
17


.946
F
17


1.270
F
17


.414
M
17


.434
M
17


1.624
F
18


2.292
F
18


2.966
F
18


0.52
M
18


1.285
F
18









EXAMPLE III
Production and Secretion of Beta-galactosidase in Yeast
In the previous example, exogenous GUS was employed to cleave the estrogen-glucuronide conjugates. This example shows that the active GUS enzyme can be produced in yeast and effectively used to cleave estrogen conjugates present in allantoic fluids. A series of plasmids were constructed as described in Table 8. In the assays shown, plasmid pRS425-GPDp-Ub-GUS that secretes GUS at high concentrations was employed.
GUS activity inside and outside the cell was measured as follows: Yeast cells were grown in synthetic complete media without leucine to OD600=0.5. 50 μL of cell suspension was transferred to 96-well plates. The cell suspension was transferred into eppendorf microfuge tubes and centrifuged at maximal speed on bench top centrifuge for 5 minutes. 50 μL of media (cell-free media) was transferred to 96 microplates wells. 50 μL of PNP-Gluc-2×“Z” Sarcosine-buffer [120 mM Na2HPO4, 80 mM NaH2PO4, 20 mM KCl, 2 mM MgSO4, 100 mM beta-mercaptoethanol (Sigma), pH 7.0, 0.4% lauroyl sarcosine (Sigma), 0.5 mg/mL PNP-Gluc (PNP-Gluc is an abbreviation of p-NITROPHENYL-BETA-D-GLUCURONIC ACID;, Diagnostic Chemicals limited)] was added to each well. The buffer was freshly prepared. After incubation at 37° C. for 1 hour . The reaction was stopped by adding 100 μL of quenching solution 0.5 M Na2CO3. OD405 was measured in a micro plate reader (Biotek) at a wavelength of 405–450 nanometers to estimate the relative enzymatic activity of GUS.
It has been previously established that attachment of ubiquitin, a 76 amino acid, highly conserved protein to the N-terminus of other proteins enhances the expression of fused proteins several fold. However, it is not known whether ubiquitin plays any role in the extracellular secretion of proteins. Accordingly, as described herein, several GUS yeast expression vectors have been designed wherein the C-terminus of ubiquitin was attached to the N-terminus of GUS (GDPp-UB-GUS). In addition, a factor secretory sequences were attached at the N-terminus of the ubiquitin-GUS fusion (GPD-α-factor-GUS) as well as between ubiquitin and the GUS gene (GDP-UB-α-factor-GUS). The fusion of ubiquitin increases the stability of the recombinant proteins and preserves their biological properties, thus enhancing the quality and quantity of the proteins that are expressed in yeast and E. coli [28, 29].







TABLE 8







GUS Expression in the Cells and Secretion of the Protein.














GUS
GUS





Activity
Activity


Vector

Signal
Inside
Outside the


(pRS425)
Promoter
Sequence
Cells
Cells





ADH1-GUS1
ADH1
—
+++
−


ADH1-α-factor-
ADH1
α-factor
+
−


GUS1


GPD-α-factor-
GPD
α-factor
++
−


GUS1


GPD-Ub-GUS1
GPD
Ubiquitin
++++
++++


GPD-Ub-α-factor-
GPD
Ubiquitin-α-
++++
+


GUS1

factor


GPD-α-factor-
GPD
α-factor-
+/++
+


Ub (pro)-GUS1

Ubiquitin(pro)


GPD-α-factor-
GPD
α-factor-
+
+


Ub (met)-GUS1

Ubiquitin(met)


CUP1-Ub-GUS1
CUP1
Ubiquitin
++++
++









The data described in Table 8 show that attachment of ubiquitin increases expression levels of GUS several fold. The data also shows that only the ubiquitin fusions (with or without α-factor secretory sequences) efficiently secrete GUS. The small amount of GUS activity observed in non-secretory GUS expression strains may be due to release following lysis of dead cells.
EXAMPLE IV
Gender Sorting Sensor Development
The plasmid pRS425-GPDp-Ub-GUS was assessed in additional experiments. This plasmid was used due to its robust GUS expression and high enzymatic activity of GUS in yeast culture media. The GUS producing plasmid was transformed into several yeast strains along with plasmid for expression of human estrogen receptor (YEpE12; See FIG. 4) and estrogen-inducible reporter gene (YRpE2; See FIG. 3A). The strains were screened for growth and GUS production. A yeast strain (BJ 1991) has been identified that grew rapidly with GUS plasmid and produced the highest levels (units) of GUS per mg protein. In addition, this strain was also tolerant of estrogen receptor expression and provides excellent response to estrogen in estrogen transactivation assays.
Synthetic yeast selective growth media have pH ranges between 4.5 to 5.5. The pH optimum for E. coli GUS was 6.8 as indicated by the Sigma protocol for GUS assay. To maximize growth of yeast and GUS activity, new media conditions were developed that allow optimum growth of yeast as well as robust allantoic estrogen-conjugate responsiveness.
The yeast strain BJ 1991 was transformed with plasmid pRS425-GPDp-Ub-GUS and grown in the standard yeast media (complete media without leucine, 22). The culture media was supplemented with different salt and buffering conditions by diluting 1:9 with NEB buffer 1, 2, 3, 4 10×-buffers (New England Biolabs). The yeast cells for all the buffering conditions were inoculated at the same OD600 0.1 and grown overnight. No differences were observed in growth rates of the culture. GUS activity was determined after 24 hours of growth in media, as well as in cell lysates using PNP-gluc as a substrate. The GUS activities were measured as described in EXAMPLE III. The data are provided in Table 9.







TABLE 9







GUS secretion and activity in cells grown in


different media (NEB1–NEB4)












GUS activity
GUS activity



Media
in media
in cells















NEB1
0.117
2.693



NEB2
0.107
2.337



NEB3
0.138
2.963



NEB4
0.945
2.939



Standard
0.87
2.535










As shown in Table 9, NEB buffer 4 and salt conditions provide the best GUS expression and secretion levels in the media. As demonstrated herein, the final pH of the media is 6.4 after dilution (1:10) of NEB4 (10×) with standard media. Hence, for optimal GUS activity in gender sorting assays, all the experiments and cell growth were carried out in NEB 4 buffer and media conditions. The details of the media are described below.
Mixing Transactivator and GUS Producing Strains to Monitor Gender
As shown above for the test media assessed, use of the NEB4 media provided optimized results for extracellular secretion of GUS from yeast. In the next step, NEB4 media was used to grow yeast and for monitoring gender. There were two approaches to monitor gender: a) using two yeast strains—one for GUS production and the other as the estrogen sensor and b) using a single yeast strain that has been transformed to express both GUS and the necessary components of the estrogen sensor. For the first approach, BJ 1991 cells harboring GPD-Ub-GUS1 were grown independently (GUS producer strain) or transformed with only the human estrogen receptor and ERE-beta-Galactosidase reporter vectors (estrogen transactivator strain). Two batches of cells were thereby generated, one producing secretory GUS and the other strain harboring only the transactivator and reporter gene. Cells were grown in liquid modified media following the brief protocol set forth below.

    1. GUS producer cells were grown in liquid synthetic complete NEB4 media without leucine to OD600=1–2
    2. Transactivator cells were grown in liquid synthetic complete NEB4 media without uracil and tryptophan media overnight to OD600=1–2.
    3. Transactivator cells were diluted 1/10 or 1/20 in NEB4 complete media (pH=6.4) to OD600=0.1
    4. 50 μL of GUS producer from (1), 50 μL of Transactivator in NEB4 from (3) and 8 μL of Male or Female allantoic fluid were added to wells in a microtiter plate.
    5. Plates were covered with sealer and incubated at 30° C. in a shaker for 4 h.
    6. 100 μL of 2×“Z”-ONPG buffer was then added.
    7. Incubate at 37° C. for 10 minutes to 1 h.
    8. Add 100 μL of quenching solution 0.5M Na2CO3.
    9. Measure at OD405 


For the second approach, employing a single yeast strain, the BJ1991 strain was transformed with three plasmids: 1) a GUS enzyme expression vector (pRS425-GPDp-Ub-GUS); 2) an expression vector for human estrogen receptor alpha (YEpE12); and 3) a vector expressing an estrogen inducible reporter (YrpE2). Briefly, the BJ1991 yeast strain was transformed with all three expression vectors and grown in liquid synthetic complete NEB4 media without leucine, uracil and tryptophan to OD600=1–2. The cells were diluted 1/10 or 1/20 in NEB4 complete media (final pH=6.4) to OD600=0.2. To each well of 96-well plate, 4 μL of Male or Female allantoic fluid were added. 100 μl of the diluted cell suspension was then added. Plates were covered with sealer and incubated at 30° C. at shaker for 4 h. 100 μL of 2×“Z”-ONPG buffer was then added and the samples incubated an additional 10 minutes to 1 hour at 37° C. 100 μL of quenching solution (0.5M Na2CO3) was then added and the OD405 measured. Both approaches efficiently distinguished male from female embryos.
Table 10 presents data of gender monitoring using 1 yeast strain harboring all three plasmids.















Allantoic fluids
OD405 × 1000
Sex




















DMSO
449




estrogen 1 nM
2982.5



N191
711
Male



N219
719.5
Male



N212
632
Male



N216
696.5
Male



N215
661.5
Male



N224
836
Male



N229
1342
Female



N207
1598.5
Female



N228
1737
Female



N230
1630.5
Female



N199
2638.5
Female



N200
2812.5
Female



N201
2508.5
Female



N202
1509
Female



N203
2544.5
Female



N209
2695
Female



N210
1625.5
Female



N211
2359.5
Female



N214
2894.5
Female



N218
1973
Female



N220
2950
Female







Note:



the sex Male or Female was identified independently by open dissection of the embryos.






EXAMPLE V
Male/Female Sex Sorting Using Superior Substrates for Measuring Enzymatic Activity of Beta-galactosidase
Many substrates for measuring beta-galactosidase (beta-Gal) activity are commercially available. Examples include ONPG and PNPG (Diagnostic chemicals, Limited) which are colorimetric substrates for beta-Gal. End products of these compounds have yellow color and are measured at absorption wave length 405 nM. Similar to ONPG, PNPG can be used as a beta-galactosidase substrate to distinguish male verse female eggs. MuGal (Diagnostic chemicals, Limited) is another substrate, whose end product emits yellow fluorescence that can be measured using the appropriate excitation/emission filter. Resorufin-beta-D-galactopyranoside (Sigma) is a red fluorescent substrate for beta-Gal. The advantage of using substrates that emit red fluorescence is that living organism do not emit red autofluorescence. Therefore any autofluorescent background due to cell debris, or blood contamination of the allantoic fluids should be zero.
One advantage of the present transactivation system is that the assay is relatively inexpensive to perform. To enhance cost-effectiveness, serial dilutions of substrates were tested in the transactivation assays described herein to ascertain the effects of such cost-saving measures and the ability to distinguish males from females. Ten-fold dilution of ONPG and PNPG compromised the male/female ratio readout (data not shown). A ten-fold dilution of the resorufin substrate, however, did not affect the male/female signal ratio. A 5–10 fold difference in readout between male and female samples was maintained despite sample dilution.
Accordingly, assays were performed using resorufin as substrate at low concentration and allantoic fluids from 30 eggs at 17-Days of incubation. Table 11 shows the results for 24 samples in comparison with sexing data obtained by open egg dissection. Clearly, the method of the present invention provides the accuracy of conventional, time-consuming methods, but in a fraction of the time.
Table 11 shows sec identification using Lifesensor with resorufin-beta-D-galactopyranoside as a substrate in comparison with sex identification using open dissection of embryos.















Egg number
















582
583
584
585
586
587
588
589





Sex ID
Female
Male
Male
Female
Male
Female
Male
Male


Readout
0.1358
0.0000
0.0000
0.0865
0.0000
0.1106
0.0000
0.0035












Egg number
















590
591
592
593
594
595
596
597





Sex ID
Female
Male
Female
Female
Female
Female
Male
Female


Readout
0.1057
0.0032
0.1143
0.1240
0.1229
0.1059
0.0000
0.1236












Egg number
















598
599
600
601
602
603
604
605





Sex ID
Male
Female
Female
Male
Male
Male
Male
Female


Readout
0.0019
0.1096
0.1126
0.0120
0.0270
0.0000
0.0000
0.1060





The final concentration of resorufin was 0.00025 mg/mL. The assay used 20 μL of allantoic fluids from Day 17 incubated eggs diluted in 80 μL of yeast cells, incubated at 30° C. for 4 hours. Substrates were added and incubated for 10 minutes at 37° C. The readout was measured using a 544 nm excitation filter and a 590 nm emission filter.






The results depicted in Table 11 are graphed in FIG. 7. There is 100% match between sex and red fluorescent readout: the females have a high readout (higher than 0.0600), and the males have a low readout (lower than 0.01) reflecting differences in estrogen level. Thus, using resorufin-beta-D-galactopyranoside as beta-galactosidase substrate, the background signal has been diminished, thereby generating a higher signal to noise ratio.
EXAMPLE VI
Modification of Gender Sorting Sensor to Shorten Assay Time and Increase Robustness
The present inventors have modified the gender sorting assay system to include the use of heat shock promoters. Yeast heat shock promoters such as HSP104, HSP26 and SSA4 are robust promoters which are activated rapidly in response heat shock stress (see Tijirina and Sayer 1998). High levels of these heat shock proteins have been observed within fifteen minutes of exposure of yeast cells to either high temperature (38° C.) or osmotic pressure. Transcriptional activation of HSP26, HSP104 and SSA4 increased 55-, 39-, and 167-fold, respectively, after 15 minutes of exposure to heat shock conditions (Tijirina and Sayre, 1998). Accordingly, an SSA4-based estrogen inducible reporter has been constructed that responds to activation signals with faster kinetics (at least twice as fast) than the CYC1-based reporter described above.
The SSA4-based estrogen inducible reporter plasmid YRpE2SSA4-lacZ was constructed using the ecdysone inducible reporter plasmid pBRSS 6×EcRE-lacZ as the backbone construct (Tran et al., 2001). The Sal1-BamH1 fragment comprising the 6×EcRE ecdysone response elements along with CYC1 promoter of pBRSS 6×EcRE-lacZ have been replaced with a Sal1-BamH1 fragment that contains 2×EREs plus part of the SSA4 promoter. The Sal1-BamH1 DNA fragment comprising 2×EREs plus part of the SSA4 promoter was generated by PCR amplification of yeast Saccharomyces cerevisiae genomic DNA using two primers: SSA4-forward primer containing two ERE elements: 5-GCGTCGACTCTGGTCACAGTGACCggtcacagtgaccTTATGGAAGCACCAAG-3′ (SEQ ID NO: 5; the Sal1 site is underlined and the two estrogen response elements are indicated in italics) and SSA4-reverse primer starting from the ATG codon: 5′-GTGGATCCCATGATTATTGTTTTG-3′ (SEQ ID NO: 6; the BamH 1 site is underlined). Of note, SSA4 is a heat shock inducible gene, the transcription of which is rapidly and robustly induced upon heat shock exposure (Tijirina and Sayre, 1998). A PCR product generated using the-above template and primers would, therefore, comprise the SSA4 heat shock promoter operably linked to two EREs and flanked by Sal1 and BamH1 restriction enzyme sites.
The data presented in the Table 12 demonstrated that the response of the SSA4-based estrogen inducible reporter plasmid YRpE2SSA4-lacZ to estrogen activation was at least twice as fast as that of the CYC1 promoter-based reporter. The development of the SSA4-ERE reporter system, therefore, significantly expedited the gender sorting ability of the yeast sensor assay. See Tables 13 and 14.
The following is a brief protocol of the estrogen transactivation assays used to compare the CYC1 promoter-based reporter to the SSA4 promoter-based reporter.

    
    
        1. BJ1991 yeast cells containing YEpE12 (estrogen expressing vector), pRS425-GPDp-Ub-GUS and either reporter plasmid YRpE2 or YRpE2SSA4-lacZ were grown in liquid synthetic MP medium to OD600=1–2.
        2. Yeast culture was diluted in liquid MP media to OD600=0.2.
        3. 100 μL of yeast cell suspension were spiked into each well of a 96-well microtiter plate. Estrogen (diluted in DMSO) was added to the wells to a final concentration 5 nM.
        4. Plates were covered with sealer and incubated at 30° C.
        5. At intervals of 15 minutes, 100 μL of 2×“Z”-ONPG buffer (without beta-mercaptoethanol) was added.
        6. Incubations were performed at 30° C. for 15 minutes.
        7. Optical densities were determined at OD405.
    
    









TABLE 12







Time/response comparison of two estrogen inducible


reporters (CYC1 promoter-based and SSA4 promoter-based)


in response to 5 nM estrogen. The data shown is the average


of three independent measurements.











Time of
CYC1-
SSA4-



incubation
reporter
reporter







 0 minutes
0.144
0.157



 15 minutes
0.133
0.167



 30 minutes
0.228
0.241



 45 minutes
0.185
0.535



 60 minutes
0.206
0.720



 75 minutes
0.250
0.805



 90 minutes
0.378
0.857



105 minutes
0.268
1.041



120 minutes
0.338
0.991










As shown in FIG. 8 and Table 12, the SSA4-based reporter responded to estrogen in only 30–45 minutes, whereas the SSA4-based sensor required approximately 1.5 to 2 hours of incubation time to distinguish male and female allantoic fluids. These results suggest that the cleavage of estradiol glucuronide conjugates to free estradiol is a time limiting factor in such assays. The amount of GUS secreted by yeast cell suspensions (OD600=0.2) into the growth media after four hours of growth was less than 0.01 GUS activity units, as compared to a standard titration series of purified E. coli GUS (Sigma) using PNPG as a substrate. The present inventors have discovered that supplemention of 100 μl cell suspension reactions with 0.1–1 activity units of GUS (Sigma) reduces the assay time from 2 hours to less than 1 hour.
The following protocol was performed to titrate the GUS activity in allantoic fluid assays.

    
    
        1. BJ1991 yeast cells comprising YEpE12 (estrogen expressing vector), pRS425-GPDp-Ub-GUS and reporter plasmid YRpE2 or YRpE2SSA4-lacZ were grown in liquid synthetic MP medium to OD600=1–2.
        2. Yeast cell cultures were diluted in liquid MP media to OD600=0.2.
        3. E. coli GUS (Sigma #G-7396) was added to yeast suspensions to achieve different final concentrations of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 enzymatic activity units per 100 μl of yeast suspension.
        4. Eighty microliters of yeast cell suspension (containing exogenous GUS) were spiked into individual wells of a 96-well microtiter plate. In each well there was either 20 μl of male or female allantoic fluids.
        5. Plates were covered with sealer and incubated for 1 hour at 30° C.
        6. One hundred microliters of 2×“Z”-ONPG buffer (without beta-mercaptoethanol) was then added.
        7. Incubation was performed at 30° C. for 15 minutes.
        8. The number of pixels in each well was measured using a CCD camera.
    
    









TABLE 13







GUS enzyme titration and the ability of the SSA-4


based sensor to distinguish between male and


female allantoic fluids as measured using a


CCD camera. The GUS titration was performed as


described above.













GUS
CYC1
CYC1
SSA4
SSA4
SSA4
SSA4


(units)
Male
Female
Male
Female
Male
Female
















100
0
20
7
400
7
420


10
0
7
0
500
0
400


1
0
7
0
620
0
450


0.1
0
2
0
560
0
400


0.01
0
0
0
320
0
50


0.001
2
0
0
40
0
5


0.0001
1
0
2
5
5
30


0
1
2
10
10
10
40









The data presented in the Table 13 demonstrated that 0.1–1 units of GUS were sufficient to enhance the sensitivity of gender sorting assays. The cost of 25,000 units of E. coli GUS (Sigma) is $16, which means that the additional cost associated with the above-modified assays was less than 0.0075 cents (0.1 unit) of exogenous GUS per assay well to effect a 50% reduction in assay time (reduced from 2 to 1 hour).
The results shown in Table 14 demonstrate the ability of the modified gender sorting assay to accurately distinguish male and female allantoic fluid samples. As shown, five male and five female allantoic fluid samples were tested in the modified gender sorting assay. The assay was performed with the indicated allantoic fluids, using the SSA4-reporter construct in the presence of exogenous GUS (0.1 units per assay) for one hour of incubation time, which was followed by a substrate reaction of 15 minutes.







TABLE 14







Ability of the SSA-4 based sensor in combination


with 0.1 unit of exogenous GUS to distinguish


between male and female allantoic fluids using either


a colorimetric microplate reader or CCD camera.










SSA4, clone1
SSA4, clone 2












OD405
CCD
OD405
CCD


Allantoic
measurement
measurement
measurement
measurement














Male
0.200
5
0.250
6


Male
0.209
7
0.221
0


Male
0.254
7
0.284
9


Male
0.329
6
0.337
6


Male
0.232
9
0.257
20


Female
0.672
520
0.68
600


Female
0.607
600
0.615
675


Female
0.506
600
0.487
700


Female
0.514
590
0.506
640


Female
0.561
600
0.574
650









EXAMPLE VII
Detection of Steroid and Drug-conjugates in Biological Fluids
The human body actively glucuronidates a variety of steroids and xenobiotics. The liver contains several UDP-glucuronsyltranferases (UGT) that glucuronidate many drugs and steroids (Burchell et al, 1994). Glucuronidation of steroids and drugs is a widely used mechanism for the inactivation and removal of these agents from the body. Drug and steroid-glucuronides or sulfates have lower affinity for their cognate receptors or targets. If one can de-conjugate a particular drug or xenobiotic, a receptor or drug-target driven sensor can then be used to estimate the amount of the steroid or drug released from the conjugate. The glucuronidases have rather broad specificity for glucuronidated molecules. Hence, it is possible to use a selected series of the glucoronidase expression vectors that are transformed into a yeast strain harboring the target receptor or enzyme. Incubation of biological fluids, including without limitation, sera, urine or saliva will convert low affinity inactive drug steroid-conjugate into a high affinity active drug that will bind to the target receptor to activate the receptor and promote the transactivation signal.
An exemplary drug detection assay can be performed using the following steps:

    
    
        1) The appropriate GUS producing cells are transformed with the target receptor expression vector and the reporter gene.
        2) The GUS secretory cells are grown in selective media achieving an O.D=0.2 or alternatively, an O.D that allows maximal cleavage of the drug conjugate. The cells, 20–100 μL are transferred to 96 or 384 well plates.
        3) To each well 1–10 μl of a biological ample is added. The salt and buffer conditions are optimized in cell suspension to maximize GUS enzyme function. The suspension is approximately 5–30 microliters. Different volumes of biological fluids are assayed in duplicate.
        4) To each well, appropriate substrate is added to determine the induction of the reporter gene as described above for Examples III, IV and V.
        5) The amount of the de-conjugated target receptor ligand is estimated by comparing the signal obtained from the biological fluid to a standardized control ligand assayed at the same time.
        6) Standard control reactions, e.g., with ligands that target the receptor under investigation, are performed on the same plate in which the conjugated ligand or xenobiotics are assayed. Appropriate software is then utilized to facilitate calculation of the concentration of the conjugated xenobiotics following the computation of the signal detected from the control wells.
    
    


Detection of conjugated xenobiotics by the receptor-based sensors of the invention has several applications in determining the sensitivity and toxicity of various drugs, ligands and steroids in the human population.
For example, several drugs become more toxic once they are glucuronidated or modified by drug metabolism pathway such as the cytochrome P450 series of enzymes. A rapid sensor that efficiently assesses the level of glucuronidated drugs will aid the clinician in the development of drug tolerance studies as well as toxic dose determinations in selected patients.
Conversely, conjugation of xenobiotics is often attributed to the loss of activity and clearance from the human system. This is due to the ability of different individuals to glucuronidate certain steroid and other xenobiotics and specifically the function of human UDP-glucuronsyltranferases. It is also feasible that the glucuronidation pattern of an individual is related to drug sensitivity and toxicity. In this respect the receptor and GUS encapsulated sensors are tools for studying pharmocogenomics of populations. Thus the sensors can be used as tools to stratify populations for clinical trials.
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While certain preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made to the invention without departing from the scope and spirit thereof as set forth in the following claims.

## Claims

1. An estrogen dependent transactivation system for gender-sorting in an avian test animal, comprising;
a) a first DNA construct comprising a nucleic acid molecule encoding an estrogen receptor having specific binding affinity for said estrogen operably linked to a promoter;
b) a second DNA construct comprising a promoter containing a plurality of estrogen response elements, said promoter containing a plurality of estrogen response elements being operably linked to a reporter gene;
c) a third DNA construct comprising a nucleic acid sequence encoding a secreted fusion protein comprising beta-glucuronidase and ubiquitin for cleaving an estrogen glucuronide said estrogen glucuronide comprising said estrogen, said nucleic acid sequence encoding said fusion protein being operably linked to a promoter sequence; and
d) a host cell comprising said first, second and third DNA constructs, wherein expression of said reporter gene in said host cell is dependent upon cleavage of said estrogen from said estrogen glucuronide present in a biological sample isolated from said avian test animal, reporter gene expression being indicative of the presence of an estrogen which indicates the sex of said avian test animal.

2. The system as claimed in claim 1, wherein said estrogen receptor is selected from the group consisting of estrogen receptor alpha and estrogen receptor beta.

3. The system as claimed in claim 1 wherein said avian test animal is selected from the group consisting of chickens and turkeys.

4. The system as claimed in claim 1, wherein said estrogen glucuronide is selected from the group of conjugates consisting of estrone glucuronide and estradiol 17-beta-glucuronide.

5. The system as claimed in claim 1, wherein cleavage of said estrogen glucuronide releases an estrogen selected from the group consisting of 17-beta-estradiol and estrone.

6. The system as claimed in claim 1, wherein at least one of said promoters in said DNA constructs of parts 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.

7. The system as claimed in claim 1 wherein at least one of said promoters in said DNA constructs of parts a), b) and c), is a constitutive promoter selected from the group consisting of ADH1, and GPD.

8. The 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 of said expression vectors in both prokaryotes and eukaryotes.

9. The 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.

10. The system as claimed in claim 9, wherein said yeast cell is Saccharomyces cerevisiae.

11. The 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.

12. The 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 the avian test animal tissue and extract from the avian test animal tissue.

13. A method for detecting the presence of an estrogen in an avian biological sample, comprising:
a) providing a host cell containing 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 second promoter being operably linked to a reporter gene; and a third DNA construct comprising a nucleic acid sequence encoding a secreted fusion protein comprising beta-glucuronidase and ubiquitin for cleaving an estrogen glucuronide said estrogen glucuronide comprising said estrogen, said nucleic acid sequence encoding said fusion protein being operably linked to a third promoter sequence;
b) contacting said host cell with said avian biological sample suspected of containing estrogen; and
c) assessing levels of expression of said reporter gene in said host cell, said level of expression being dependent upon cleavage of said estrogen from said estrogen glucuronide conjugate, if present, in said avian biological sample.

14. The method as claimed in claim 13, wherein said estrogen receptor is selected from the group consisting of estrogen receptor alpha and estrogen receptor beta.

15. The method as claimed in claim 13, wherein said estrogen glucuronide is selected from the group of conjugates consisting of estrone glucuronide and estradiol 17-beta-glucuronide.

16. The method as claimed in claim 13, wherein cleavage of said estrogen glucuronide releases an estrogen selected from the group consisting of 17-beta-estradiol and estrone.

17. The method as claimed in claim 13, wherein at least one of said promoters in said DNA constructs of step a) is an inducible promoter selected from the group consisting of CUP1, HSP70, HSP26, HSP104, SSA4, galactose-inducible promoters, GAL1 and GAL10.

18. The method as claimed in claim 13, wherein at least one of said promoters in said DNA constructs of step a) is a constitutive promoter selected from the group consisting of ADH1 and GPD.

19. The method as claimed in claim 13, wherein said first, second and third DNA constructs are expression vectors, wherein each of said expression vectors comprises sequences which enable replication of said expression vectors in both prokaryotes and eukaryotes.

20. The method as claimed in claim 13, wherein said host cell is selected from the group consisting of a yeast cell, an insect cell, a mammalian cell, and a bacterial cell.

21. The method as claimed in claim 20, wherein said yeast cell is Saccharomyces cerevisiae.

22. The method as claimed in claim 13, 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.

23. The method as claimed in claim 13, wherein said biological sample is selected from the group consisting of allantoic fluid, blood, urinates, saliva, culture media of avian test animal tissue, and extract from avian test animal tissue.

24. The method as claimed in claim 13, wherein the level of expression of said reporter is assessed using a method selected from the group consisting of determination of enzymatic activity using enzymatic substrates and detection of protein encoded by said reporter gene using an antibody immunologically specific for said protein encoded by said reporter gene.

25. The method as claimed in claim 13, wherein said reporter gene encodes a fluorescent reporter protein.

26. A kit comprising the test system of claim 1.

27. 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 containing a plurality of estrogen response elements being operably linked to a reporter gene;
c) a third DNA construct comprising a nucleic acid sequence encoding a secreted fusion protein comprising beta-glucuronidase and ubiquitin for cleaving a naturally occurring estrogen-glucuronide conjugate, said estrogen-glucuronide conjugate comprising estrogen, said nucleic sequence encoding said fusion protein being operably linked to a promoter sequence;
d) a yeast cell comprising said first, second and third DNA constructs, wherein expression of said reporter gene in said yeast cell is dependent upon release of estrogen from said estrogen-glucuronide conjugate present in an allantoic sample of said avian, reporter gene expression levels being correlated with the presence of said estrogen which indicates a sex of said avian;
e) media for yeast cell growth and enhancement of the glucuronidase-ubiquitin fusion protein expression, secretion and activity; and
f) a substrate and protocol to assess said reporter gene expression.

28. A method for detecting the presence of estrogen 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 second promoter being operably linked to a reporter gene; and a third DNA construct comprising a nucleic acid sequence encoding a secreted fusion protein comprising glucuronidase and ubiquitin for cleaving naturally occurring estrogen-glucuronide conjugates, said nucleic acid sequence encoding said fusion protein 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 estrogen from said estrogen-glucuronide conjugate present in said biological sample, reporter gene expression levels being correlated with the presence of estrogen in said biological sample.

29. The method of claim 28, 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.