SAME

FILING DATA

[0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2009900452, filed on 8 February, 2009, entitled "Sex-specified avians and methods of producing same", the entire contents of which, are incorporated herein by reference.

FIELD

[0002] The present invention relates generally to the field of sex determination. More particularly, the present invention provides methods and agents to manipulate sex determination in species having a male chromosome-linked sex regulatory gene.

BACKGROUND

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0005] Sex is generally chromosomally determined in most animal species (Ellegren, Trends Ecol Evol 75:188-192, 2000; Mizuno et al, Cytogenet Genome Res PP:236-244, 2002). In poultry birds and other avian species, the homogametic sex is male (ZZ) and the heterogametic sex is female (ZW). The mechanism of sex determination in an avian embryo has remained elusive with one hypothesis being that the W (female) chromosome carriers a dominant-active ovary determinant (Arit et al, Proc. Biol. ScL 271 Suppl. 4:S249-251, 2004; Nakagawa, Trends Genet 20:479-480, 2004). An alternative hypothesis is the dosage of a Z (male)-linked gene wherein two doses (ZZ) determines masculinity (Smith and Sinclair, BioEssays 26:120-132, 2004).

[0006] The ability to be able to determine and manipulate sex determination in a range of species is particularly important in the agricultural industry. To selectively produce female chickens, for example, would facilitate increased economic production of eggs and minimize the unnecessary rearing of male birds. Conversley, male birds are preferred for meat production. By producing single sex lines of chickens it obviates the need for individually sexing every hatchling. It also means that 50% of the hatchlings do not need to be culled because they are not of the required sex. Sex is currently determined in chickens manually by visual inspection but this is time-consuming, tedious and can be inaccurate.

[0007] Sex can also be determined by assaying for certain genetic or protein markers. Such methods of sex determination generally require a level of technical expertise and facilities which are not readily available in commercial operations. Furthermore, such methods do not lend themselves to automation, especially in agricultural environments.

[0008] There is a need to be able to provide non-human species with a specified sex.

SUMMARY

[0009] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0010] In accordance with the present invention, it is determined that the Z-linked gene, DMRTl, in its homozygous form, confers testis development in avians. Down-regulation of DMRTl leads to ovarian development in avian embryos. When DMRTl expression is reduced, affected male embryos exhibit sex reversal, characterized by a feminized left gonad and right testis. The feminized gonad exhibits reduced DMRTl expression, disorganized testis cords and a decline in testicular biomarkers (e.g. SOX9). Hence, DMRTl is shown to be a Z chromosome-linked testis (male) regulatory gene and plays a pivotal role in avian male sex determination. The DMRTl gene has homology in a number of species including fish, reptiles and amphibians. The ability to induce feminization in avians enables efficient production of sex-specified animals as required, such as for use in the agricultural industry.

[0011] Accordingly, one aspect of the present invention provides a sex specified animal, the animal or its parent genetically modified to either (i) inhibit expression of DMRTl or a male chromosome-linked homolog thereof or reduce DMRTl protein-activity; or (ii) to elevate expression of an DMRTl or its homolog or elevate DMRTl protein activity wherein reduced expression of DMRTl or DMRTl protein activity leads to an animal with female characteristics and elevated expression of DMRTl or DMRTl protein-activity leads to an animal with male characteristics.

[0012] By "elevated expression" of DMRTl means that the level of DMRTl protein produced are at least equivalent to the expression of two alleles of DMRTl, i.e. in a normal ZZ male. Similarly, a "reduced expression" of DMRTl means a reduction from the normal ZZ male level. In a particular embodiment, the animal is an avian animal. - A -

[0013] Accordingly, another aspect of the present invention provides a sex-specified avian animal, the avian animal or its parent genetically modified to change the level of expression of DMRTl or its homolog or change the level of activity of DMRTl protein or its homolog, which level of DMRTl expression or DMRTl protein activity determines the sex of the avian animal.

[0014] More particularly, the present invention provides a sex-specified avian animal, the avian animal or its parent genetically modified to minimise a modified level of expression of DMRTl or its homolog or minimize the level of activity of DMRTl protein or its homolog, wherein a reduced level of DMRTl expression or DMRTl protein activity leads to an avian animal with female characteristics.

[0015] A further aspect of the present invention provides a sex-specified- avian animal, the avian animal or its parent genetically modified to elevate the level of expression of DMRTl or its homolog or elevate the level of activity of DMRTl protein or its homolog wherein elevated expression of DMRTl or of DMRTl protein activity leads to an avian animal with male characteristics.

[0016] As indicated above, in terms of avian species such as chickens, an elevated level of DMRTl means to a level similar to a normal (ZZ) male. A reduced level means a similar to a normal (ZW) female.

[0017] Another aspect of the present invention is directed to a method for generating a sex-specified animal, the method comprising introducing into a blastoderm or developing embryo of the animal an agent which modulates the level of expression of DMRTl or a male chromosome-linked homolog thereof or modulates the activity of DMRTl protein and allowing the embryo to develop into a postnatal animal wherein an agent which reduces expression of DMRTl or DMRTl protein activity results in an animal with female characteristics and an agent which elevates expression of DMRTl or DMRTl protein activity results in an animal with male characteristics. [0018] In a particular embodiment, the animal is an avian animal.

[0019] Consequently, another aspect of the present invention provides a method for generating a sex-specific avian animal, the method comprising introducing into a blastoderm or developing embryo of the avian animal an agent which modulates the level of expression of DMRTl or its homolog or the level of activity of DMRTl protein, allowing the embryo to develop to a hatchling wherein the hatchling having female characteristics comprises a reduced expression of DMRTl or reduced DMRTl protein activity and a hatchling having male characteristics comprising elevated expression of DMRTl or elevated DMRTl protein-activity.

[0020] Still another aspect of the present invention is directed to a method for generating a sex-specified avian animal, the method comprising modulating the level of expression of DMRTl or its homolog or the activity of DMRTl protein in the avian animal for a time and under conditions sufficient to decrease DMRTl expression to generate a female avian animal or to induce DMRTl expression to generate a male avian animal.

[0021] Yet another aspect of the present invention contemplates a method of inducing feminization of an avian embryo, the method comprising introducing to the embryo an agent which reduces the functional level of DMRTl expression or DMRTl protein activity for a time and under conditions sufficient for the embryo to develop female characteristics.

[0022] Still yet another aspect of the present invention contemplates a method of inducing masculization of an avian embryo, the method comprising introducing to the embryo an agent which comprises DMRTl protein or its functional homolog or analog or which facilitates expression of DMRTl or its functional homolog for a time and under conditions sufficient for the embryo to develop male characteristics.

[0023] In an embodiment, the agents of the present invention are genetic constructs or compounds which either down-regulate expression of an endogenous DMRTl gene or within elevated expression of DMRTl. Examples include viral vectors, microRNAs (miRNA), antisense oligonucleotides or compounds, RNAi molecules, siRNA molecules, sense oligonucleotides, ribozymes, dsRNA molecules and oligonucleotides comprising hairpin loops. Such molecules are proposed to target DMRTl expression and to reduce levels of DMRTl protein. In another embodiment, the agents are genetic constructs which, when expressed or introduced into the avian chromosome, produce DMRTl protein. In yet another embodiment, the agents are chemical molecules which inhibit the function of DMRTl protein or which inhibit the expression of the DMRTl gene. Still, in another embodiment, DMRTl protein or DNA or RNA is injected into the embryo to induce mascularization of an avian embryo.

[0024] The present invention further contemplates genetically modified fertilized avian eggs, the eggs comprising a blastoderm or developing embryo genetically modified to either reduce expression of DMRTl to a level less than a homozygous DMTRl male or to elevate expression of DMRTl to the level of a homozygous DMRTl male. Such eggs are then provided to a consumer on the basis that they will substantially give rise to all female or all male hatchlings.

[0025] Reference to avian species or animals herein includes inter alia chickens, ducks, geese, turkeys, bantams, pheasants and quail. However, the present invention extends to any avian species including penguins, aviary birds, game birds, bird pests and the like. The present invention further extends to non-human animals other than avian species such as fish, reptiles and amphibians.

[0026] Reference to "sex" includes gender.

[0027] Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>l (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims. TABLE 1

List of Sequences

BRIEF DESCRIPTION OF THE FIGURES

[0028] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0029] Figure 1 is a photographic and diagrammatic representation of knockdown of DMRTl protein in vitro, using RCASBP virus to deliver miRNA against DMRTl. Immunofluorescent detection of DMRTl protein, and GFP fluorescence, (a) Chicken fibroblastic DFl cells infected with RCASBP. A.DMRT1 only, showing no GFP expression but robust over-expression of DMRTl protein in cell nucleic (red), (b) Cells infected with RCASBP.A.DMRT1 after infection with virus carrying a non-silencing control scrambled sequence with GFP reporter (scrambled control), showing widespread GFP and DMRTl protein expression (i.e. no knockdown). In the merged image, some cells appear yellow or orange, because the GFP can have both cytoplasmic and nuclear localization, (c) Cells infected with RCASBP.A.DMRT1 following infection with virus carrying DMRTI miRNA, showing widespread expression of GFP (green) and knockdown of DMRTl protein (red). Note that a few cells lacking GFP expression (and hence no miRNA) show strong DMRTl expression (arrow), (d) Knockdown of DMRTl mRNA expression in DFl cells treated with RCASBP.B.GFP.DMRT1 compared to cells treated with RC ASBP.B. scrambled control Uninfected DFl cells show no endogenous DMRTl expression.

[0030] Figure 2 is a photographic representation of abnormal gonadal development in male gonads treated with DMRTl miRNA. Urogenital systems from day 10 chicken embryos treated at day 0 with scrambled control or DMRTl miRNA. Gonads are outlined for clarity, (a) Control male (ZZ), showing bilateral development of symmetrical testes, (b) Control female (ZW), showing typical asymmetric development, characterized by larger left ovary and smaller right gonad, (c) Genetic male (ZZ) infected with virus carrying DMRTl miRNA, showing abnormal (female-like) asymmetry, with a larger left and smaller right gonad, (d) Female (ZW) infected with virus carrying DMRTl miRNA, showing normal asymmetric development. Ms - paired mesonephric kidneys.

[0031] Figure 3 is a photographic representation of feminization of genetic male (ZZ) chicken embryos treated with DMRTl miRNA. (a) Normal expression of DMRTl protein in a 10 day old genetic male embryo (ZZ) treated with non-silencing scrambled control sequence. DMRTl is strongly expressed in the Sertoli and germ cells of the organizing testis cords (arrows), (b) Internal distribution of germ cells within the testis cords of a control male, as assessed by staining for Chick Vasa Homologue (CVH). (Still yet another aspect of the present invention contemplates a method of inducing masculization of an avian embryo, the method comprising introducing to the embryo an agent which comprises DMRTl protein or its functional, homolog or analog or which facilitates expression of DMRTl protein or its functional homolog for a time and under conditions sufficient for the embryo to develop male characteristics, (c) Widespread expression of GFP in a control male gonad, (d) Reduced DMRTl protein and disorganized cords in a left male (ZZ) gonad treated with DMRTl miRNA. Compare with (a) above. Some areas show normal DMRTl expression (e.g. arrows), but expression is weak in other areas and cord formation is lost, (e) Cortically biased (female-like) germ cell distribution in a male gonad treated with DMRTl miRNA (e.g. arrows), (f) Widespread GFP expression in a gonad treated with DMRTl miRNA, characterized by normal DMRTl expression in a small right testis and greatly reduced DMRTl expression in a left ovary, (h) Female-like germ cell distribution in the left ovary shown in (g). (i) GFP expression in the left ovary shown in (h). (j) Left ovary of a control female (ZW), showing low DMRTl expression throughout the gonad, except for high level expression in cortical germ cells, (k) Typical cortical germ cell distribution in left ovary of control female, as assessed by CVH. (1) Widespread GFP expression in a control female gonad.

[0032] Figure 4 is a photographical representation of down-regulation of male markers and activation of female markers in male gonads treated with DMRTl miRNA. (a) Lack of SOX9 expression in a ZW control female, (b) Normal S OX9 expression in organized testis cords of a control male (ZZ) treated with scrambled control miRNA sequences, (c) Reduced SOX9 expression and disorganized testis cords in a ZZ male treated with DMRTl miRNA. (d) Normal Aromatase enzyme expression in the paired gonads of a control female (ZW) treated with scrambled miRNA. (c) Lack of aromatase expression in the (left) gonad of a control male (ZZ) treated with scrambled miRNA sequence, (f) Ectopic activation of aromatase in the left gonad of a ZZ male treated with DMRTl miRNA. (g) Higher power view of aromatase expression in the left gonad of a control female (ZW), showing expression in cells surrounding the characteristic lacunae (cavities) of the medulla (arrows), (h) Partial DMRTl expression in a ZZ male treated with DMRTl miRNA. DMRTl expression is reduced and testis cords are disrupted in the bracketed area, which now ectopically expresses aromatase, shown in (i). The region expression aromatase shows female-like lacunae (e.g. arrowheads).

[0033] Figure 5 is a diagrammatic representation of the design of shuttle plasmid for cloning cGFP.shRNA into RCASBP.B viral vector, Plasmid pRmiR (RCAS miRNA) is derived from pcDNA6.2-GW/EmGFP-miR (Invitrogen). Three ds-oligos were cloned into the parent vector to allow for cloning of siRNA ds-oligos into the miRNA cassette {via the back to back Bsal sites) and provision of two Clal sits up- and down-stream of the eGFP/miR cassette to allow cloning into RCAS. Once cloned into RCAS, a single polyadenylated cGFP/miRNA transcript is .produced from the viral LTR resulting in expression of eGFP protein and mature siRNA within the same cell.

DETAILED DESCRIPTION

[0034] In accordance with the present invention, sex determination in avian species is specified by expression of the Z-linked gene, DMRTl to produce DMRTl protein. The DMRTl gene is described by Shan et al, Cytogenetics Cell Genetics 89:252-251, 2000; Smith et al, Nature 402:601-602, 1999. It is proposed herein that dosage dependent (i.e. two alleles in a homozygous [ZZ] male) expression of DMRTl leads to masculinization of an avian embryo. Similarly, down-regulating levels of DMRTl expression or DMRTl protein leads to feminization of an avian embryo.

[0035] DMRTl is, therefore, regarded as a male chromosome-linked sex regulatory gene. In generic terms, if M is considered a male gamete and F is considered a female gamete, a homogametic sex (MM) is male and the heterogametic sex (MF) is female. It is proposed that an M chromosome-linked homolog of DMRTl confers masculinity in the homogametic state. Hence, a dose of DMRTl conferred by an MM male is considered here as a 2x dose.

[0036] A female animal (MF) may exhibit a Ix dose. Hence, it is proposed herein that feminization occurs by manipulating a MM chromosome to exhibit less than a 2x (<2x) dose of DMRTl or its M-linked homolog. By "less than 2x" or "<2x" includes meant from Ox to 1.5x which encompasses amounts such as 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 and 1.5x, or amounts inbetween.

[0037] Hence, the present invention contemplates targeting DMRTl expression or DMRTl protein activity or a male chromosome-linked homolog thereof in order to manipulate and specify animal gender. Reduced expression of DMRTl or its male chromosome-linked homolog to less than the level of expression of two alleles in a normal male results in feminization. Elevated expression of DMRTl or its male chromosome-linked homolog to the level of expression of two alleles in a normal male results in mascularization.

[0038] Genetic, proteomic and chemical approaches are therefore contemplated herein to either reduce levels of DMRTl expression or DMRTl protein activity in order to generate female animals or to induce DMRTl expression or DMRTl protein production to favor generation of male animals.

[0039] Accordingly, one aspect of the present invention provides a sex specified animal, the animal or its parent genetically modified to either (i) inhibit expression of DMRTl or a male chromosome-linked homolog thereof or reduce DMRTl protein-activity; or (ii) to elevate expression of an exogenous DMRTl or its homolog or elevate DMRTl protein activity wherein reduced expression of DMRTl or DMRTl protein activity leads to an animal with female characteristics and elevated expression of DMRTl or DMRTl protein- activity leads to an animal with male characteristics.

[0040] By "elevated expression" of DMRTl means that the level of DMRTl protein produced is at least equivalent to the expression of two alleles of DMRTl, i.e. in a normal DMRTl male (i.e. 2zx dose). Similarly, a "reduced expression" of DMRTl means less than that of a normal male (i.e. <2x dose). In a particular embodiment, the animal is an avian animal.

[0041] The present invention provides, therefore, a gender specified non-human animal and a method of producing same wherein the sex of the animal is determined by the dose (x) of a male chromosome (M)-linked sex regulatory gene and wherein a homogametic (MM) male has a 2x dose of the regulatory gene and a heterogametic (MF) female, wherein F is the female gamete, has a Ix dose of the regulatory gene, the gender specified animal or its parent genetically modified by a means selected from:

(i) down-regulating expression of one or both alleles of the sex regulatory gene on the M chromosome to a dose of <2x, wherein the gender is female;

(ii) up-regulating expression of the sex regulatory gene on a M chromosome in a MF female to a dose level of 2x or more wherein the gender is male; and (iii) introducing and expressing an exogenous sex regulatory gene to the F or M chromosome, wherein the gender is male;

wherein the sex regulatory gene is DMRTl or a male chromosome-linked homolog thereof.

[0042] Another aspect of the present invention is directed to a method for generating a sex-specified animal, the method comprising introducing into a blastoderm or developing embryo of the animal an agent which modulates the level of expression of DMRTl or a male chromosome-linked homolog thereof or modulates the activity of DMRTl protein and allowing the embryo to develop into a postnatal animal wherein an agent which reduces expression of DMRTl or DMRTl protein activity results in an animal with female characteristics and an agent which elevates expression of DMRTl or DMRTl protein activity results in an animal with male characteristics.

[0043] In a particular embodiment, the animal is an avian animal.

[0044] Consequently, another aspect of the present invention provides a method for generating a sex-specific avian animal, the method comprising introducing into a blastoderm or developing embryo of the avian animal an agent which modulates the level of expression of DMRTl or its homolog or the level of activity of DMRTl protein, allowing the embryo to develop to a hatchling having female characteristics comprises a reduced expression of DMRTl or reduced DMRTl protein activity and a hatchling having male characteristics comprising elevated expression of DMRTl or elevated DMRTl protein-activity.

[0045] Accordingly, another aspect of the present invention provides a sex-specified avian animal, the avian animal or its parent genetically modified to change the level of expression of DMRTl or its homolog or change the level of activity of DMRTl protein or its homolog, which level of DMRTl expression or DMRTl protein activity determines the sex of the avian animal. [0046] Whilst the present invention extends to any non-human animal having a male chromosome-linked DMRTl homolog such as fish, reptiles and amphibians, it is particularly directed to avian animals.

[0047] More particularly, the present invention provides a sex-specified avian animal, the avian animal or its parent genetically modified to minimise a modified level of expression of DMRTl or its homolog or minimize the level of activity of DMRTl protein or its homolog, wherein a reduced level of DMRTl expression or DMRTl protein activity leads to an avian animal with female characteristics.

[0048] A further aspect of the present invention provides a sex-specified avian animal, the avian animal or its parent genetically modified to elevate the level of expression of DMRTl or its homolog or elevate the level of activity of DMRTl protein or its homolog wherein elevated expression of DMRTl or of DMRTl protein activity leads to an avian animal with male characteristics. In this regard, an endogenous DMRTl on a heterogametic female may be manipulated to enhance its expression to a 2x dose amount or a further copy introduced to increase copy number. An "introduced" DMRTl is referred to as an "exogenous DMRTl".

[0049] Still another aspect of the present invention is directed to a method for generating a sex-specified avian animal, the method comprising modulating the level of expression of DMRTl or its homolog or the activity of DMRTl protein in the avian animal for a time and under conditions sufficient to decrease DMRTl expression to generate a female avian animal or to induce DMRTl expression to generate a male avian animal.

[0050] Yet another aspect of the present invention contemplates a method of inducing feminization of an avian embryo, the method comprising introducing to the embryo an agent which reduces the functional level of DMRTl expression or DMRTl protein for a time and under conditions sufficient for the embryo to develop female characteristics.

[0051] Still yet another aspect of the present invention contemplates a method of inducing masculization of an avian embryo, the method comprising introducing to the embryo an agent which comprises DMRTl protein or its functional, homolog or analog or which facilitates expression of DMRTl or its functional homolog for a time and under conditions sufficient for the embryo to develop male characteristics.

[0052] In one embodiment, the agent is a genetic molecule which inhibits DMRTl expression or which facilitates the generation of a DMRTl deletion or inactivation such as by introducing a stop codon or insertion.

[0053] Examples of such molecules include micro(mi)RNAs, RNAi, siRNA, dsRNA, oligonucleotides comprising hairpin loops, antisense oligonucleotides, sense oligonucleotides or their chemically modified forms including antagomers. In particular, the disruption of the DMRTl gene may be transient or rendered permanent. In a particular aspect, avian animals are generated with an inability to express DMRTl.

[0054] Hence, another aspect of the present invention provides a genetically modified avian animal, the avian animal substantially incapable of expressing DMRTl or its functional homolog or progeny of the avian animal.

[0055] Such an avian animal would exhibit female characteristics and be able to lay eggs.

[0056] Consequently, this aspect of the present invention provides a genetically modified female egg laying avian animal, the avian animal substantially incapable of expression DMRTl or its functional homolog or progeny of the avian animal.

[0057] In an embodiment of the present invention, the DNA encoding the endogenous DMRTl gene in an avian is deleted. Methods for deleting an endogenous gene such as DMRTl in avians are well known to a person skilled in the art and generally comprise inserting a genetic construct into a pluripotent cell and transferring the cell into an embryo to yield a chimera. Through breeding, the construct becomes integrated into the germline of a resulting animal and ultimately results in the disruption of the production of endogenous DMRTl. The disruption of endogenous DMRTl production may occur by targeted disruption of a specific DMRTl gene locus, the substantial deletion of a DMRTl gene locus, or the insertion of an engineered construct (e.g. stop codon) that, through ordinary processes of cell division, replaces an intact endogenous locus in an embryonic stem cell or in the resulting animal. The construct may also induce gene silencing by, for example, miRNA, RNAi, siRNA and the like. Methods for inactivating genes in avians are further described in, for example, International Patent Publication No. WO 03/081992.

[0058] Other mechanisms for silencing DMRTl expression include gene silencing through epigenetic processes such methylation of all or part of the DMRTl genetic locus. As indicated above, gene silencing may be induced in any number of ways including the use of miRNA, RNAi, siRNA, siRNA, Zinc-finger nucleases and various dicer-comprising constructs. Hence, the present invention provides an oligonucleotide compound which selectively inhibits expression of an endogenous DMRTl gene or all or part of the DMRTl genetic locus. Generally, the inhibition of expression is permanent and is passed on to future generations. However, transient inhibition is also contemplated herein. By "transient" includes days, weeks, months or for the life of the avian animal.

[0059] Hence, the present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of DMRTl in animal species. This is accomplished by providing oligonucleotides which specifically target DNA or RNA encoding DMRTl. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding DMRTl" have been used for convenience to encompass DNA encoding DMRTl, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. This aspect encompasses antisense and sense-suppression of DMRTl.

[0060] The functions of DNA to be interfered with can include replication and transcription. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One particular result of such interference with target nucleic acid function is a reduction in expression of DMRTl.

[0061] It is understood in the art that the sequence of a sense or antisense oligonucleotide compound need not be 100% complementary to that of its target nucleic acid to be effective in inducing inhibition. Moreover, an oligonucleotide may comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more particularly comprise 90% sequence complementarity and even more particularly comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which it is targeted.

[0062] DMRTl levels may also be reduced functionally by the introduction of an inhibitor of DMRTl activity or DMRTl expression. Generally, the inhibitor is produced by genetic means introduced into the avian animal.

[0063] Hence, another aspect of the present invention contemplates a genetically modified animal including an avian animal, the animal genetically modified to express genetic material which encodes an inhibitor of DMRTl activity or DMRTl expression or of a male chromosome-linked homolog thereof.

[0064] Accordingly, the inhibitor may be a protein inhibitor of DMRTl activity or may be an antisense or sense oligonucleotide which down-regulates DMRTl expression.

[0065] Examples of protein inhibitors include antibody chains, marine animal-derived single chain antibodies (IgNARs) and a peptide inhibitor of DMRTl.

[0066] In another embodiment, DMRTl activity is introduced or enhanced to ensure the generation of male animals including avian animals. Generally, this is accomplished by introducing genetic material which encodes a DMRTl protein or its functional homolog. Generally, an introduced DMRTl gene is referred to as an "exogenous" DMRTl. This also still applies to subsequent progeny.

[0067] Accordingly, another aspect of the present invention provides a genetically modified animal including an avian animal, the animal genetically modified to express a DMRTl protein or its homolog or an enhancer of DMRTl expression or of a male chromosome-linked homolog.

[0068] In a further embodiment, genetically modified animals including avian animals are produced which have been genetically modified to enable the DMRTl gene or gene locus to be selectively disrupted, generally in ovo. For example, the DMRTl gene or genetic locus may be inducibly inactivated upon certain conditions. Hence, if a female animal is required, inducible inactivation occurs to inhibit DMRTl expression. However, if a male animal is required, the embryo is not subject to conditions resulting in inactivation of the DMRTl gene.

[0069] A useful vector to genetically manipulate embryos, is depicted in Figure 5. Once either DMRTl expression is disrupted or induced, the level of expression can be determined.

[0070] There are many methods which may be used to detect a DMRTl expression including determining the presence to DMRTl mRNA via sequence identification. Direct nucleotide sequencing, either manual sequencing or automated fluorescent sequencing can detect the presence of a particular mRNA species.

[0071] Techniques for detecting nucleic acid species include PCR or other amplification techniques.

[0072] Nucleic acid analysis via microchip technology is also applicable to the present invention. In this technique, distinct oligonucleotide probes are built up in an array on a silicon chip. Nucleic acids to be analyzed are fluorescently labeled and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips. Using this technique, one can determine the presence of DMRTl mRNA species or the level of mRNA as well as the expression of levels of DMRTl .

[0073] Hence, alteration of mRNA expression from a DMRTl genetic locus can be detected by any techniques known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of an affected gene. Alteration of DMRTl expression can also be detected by screening for alteration of expression product such as a protein. For example, monoclonal antibodies immunoreactive with a DMRTl protein can be used to screen a tissue. Lack of cognate antigen or a reduction in the levels of antigen would indicate a reduction in expression of DMRTl or a male chromosome-linked homolog thereof. Such immunological assays can be done in any convenient formats known in the art. These include Western blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered protein can be used to detect alteration of the wild-type protein. Functional assays, such as protein binding determinations, can be used.

[0074] Hence, the present invention further extends to a method for identifying a genetic basis behind sex in an animal, the method comprising obtaining a biological sample from the animal and detecting the level of expression of DMRTl, wherein the presence of a reduced level of DMRTl expression of its homolog is instructive as to a female or male animal, respectively.

[0075] The biological sample is any fluid or cell or tissue in which DMRTl is expressed. In one embodiment, the biological sample includes blood, lymph, urine and saliva or cells from these samples.

[0076] Consequently, the present invention provides genetic, chemical and proteinaceous agents which either inhibit DMRTl gene expression or DMRTl protein activity or which facilitate DMRTl activity. [0077] Another aspect of the present invention is directed to the use of an agent which inhibits DMRTl gene expression or DMRTl protein activity or a male chromosome-linked homo log thereof in the manufacture of a feminized animal, such as an avian animal.

[0078] One such agent is the vector in Figure 5.

[0079] Still another aspect of the present invention contemplates the use of an agent which facilitates DMRTl expression or protein activity or a male chromosome-linked homolog thereof in the manufacture of a mascularized animal such as an avian animal.

[0080] Reference to the "DMRTl" gene includes the DMRTl genetic locus and further includes functional variants (e.g. polymorphic variants) and functional male chromosome- linked homologs thereof. A functional variant or homolog includes a gene having at least 80% identity to the cDNA sequence encoding DMRTl. By at least 80% identity means at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity. This can be conveniently determined by any number of algorithmic means. Determination of identity is generally after optimal alignment. Similarly, a functional variant or homolog of DMRTl includes genes having a DNA strand which hybridizes under medium to high stringency conditions to a strand of DMRTl DNA.

[0081] Reference herein to hybridization includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out T _m = 69.3 + 0.41 (G+C)% (Marmur and Doty, J MoI. Biol. 5: 109, 1962). However, the T _m of a duplex DNA decreases by I ⁰ C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25°-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.

[0082] Similarly, reference to the DMRTl protein, includes all functional variants and homologs thereof such as proteins having at least 80% amino acid similarity to DMRTl after optimal alignment.

[0083] Another aspect of the present invention contemplates a business model, such as for the poultry industry. In the business model, poultry birds are genetically modified to inactivate expression of DMRTl. Fertile eggs produced by such poultry birds will give rise to female birds only and hence the level of loss due to unnecessary rearing of male birds substantially reduced.

[0084] Hence, a business model is provided for the generation of female poultry birds with enhanced economic returns, the model comprising generating female poultry birds with an inactivated DMRTl gene or gene locus, mating these birds to one or more male birds to generate fertilized eggs, allowing the eggs to hatch wherein the resulting hatchlings are all female and wherein the female birds are reared and introduced in an existing poultry bird operation.

[0085] Such a business model can result in enhanced economic gains by reducing the number of male birds which need to be reared and increasing the number of birds which can, for example, be used for egg production or meat production. [0086] Reference herein to an "avian animal" or "avian species" includes any member of the Class Aves including poultry birds such as chickens, ducks, geese, turkeys, bantams, pheasants and quail. The methods of the present invention are also applicable to game birds, aviary birds and as a means to control avian animals regarded as pests. As indicated above, the present invention extends to non-human animals other than avian species such as fish, reptiles and amphibians.

[0087] The present invention further contemplates genetically modified fertilized avian eggs, the eggs comprising a blastoderm or developing embryo genetically modified to either reduce expression of DMRTl to a level less than a homozygous DMRTl male or to elevate expression of DMRTl to the level of a homozygous DMRTl male. Such eggs are then provided to a consumer on the basis that they will substantially give rise to all female or all male hatchlings.

[0088] Again, the present invention provides a business model to enhance economic returns for the poultry industry, the model comprising supplying genetically modified fertilized eggs with an indication whether the eggs will give rise to female hatchlings or male hatchlings, each egg comprising a genetically modified blastoderm or developing embryo in which either expression of DMRTl is reduced to a level less than a normal ZZ male or expression of DMRTl is elevated to the level of a normal ZZ male.

[0089] As indicated above, reduced expression of DMRTl means at a level wherein the embryo will develop female characteristics. An elevation of expression of DMRTl means to a level wherein the embryo exhibits male characteristics.

[0090] The present invention further contemplates genetic elements such as a retroviral vector for use in inducing reduction or elevation in expression of DMRTl or its male chromosome-linked homolog.

[0091] The present invention extends to all progeny of genetically modified animals and to stem cell lines therefrom. Genetically modified hatchlings are also contemplated herein. [0092] The present invention is further described by the following non-limiting Examples. Aspects of the present invention have been published in Smith et al, Nature Letters 46:261-271, 2009, the contents of which, are incorporated herein by reference. In the Examples, the following materials and methods were employed.

Materials and Methods

Preparation of viruses:

[0093] RCASBP.B virus was modified to carry the open reading frame of GFP together with a recombinant microRNA directed against chicken DMRTl in its 3' UTR. The sequence 5 was designed using the Invitrogen Block-It (Trade Mark) system, and generated in a short hairpin format. The sequence was directed against exon three of chicken DMRTl mRNA, and did not show significant homology to other sequences in the chicken genome (equal to or greater than 16/21 bases). The hairpin sequence was cloned into an engineered shuttle plasmid carrying GFP (pRmiR - Figure. 5). The GFP- microRNA was then subcloned into RCASBP.B strain virus. High quality DNA was then used to transfect chicken DFl cells. Virus was harvested from DFl cells and purified as described previously (Smith et al, Int. J. Dev. Biol. 53:59-67, 2009). Viral titres of approximately 108 infectious units/mL were obtained. For controls, a scrambled sequence of the same bases was used, cloned into RCASBP.B and propagated in the same way (= RCASBP.B.GFP.scrambled control).

Knockdown of DMRTl expression in vitro:

[0094] To test the efficacy of knockdown, DFl cells were infected with RCASBP.B. GFP. DMRTl or scrambled control virus, grown for several days, and then infected with RC ASBP. A. strain virus carrying DMRTl open reading frame. (DFl cells do not produce endogenous DMRTl . Cells can be infected with two viruses provided they are of different strains). After fours days of co-infection, cells were processed for GFP and DMRTl protein expression. Immunofluorescence was employed, using a DMRTl antibody (in-house) and Alexfluor secondary antibodies, as described previously (Smith et al, Biol Reprod 55:560-567, 2003). Knock down was also assessed by quantitative real time PCR. RNA was extracted from the cells and cDNA synthesized as previously described (Smith et al, BMC Dev Biol 8:72, 2008). Probes were designed using the UPL Assay Design Centre (https://www.roche-applied-science.conϊ) and are as follows; DMRTl probe # 59 For 5'-AGCCTCCCAGCAACATACAT-S' (SEQ ID NO:1) and rev 5'-GCGGTTCTCCATCCCTTT-S' (SEQ ID NO:2); HPRT probe # 38 For 5'- CGCCCTCGACTACAATGAATA-3' (SEQ ID NO:3), Rev 5'- CAACTGTGCTTTCATGCTTTG-3 (SEQ ID NO:4)'. Analysis was performed using LightCycler 480 instrument and software (Roche).

Embryos:

[0095] Fertile chicken eggs (Gallus gallus domesticus) were obtained from SPAFAS, Woodend, Victoria, Australia. Four hundred day 0 blastoderms were injected with DMRTl knockdown or scrambled control virus using a fine glass capillary needle. Eggs were sealed and incubated until day 10. Survival to day 10 was 40%. Embryos showing 10 GFP fluorescence in the urogenital system (28/160) were selected for further analysis. GFP expression in the urogenital system varied from strong to weak. Embryos were genotypically sexed by PCR as described previously (Smith et al, 2003, supra). Of the 28 embryos, five males that showed strong GFP in the gonads were found to have varying degrees of DMRTl knockdown and evidence of feminisation. Several females showed strong GFP 15 in the gonads but normal ovarian development. All other embryos (15) had variable GFP in the gonads, normal gonadal sex differentiation and no DMRTl knockdown. It as hypothesized that lower levels of microRNA delivery (as assessed by lower GFP expression) may be insufficient to influence endogenous DMRTl. (There was a good inverse correlation between GFP expression level and DMRTl expression; embryos 20 with stronger GFP showed stronger knockdown of DMRTl, as anticipated). All genetic male embryos that showed feminization were re-sexed, using tissue derived directly form the urogenital system. In all cases, the originally assigned genotypic sex was confirmed. Immunofluorescence:

[0096] Urogenital systems were fixed in 4% v/v paraformaldehyde/PBS, cryo-protected in 30% w/v sucrose/PBS, embedded in OCT compound, and sectioned, as described (Smith et al, 2008, supra). Immunofluorescence was used to assess protein expression. Rabbit DMRTl, anti-aromatase and anti-SOX9 (1 :6000) were all raised in-house and have been described (Smith et al, 2003, supra).

EXAMPLE 1 Down-regulation of DMRTl

[0097] Recombinant microRNA (miRNA) directed against the DMRTl transcript was delivered into living chicken embryos via the avian retroviral vector, RCASBP.B (Replication Competent Avian Sarcoma leukosis virus, high titre Bryan Polymerase, strain B). The virus was engineered to carry the open reading frame of GFP, to monitor viral spread, with the DMRTl miRNA in the 3' UTR of the transgene. The strong viral LTR promoter rather than an internal U6 promoter drove miRNA expression. This virus delivered robust GFP expression and knockdown of exogenous DMRTl protein in cultured chicken DFl cells (Figure 1). Cells infected with RCASBP. A strain virus expressing only the DMRTl cDNA showed strong DMRTl over-expression (Figure Ia). Cells co-infected with virus carrying DMRTl cDNA and virus expressing a non-silencing RCASBP.B.GFP.scrambled miRNA showed robust DMRTl protein expression (Figure Ib). In contrast, cells co-infected with DMRTl and the DMRTl miRNA sequence showed knockdown of DMRTl protein (Figure Ic), and a 70% reduction in DMRTl transcript compared to cells treated with scrambled control miRNA (Figure Id).

EXAMPLE 2 Generation of genetically modified embryos

[0098] Virus carrying the GFP reporter together with DMRTl miRNA was used to infect day zero chicken blastoderms, and embryogenesis was allowed to proceed until day 10. Control embryos were infected with virus carrying GFP and the scrambled non-silencing miRNA sequence. All embryos were genotypically sexed by PCR amplification of a W (female)-specifϊc Xhol repeat sequence. In the chicken embryo, the gonads form on the mesonephric kidneys around day 3.5 of incubation. Sexual differentiation into testes or ovaries begins at day 6 and is normally advanced by day 10. Embryos infected with virus at day zero showed robust global expression of GFP by day 10, including widespread expression in the urogenital system and in sectioned 25 gonads. Overall embryonic development was normal. Gonadal development in embryos treated with control miRNA was normal, with bilateral testes in males and typical asymmetric ovarian development in females (Figures 2a and b) [n =10]. In all control cases, gonadal sex matched genotypic sex. However, among those embryos treated with DMRTl miRNA, five males that showed high GFP expression also showed disrupted testicular development. Macroscopically, three of these males showed abnormal (female-like) asymmetry (larger left and smaller right gonads) [Figure. 2c]. Females (ZW) treated with DMRTl miRNA showed normal asymmetric ovarian development (Figure 2d) [n= 5]. Gonads from embryos with high levels of GFP (and hence miRNA delivery) were sectioned and assessed for DMRTl and marker gene expression. In control embryos infected with virus carrying the non-silencing scrambled miRNA, DMRTl expression was not affected and gonadal histology was normal. In control males, DMRTl protein was uniformly expressed in developing Sertoli and germ cells of testis cords (Figure 3 a). Expression was strong, bilateral (in both left and right gonads) and indistinguishable from staining in uninfected male embryos. Germ cells were distributed 10 within testis cords in the medulla of both gonads, as assessed by staining for Chicken Vasa homologue (CVH) [Figure 3b]. GFP immunofluorescence confirmed widespread expression of the scrambled miRNA sequence in control males (Figure 3 c). [0099] In contrast, five male embryos (ZZ) treated with DMRTl miRNA showed variably reduced DMRTl protein expression in the left gonad, disrupted testis cord formation and ectopic female gene expression. The extent of DMRTl knockdown and testis cord disruption varied among embryos. Some individuals showed disrupted DMRTl expression, with disorganized cords and a cortical (female-like) pattern of germ cell distribution (Figures 3d and e). As in control males, these individuals showed strong GFP expression (Figure 3f). Other ZZ embryos showed stronger feminization, characterized by normal DMRTl expression and cord formation in the right gonad, but greatly reduced DMRTl expression, loss of cord organization and ovarian-type left gonad (Figure 3g). The germ cells of the left feminized male gonads again exhibited a female-like distribution (i.e. concentrated in the outer part of the gonad, the cortex, rather than within testis cords) [Figure 3h]. Female-type morphology of ZZ male gonads treated with DMRTl miRNA was also revealed by fibronectin immunofluorescence, which delineated the presence of a thickened ovarian-like cortex and poorly formed cords. The strongest examples of ZZ feminization were observed in those gonads showing the strongest GFP expression (i.e. the highest delivery of the knockdown 30 miRNA) [Figures 3i and I].

[0100] In control females (ZW), ovarian development was normal. DMRTl was lowly expressed in the developing (left) ovary, with the exception of higher expression in cortical germ cells (Figure 3j). CVH staining revealed normal cortical germ cell distribution in controls (Figure 3k). As in males, GFP expression was widespread in control females (Figure 31). In genetic females treated with DMRTl microRNA, endogenous DMRTl expression appeared lower, but the gonads nevertheless appeared normal, with typical asymmetry. This indicates that DMRTl is not essential for chicken ovarian development. EXAMPLE 3 Characterization of embryos

[00100] Gonads were further examined for the expression of male and female markers. A key gene involved in testicular differentiation is SOX9. In mammals, SOX9 is activated by SRY and is required for Sertoli cell differentiation and proper testis development. SOX9 is male up-regulated in all vertebrates that have been examined, including birds, pointing to a conserved role in testicular development. In day 10 control embryos infected with scrambled miRNA, SOX9 was expressed normally in male gonads. Female gonads lacked SOX9 expression, as expected (Figures 4a and b). In genetic males (ZZ) treated with DMRTl miRNA, SOX9 protein expression was variably reduced, reflecting disrupted testis cords (Figure 4c). DMRTl may therefore play a role in the activation or maintenance of SOX9 expression during testis determination in the chicken embryo (a role supplanted by SRY in mammals). Genetic males treated with DMRTl miRNA also showed ectopic activation of the robust female marker, aromatase. Aromatase enzyme is normally expressed only in female gonads, where it synthesizes the oestrogen that is required for ovarian differentiation in the chicken. Aromatase enzyme is never detected in normal male embryonic gonads. In control and knockdown female (ZW) embryos, aromatase was expressed normally in the medulla of both left and right gonads (Figure 4d). No expression was seen in male controls (Figure. 4e). However, in the five genetic males feminized with DMRTl miRNA, aromatase was activated in the left (but not right) gonad (Figure 4f). In some genetic males (ZZ) with partial knockdown, areas of reduced or absent DMRTl expression correlated with ectopic aromatase expression and female-like lacunae (Figures 4 g, h, i). These findings suggest that elevated DMRTl in male gonads normally suppresses aromatase and hence female development. This effect could be direct, or indirect via repression of the 5 FOXL2 gene, which is thought to activate aromatase. EXAMPLE 4

Mechanism of action

[00101] The results of research underlying the invention support the Z dosage hypothesis for 10 avian sex determination (Nanda et al, Cytogenet Genome Res 122:150- 156, 2008). A higher dosage of DMRTl initiates testicular differentiation in male embryos, activating SOX9 expression and suppressing aromatase, which is essential for female development. DMRTl fulfils the requirements expected of an avian master sex- determining gene. It is sex-linked, conserved on the Z sex chromosome of all birds examined, including the basal ratites (ostriches, emus, etc) [Sheety et al, cytogenet Genome Res 99:245-251, 2002]. It is expressed exclusively in the urogenital system prior to gonadal sex differentiation in chicken embryos, with higher expression in males (Smith et al, 2003, supra), and knockdown leads to gonadal sex reversal. In the other vertebrates, DMRTl is also implicated in testis determination. In reptiles with temperature sex determination, DMRTl expression is up-regulated during the thermosensitive period when sex is being determined, and only at male-producing temperatures (Shoemarker et al, Dev Dyn 25(5:12055-1063, 2007; Kettlewell et al, Genesis 25:174-178, 2000). In the medaka fish, Oryzias latipes, a duplicated copy of DMRTl, DMY, is the master testis determinant (Matsuda et al, Nature 417:559-562», 2002), while a W-linked copy, DMW, is involved in ovarian development in an amphibian, Xenopus laevis (Yoshimoto et al, Proc Natl Acad Sci USA 105:2469-2474, 2008). The data herein provide evidence that DMRTl is the elusive male sex determinant in birds.

[00102] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY

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