Regulation of productive characteristics in birds

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Method includes introduction of RNA molecule into a bird egg. Introduced RNA molecule contains double-stranded region and results in reduction of the level of molecule of RNA and/or protein, included into determination of sex in birds, in the egg. Invention can be used in poultry breeding.

EFFECT: claimed is method of changing sex characteristics in birds.

7 cl, 3 dwg, 6 tbl, 2 ex

The technical field to which the invention relates

The present invention relates to methods of regulating signs, especially productive traits in birds, such as chickens. In particular, the invention relates to shippingin ovomolecules dsrnas (dsRNA), especially Mirna molecules (siRNA), to regulate productive traits in commercial importance of birds.

Prior art

People have changed the phenotypic characteristics of domestic animals through breeding breeding stock for many generations since then, the animals have been domesticated. This resulted in the improvement of quantitative parameters of productivity, such as body size and muscle mass. Many innovations recently in the regulation of productive traits in poultry and/or increase resistance to pathogens have been focused on transgenic approaches, however, many consumers are worried about the use of genetically modified organisms.

Suppliers of chickens needed to be an effective and economical way of determining the sex of one-day Chicks. Different suppliers have used the determining the sex of chickens by examining the cloaca and by examining plumage, however, these methods proved to be extremely uneconomical, as required significant the different costs of time and labor when splitting chickens, male and female. The use of probes (US 5508165) is also expensive and impractical from an economic point of view. Another way of determining the sex of chickens is the anal probing areas chickens (US 4417663), but it is also expensive and requires time-consuming, because each chick take for the procedure. Use of experts who can determine the sex of a chicken feather, but such experts are expensive, and sex determination of a feather takes a long time.

There is a need for methods of control of productive traits in poultry, which will not lead to transformation of the genome of a bird, but can be intensified in the implementation process.

The invention

The authors of the present invention surprisingly found that the introduction of a suitable nucleic acid molecule containing a double-stranded region, bird egg can modify the phenotype of the developing embryo.

Thus, in the first aspect of the present invention relates to method changes the sign of the bird, including the introduction in bird egg at least one nucleic acid molecule containing a double-stranded region, where the molecule of nucleic acid reduces the level of at least one RNA molecule and/or protein in the egg.

In another and is the aspect the present invention relates to method changes the sign of the bird, includes introduction to bird egg at least one RNA molecule containing a double-stranded region, where the RNA molecule reduces the level of at least one RNA molecule and/or protein in the egg and where the method does not include electroporation eggs.

In the following aspect the present invention relates to method changes the sign of the bird, including the introduction in bird egg at least one RNA molecule containing a double-stranded region (dsrnas), where the RNA molecule reduces the level of at least one RNA molecule and/or protein in the egg and where the RNA molecule is injected into the air SAC, yolk SAC or allantoin liquid chorion.

In a preferred embodiment, the nucleic acid molecule is a dsrnas. More preferably, dsrnas is a siRNA (siRNA) or MSRC (shRNA).

In the following a preferred embodiment, the sign is a productive sign. Examples of productive traits include, but are not limited to, muscle or floor.

In one embodiment, the implementation of the productive trait is a floor, and a molecule of nucleic acid reduces the level of protein encoded by gene DMRT1.

In one embodiment, the implementation of the productive trait is a floor, and a molecule of nucleic key is lots reduces the protein level, the encoded gene WPKCI (ASW).

In another embodiment, a productive sign is a muscle, and the molecule of nucleic acid reduces the level of protein encoded by the genome of myostatin.

Preferably, the nucleic acid molecule is administered by injection.

The bird can belong to any kind of classAves. Examples include, but are not limited to, chickens, ducks, turkeys, geese, antamok and quail. In a particularly preferred embodiment, the bird is a chicken.

In the following aspect the present invention relates to a bird, obtained using the method according to the invention.

In another aspect, the present invention relates to the chicken, obtained using the method according to the invention.

In another aspect the present invention relates to isolated and/or exogenous molecule of nucleic acids containing double-stranded region, which reduces the level of at least one RNA molecule and/or protein when introduced into a poultry egg.

Preferably, the nucleic acid molecule is a molecule dsrnas. More preferably, dsrnas represents miRNAs or MSRC.

In one of the embodiments the nucleic acid molecule reduces the level of protein encoded by gene DMRT1 or genome myostatin.

Also, a vector, to yuushi molecule of nucleic acid, or one of its chain, according to the invention. Such vectors can be used in the cell-the owner or cell-free expression system to obtain nucleic acid molecules useful for the method according to the invention.

In another aspect, the present invention relates to the cell host containing the exogenous nucleic acid molecule or one of its chain according to the invention and/or a vector according to the invention.

In another aspect, the present invention relates to compositions containing a nucleic acid molecule or one of its chain according to the invention, the vector according to the invention and/or the cell host according to the invention.

In the following aspect the present invention relates to a bird egg, containing a nucleic acid molecule or one of its chain according to the invention, the vector according to the invention and/or the cell host according to the invention.

In another aspect, the present invention relates to a kit containing the nucleic acid molecule or one of its chain according to the invention, the vector according to the invention, the cell host according to the invention and/or the composition according to the invention.

It is obvious that the preferred features and characteristics of one aspect applicable to many other aspects of the invention.

Throughout the description the word "include" or variations such as "contains" or "containing"should be understood as enabling decrees of the data item, number or step or group of elements, integers or steps but not the exclusion of any other element, number, or step or group of elements, integers or steps.

Later in this document, the invention is described by the following non-limiting examples with reference to the accompanying figures.

A brief description of the accompanying drawings

Figure 1 RT - PCR expression of polygenic clusters MSRC. Schematic representation of the PCR strategy used to obtain expression vectors MSRC. In PCR used direct primers paired with the reverse primers, with the inclusion of all components MSRC. All final PCR products consisted of a U6 promoter chickens, semantic sequence MSRC, loops, antisense sequence MSRC, consistency termination and customersXhoI.

Figure2 - Testing of selected molecules MSRC to knockdown the expression of the fused gene EGFP-Dmrt1. The average fluorescence intensity for each state transfection expressed relatively pEGFP-Dmrt1. Error bars reflect the standard error calculated for each individual experiment carried out in three repetitions.

Figure 3 - Testing of selected molecules MSRC to knockdown the expression of the fused gene EGFP-Gdf8. The DF1 cells were transicional: panel 1, only pEGFP-C; panel 2, only transcriptionally fused the I construction of pEGFP-Gdf8; panel 3-6 pEGFP-Gdf8 or pshEGFP or with expression plasmids MSRC specific to Gdf8, pshGdf8-258, pshGdf8-913 and pshGdf8-1002. Microscopic examination was performed using a fluorescent microscope (Leica DM LB (Leica Microsystems, Germany) and images were recorded at 50× magnification, using a color digital camera Leica DC300F (Leica Microsystems, Germany) and software for image processing Photoshop 7.0 (Adobe®).

The key to the list of sequences

SEQ ID no:1 - chicken, myostatin (Genbank NM_001001461).

SEQ ID no:2 is the nucleotide sequence encoding the chicken myostatin (Genbank NM_001001461).

SEQ ID no:3 is a partial sequence of a chicken DMRT1 protein (Genbank AF 123456).

SEQ ID no:4 is a partial nucleotide sequence encoding a chicken DMRT1 (Genbank AF123456).

SEQ ID no:5 - chicken WPKCI (ASW) (Genbank AF148455).

SEQ ID no:6 is the nucleotide sequence encoding chicken WPKCI (ASW) (Genbank AF148455).

SEQ ID no: 7 is the nucleotide sequence of the promoter U6-1 chickens.

SEQ ID no: 8 is the nucleotide sequence of the promoter U6-3 chickens.

SEQ ID no: 9 is the nucleotide sequence of the promoter U6-4 chickens.

SEQ ID no: 10 is the nucleotide sequence of the 7SK promoter chickens.

SEQ ID No. 11-98 and 113-122 - RNA sequences are applicable according to the invention.

SEQ ID No. 99-112 - oligonucleotide primers.

Detailed description of the invention

General methods and determined the I

Unless specifically stated otherwise, all technical and scientific terms used herein should be taken as having accepted in the field value (for example, in cell culture, molecular genetics, biology of birds, RNA interference, and biochemistry).

Unless specifically stated otherwise, the preparation of recombinant proteins, cell cultivation and immunological methods used in the present invention, are standard methods, well known to specialists in this field. Such methods are described and explained in such literary sources as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (publishers), DNA Cloning: A Practical Approach, volumes 1-4, IRL Press (1995 and 1996) and F.M. Ausubel et al. (publishers), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all reissues to date), Ed Harlow and David Lane (publishers).

The term "avian" in this document refers to any species, subspecies or types of organisms taxonomic classAvessuch as, but not limited to, chickens, turkeys, ducks, geese, quail, pheasants, parrots, finches, hawks, crows and becciliya birds, including ostrich, EMU and cassowary. The term covers a variety of known breed<> Gallus gallus(Kura), for example, white Livorno, brown Livorno, striped plymouthrock, Sussex, new Hampshire, Rhode island, australorp, Cornish, Minorca, amrox, California gray, partridge's Italian, and breed turkeys, pheasants, quail, ducks, ostriches and other poultry usually bred in commercial quantities.

In this document the term "egg" means an egg laid by birds. Generally, bird eggs consist of solid oval outer shell, egg protein or albumin, egg yolk and various thin membranes. In addition, "in ovo" means "egg".

The terms "reduce", "decrease" or variations thereof in this document refer to a tangible reduction in the number of target RNA and/or target protein in eggs compared to eggs of the same species of birds, more preferably of the genus or species of birds, and even more preferably, the same birds, which were not introduced nucleic acid as defined in this document. The term also refers to a noticeable reduction in activity of the target protein. Preferably, the reduction level of the target RNA and/or the target protein is at least about 10%. More preferably, the decrease is at least about 20%, 30%, 40%, 50%, 60%, 80%, 90% and, even more preferably about 100%.

In this paper the e phrase "nucleic acid molecule leads to the reduction or its variations indicate the presence of molecules of the nucleic acid in the egg, which causes the degradation of homologous RNA molecules in the egg in a process known in the field as "RNA interference" or "off gene". In addition, the nucleic acid molecule directly leads to a decrease and not transcribedin ovo,causing the desired effect.

"At least one RNA molecule may be any type RNA present in the eggs of birds and/or produced by them. Examples include, but are not limited to, mRNA, carnk (snRNA), microRNA and tRNA.

In this document the term "productive trait" refers to any phenotypic characteristic of birds that has commercial value, such as muscle mass, gender, and nutritional value.

In this document the term "muscle weight" means the weight of muscle tissue. The increase in muscle mass can be determined by weighing all the muscle tissue of birds that hatch from eggs, processed as described in this document, compared to a bird of the same species of birds, more preferably of the genus or species of birds, and even more preferably, with the same bird, which was not introduced nucleic acid, as specified in this document. Alternatively, specific muscles, such as the muscles of the Breasts and/or legs, can be used to identify muscle mass. Preferably, the methods according to the invention has withdrawn shall provide muscle mass at least about 1%, 2,5%, 5%, 7.5% and even more preferably approximately 10%.

"Variant" of a nucleic acid molecule according to the invention includes molecules of varying sizes and/or with one or more different nucleotides, but which can still be used to stop a target gene. For example, variants may contain additional nucleotides (e.g., 1, 2, 3, 4 or more) or fewer nucleotides. In addition, several nucleotides can be replaced without affecting the ability of nucleic acids to shut down the target gene. In one embodiment, the implementation of option contains additional 5' and/or 3' nucleotides that are homologous to the corresponding target RNA molecule and/or which increase the stability of the molecule of nucleic acid. In another embodiment, nucleic acid molecules have no more than 4, more preferably not more than 3, more preferably not more than 2 and even more preferably not more than 1 difference in nucleotides compared to the proposed in this paper sequence. In the following embodiment, nucleic acid molecules have no more than 2, and more preferably, not more than 1, additional internal and/or deletirovannykh nucleotides compared with sequences proposed in this document.

Under "isolated nucleic acid molecule" inventors understand the nucleic acid molecule that is at least partially separated from the nucleic acid molecule with which it is associated or linked in its natural state. Preferably, the nucleic acid molecule of at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which it is associated in its natural state. In addition, the term "polynucleotide" herein is used interchangeably with the term "nucleic acid".

The term "exogenous" in the context of a nucleic acid molecule means a molecule of nucleic acid present in a cell or in a cell-free expression system in the changed quantities. Preferably, the cell is a cell that normally does not contain the nucleic acid molecule. However, the cell can be a cell that contains an exogenous molecule of nucleic acids due to the increased number of molecules of nucleic acid. The exogenous nucleic acid molecule according to the invention includes nucleic acid molecules that have not been separated from other components of the recombinant cells and cell-free expression system where they are present, and nucleic acid molecules produced in such cells or cell-free systems, which are then cleaned, at least from some of the other components.

Off genes (Gene silencing)

The terms "RNA interference", "MKI" or "off genes"generally refers to the process in which a molecule of double-stranded RNAS (dsrnas) (dsRNA) reduces the expression of a nucleic acid sequence with which a molecule of double-stranded RNA has a substantial or complete homology. However, it was recently shown that switching off a gene can be achieved by using double-stranded molecules not-RNA (see, for example, US 20070004667).

RNA interference (Rnci) is particularly useful for the specific inhibition of the production of specific RNA and/or protein. Although without intent to be limited by theory, Waterhouse et al. (1998) proposed a model for the mechanism by which dsrnas (duplex RNA) can be used to reduce production of the protein. This methodology is based on the presence of molecules dsrnas, which contain the sequence, which is basically identical to the mRNA of the gene of interest or its part, in this case, the mRNA that encodes the polypeptide according to the invention. It is convenient to get dsrnas from a single promoter in a recombinant vector or cell of the host, where the sense and antisense effects the successive flanked by other sequence, which allows semantic and antimuslim sequences to gibridizatsiya to form a molecule of dsrnas with any sequence, forming a loop structure. Development and acquisition of suitable molecules dsrnas to the present invention is quite within the competence of the specialist in this area, especially considering Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619, WO 99/53050, WO 99/49029 and WO 01/34815.

The present invention encompasses nucleic acid molecules that contain and/or encoding the double-stranded region to turn off genes. Molecules of nucleic acid, usually represent RNA, but can be a DNA, chemically modified nucleotides and not nucleotides.

Double-stranded region should be the length of at least 19 consecutive nucleotides, for example, about 19-23 nucleotides, or may be longer, such as 30 or 50 nucleotides, or 100 nucleotides or more. You can use the full sequence corresponding to the full transcript of the gene. Preferably, they comprise from about 19 to about 23 nucleotides in length.

The degree of identity of the double-stranded region of the nucleic acid molecules targeted to the transcript must be at least 90% and, more preferably 95-100%. The percentage identity of a molecule of nucleic acid the acid is determined using the GAP (Needleman and Wunsch, 1970) analysis (GCG program) with the penalty for creating a gap in the sequence = 5 and with the penalty for extending a gap = 0,3. Preferably, the two sequences align along their entire length.

Of course, the nucleic acid molecule can contain extraneous sequences that can serve to stabilize the molecule.

The term "small interfering RNA" or "siRNA" in this document means a nucleic acid molecule, which includes ribonucleotides, are able to inhibit or regulate in the direction of decreasing the expression of genes, for example, Poreba of Rnci specific for the sequence follows in which double-stranded region is less than 50 nucleotides in length, preferably from about 19 to about 23 nucleotides in length. For example, miRNAs can be a nucleic acid molecule containing semicomplete sense and antisense region, where the antisense region includes a nucleotide sequence that is complementary to the nucleotide sequence in the target nucleic acid molecule or part thereof, and semantic region has a nucleotide sequence corresponding to the target nucleic acid sequence or part thereof. The Mirna molecule can be assembled from two separate oligonucleic the Dov, where one chain is a value chain, and the other is an antisense chain, where the antisense and sense chains are semicomplete.

In this paper, the term siRNA is designed to be equivalent to other terms used to describe nucleic acid molecules that are able to mediate specific for the sequence of Rnci, for example, microRNA (miRNA), small [short] hairpin RNAS (MSRC), small [short] interfering oligonucleotide, small [short] interfering nucleic acid (mink) (siNA), small interfering modified oligonucleotide, chemically-modified siRNA posttranscriptional off gene RNA (ptwrc) (ptgsRNA) and others. In addition, as used herein, the term of Rnci is intended to be equivalent to other terms used to describe a specific sequence of RNA interference, such as posttranscriptional off gene, translational inhibition, or epigenetics. For example, the Mirna molecules of the invention can be used for epigenetic off genes as posttranscriptional level and pretranslational level. In non-limiting example, epigenetic regulation of gene expression by Mirna molecules according to the invention MoE is et to be mediated by miRNAs modification of chromatin structure to modify gene expression.

Preferred small molecules interfering RNA ("siRNA") contain a nucleotide sequence that is identical to about 19 to 23 consecutive nucleotides of the target mRNA. In one of the embodiments of the invention the sequence of the target mRNA begins with dinucleotide AA, has a GC content of about 30-70% (preferably, 30-60%, more preferably 40-60%, and more preferably, about 45%-55%) and does not have a high percentage identity with any nucleotide sequence other than the target sequence in the genome of birds (preferably chicken), which will enter, for example, how to determine the standard BLAST search.

Under "MSRC" or "small hairpin RNA" refers to a molecule miRNAs, where less than about 50 nucleotides, preferably from about 19 to about 23 nucleotides form base pairs with a complementary sequence located on the same RNA molecule, and where the specified sequence and complementary sequence separated by an unpaired region of at least about 4 to 15 nucleotides that forms odnotsepochechnoi loop over the "stem" structure created by the two areas of complementary bases. Examples of sequences of single-stranded loops are 5' UUCAAGAGA 3' and 5' UUUGUGUAG 3'.

In MSRC included wojnie or two-finger and mnogopaltsevy hairpin dsrnas, in which the RNA molecule contains two or more such structures type "stem loop", split single-stranded spacer elements areas.

There are established criteria for the design of miRNAs (see, for example, Elbashire et al., 2001; Amarzguioui et al., 2004; Reynolds et al., 2004). Details can be found on the websites of some commercial suppliers, such as Ambion, Dharmacon, GenScript and OligoEngine. As a rule, you should obtain and carry out the screening of a number of miRNAs to compare their effectiveness.

After designing dsrnas for use in the method according to the present invention can be obtained by any method known in this field, for example byin vitrotranscription, recombinant or synthetic methods. miRNAs can be obtainedin vitrousing recombinant enzymes, such as RNA polymerase, T7, and oligonucleotide matrix DNA, or you can get thein vivofor example, in cultured cells. In a preferred embodiment, the nucleic acid molecule synthetic methods.

It also describes strategies for obtaining stem miRNAs from vectors containing, for example, the promoter of RNA polymerase III. Designed various vectors to obtain stem miRNAs in the cells of the host using a promoter or H1 RNA, or U6 (snU6) RNA (see SEQ ID nos 7-9). The RNA molecule, the description of the ing above (for example, the first part of the linking sequence and the second part), can functionally bind to this promoter. When transcribing RNA polymerase III, the first and second parts form a duplex stem of the hairpin, and the binding sequence forms a loop. The pSuper vector (OligoEngines Ltd., Seattle, Wash) can also be used to obtain miRNAs.

To improve properties of the nucleic acid molecules according to the invention can be modified or analogs of nucleotides. Improved properties include increased resistance to nuclease and/or increased ability to penetrate the cell membrane. Accordingly, the terms "nucleic acid molecule" and "double-stranded RNA molecule" includes synthetically modified bases, such as, but not limited to, inosine, xanthine, gipoksantin, 2-aminoadenine, 6-methyl-, 2-propyl and other alkylidene, 5-halouracils, 5-Galitsin, 6-azacytosine and 6-azathymine, pseudorutile, 4-thiouracil, 8-galadini, 8-aminoadenine, 8-teologinen, 8-tilarginine, 8-hydroxylamines and other 8-substituted adenine, 8-galogenidy, 8-aminoguanine, 8-thioguanine, 8-trialkylamine, 8-hydroxyguanine and other substituted guanine, other Aza and deazaadenosine, other Aza and deazaguanine, 5-cryptomaterial and 5-triptorelin.

Signs. Specific productive signs and responsible is e for gene

The methods according to the invention can be used to modify any characteristic of species of birds, especially signs, determined or affected at the time when the embryo develops within the egg. The preferred characteristics that can be changed are gender and muscle mass.

In one embodiment, the implementation of productive characteristic is gender, and the molecule of nucleic acid reduces the level of protein encoded by gene DMRT1. DMRT1 was the first molecule involved in sex determination, which demonstrates the conservatism sequence among taxonomic types. Avian homolog DMRT1 is located on the Z (sex) chromosome chickens and differentially expressed in the genital ridge of chick embryos, male and female (Raymond et al., 1999; Smith et al., 1999). To date DMRT1 is one of the few genes involved in sex determination in mammals, which, apparently, is strictly gonadal pattern of expression (Raymond et al., 1999).

Examples of nucleic acid molecules that can be used to reduce chicken DMRT1 protein include, but are not limited to, those that contain at least one of the following nucleotide sequences:

CCAGUUGUCAAGAAGAGCA (SEQ ID no:11)

GGAUGCUCAUUCAGGACAU (SEQ ID no:12)

CCCUGUAUCCUUACUAUAA (SEQ ID no:13)

GCCACUGAGUCUUCCUCAA (SEQ ID no:14)

CCUGCGUCACACAGAUACU (SEQ ID no:16)

GGAGUAGUUGUACAGGUUG (SEQ ID no:17)

GACUGGCUUGACAUGUAUG (SEQ ID no:18)

AUGGCGGUUCUCCAUCCCU (SEQ ID no:19)

or any of them.

In a particularly preferred embodiment, nucleic acid molecules that can be used to reduce chicken DMRT1 protein contain a sequence GCCACUGAGUCUUCCUCAA (SEQ ID no: 14) or its variant.

The following example of a gene that can be targeted exposure for change of sex as a productive trait is a gene WPKCI. It is shown that the avian gene WPKCI vysokokonservativnykh is located on the W chromosome of birds and actively expressed in chicken embryo is female before gonadal differentiation. Assume that WPKCI may play a role in differentiation of the female gonads, interferir with the function of PKCI or showing their unique function in the nucleus (Hori et al., 2000). This gene is also identified as ASW (avian-specific sex W-bound) (O'neill et al., 2000).

In another embodiment, productive symptom is muscle mass, and a molecule of nucleic acid reduces the level of protein encoded by the genome of myostatin. Myostatin, also called "factor in the growth and differentiation 8" (GDF8), is a recently discovered member of the TGFβ superfamily. It is shown that mRNA and protein of myostatin is expressed in skeletal muscle, with whom RDA and mammary gland. Target gene destruction of myostatin in mice and mutation in the third exon of the gene of myostatin the massive cattle breed Belgian blue, which is expressed nonfunctional protein myostatin, leads to an increase in muscle mass. Thus, myostatin is a negative growth regulator of skeletal muscle.

Examples of nucleic acid molecules that can be used to reduce the level of protein from chicken myostatin include, but are not limited to, those that contain at least one of the following nucleotide sequences:

AAGCUAGCAGUCUAUGUUU (SEQ ID no:20)

GCUAGCAGUCUAUGUUUAU (SEQ ID no:21)

CGCUGAAAAAGACGGACUG (SEQ ID no:22)

AAAGACGGACUGUGCAAUG (SEQ ID no:23)

AGACGGACUGUGCAAUGCU (SEQ ID no:24)

UGCUUGUACGUGGAGACAG (SEQ ID no:25)

UACAAAAUCCUCCAGAAUA (SEQ ID no:26)

AAUCCUCCAGAAUAGAAGC (SEQ ID no:27)

UCCUCCAGAAUAGAAGCCA (SEQ ID no:28)

UAGAAGCCAUAAAAAUUCA (SEQ ID no:29)

GCCAUAAAAAUUCAAAUCC (SEQ ID no:30)

AAAUUCAAAUCCUCAGCAA (SEQ ID no:3l)

AUUCAAAUCCUCAGCAAAC (SEQ ID no:32)

AUCCUCAGCAAACUGCGCC (SEQ ID no:33)

ACUGCGCCUGGAACAAGCA (SEQ ID no:34)

CAAGCACCUAACAUUAGCA (SEQ ID no:35)

GCACCUAACAUUAGCAGGG (SEQ ID no:36)

CAUUAGCAGGGACGUUAUU (SEQ ID no:37)

GCAGCUUUUACCCAAAGCU (SEQ ID no:38)

UUCCUGCAGUGGAGGAGCU (SEQ ID no:39)

CUGAUUGAUCAGUAUGAUG (SEQ ID no:40)

GACGAUGACUAUCAUGCCA (SEQ ID no:41)

CCGAGACGAUUAUCACAAU (SEQ ID no:42)

UGCCUACGGAGUCUGAUUU (SEQ ID no:43)

AUGGAGGGAAAACCAAAAU (SEQ ID no:44)

AACCAAAAUGUUGCUUCUU (SEQ ID no:45)

CCAAAAUGUUGCUUCUUUA (SEQ ID no:46)

AAUGUUGCUUCUUUAAGUU (SEQ ID no:47)

UGUUGCUUCUUUAAGUUUA (SEQ ID no:48)

GUUUAGCUCUAAAAUACAA (SEQ ID no:49)

AAUACAUAUAACAAAGUA (SEQ ID no:50)

UACAAUAUAACAAAGUAGU (SEQ ID no:51)

UAUAACAAAGUAGUAAAGG (SEQ ID no:52)

CAAAGUAGUAAAGGCACAA (SEQ ID no:53)

AGUAGUAAAGGCACAAUUA (SEQ ID no:54)

AGGCACAAUUAUGGAUAUA (SEQ ID no:55)

UUAUGGAUAUACUUGAGGC (SEQ ID no:56)

GUCCAAAAACCUACAACGG (SEQ ID no:57)

AAACCUACAACGGUGUUUG (SEQ ID no:58)

ACCUACAACGGUGUUUGUG (SEQ ID no:59)

CGGUGUUUGUGCAGAUCCU (SEQ ID no:60)

GCCCAUGAAAGACGGUACA (SEQ ID no:61)

AGACGGUACAAGAUAUACU (SEQ ID no:62)

GAUAUACUGGAAUUCGAUC (SEQ ID no:63)

UUCGAUCUUUGAAACUUGA (SEQ ID no:64)

ACUUGACAUGAACCCAGGC (SEQ ID no:65)

CCCAGGCACUGGUAUCUGG (SEQ ID no:66)

GACAGUGCUGCAAAAUUGG (SEQ ID no:67)

AAUUGGCUCAAACAGCCUG (SEQ ID no:68)

UUGGCUCAAACAGCCUGAA (SEQ ID no:69)

ACAGCCUGAAUCCAAUUUA (SEQ ID no:70)

UCCAAUUUAGGCAUCGAAA (SEQ ID no:71)

UUUAGGCAUCGAAAUAAAA (SEQ ID no:72)

AUAAAAGCUUUUGAUGAGA (SEQ ID no:73)

AAGCUUUUGAUGAGACUGG (SEQ ID no:74)

GCUUUUGAUGAGACUGGAC (SEQ ID no:75)

GAUGGAUUGAACCCAUUUU (SEQ ID no:76)

CCCAUUUUUAGAGGUCAGA (SEQ ID no:77)

ACGGUCCCGCAGAGAUUUU (SEQ ID no:78)

CGGAAUCCCGAUGUUGUCG (SEQ ID no:79)

UCCAGUCCCAUCCAAAAGC (SEQ ID no:80)

GCUUUUGGAUGGGACUGGA (SEQ ID no:81)

AAGAUACAAAGCCAAUUAC (SEQ ID no:82)

GAUACAAAGCCAAUUACUG (SEQ ID no:83)

AGCCAAUUACUGCUCCGGA (SEQ ID no:84)

UUACUGCUCCGGAGAAUGC (SEQ ID no:85)

UGCGAAUUUGUGUUUCUAC (SEQ ID no:86)

CAGGUGAGUGUGCGGGUAU (SEQ ID no:87)

AUACCCGCACACUCACCUG (SEQ ID no:88)

GCAAAUCCCAGAGGUCCAG (SEQ ID no:89)

AUCCCAGAGGUCCAGCAGG (SEQ ID no:90)

GAUGUCCCCUAUAAACAUG (SEQ ID no:91)

ACAUGCUGUAUUUCAAUGG (SEQ ID no:92)

UGGAAAAGAACAAAUAAUA (SEQ ID no:93)

AAGAACAAAUAAUAUAUGG (SEQ ID no:94)

GAACAAAUAAUAUAUGGAA (SEQ ID no:95)

CAAAUAAUAUAUGGAAAGA (SEQ ID no:96)

AUAAUAUAUGGAAAGAUAC (SEQ ID no:97)

UAUAUGGAAAGAUACCAGC (SEQ ID no:98)

CCAGAAUAGAAGCCAUAAA (SEQ ID no:113)

GCACAAUUAUGGAUAUACU (SEQ ID no:114)

GUACAAGAUAUACUGGAAU (SEQ ID no:115)

CCUAUAAACAUGCUGUAUU (SEQ ID no:116)

GCGAAUUUGUGUUUCUACA (SEQ ID no:117)

GAGUAUUGAUGUGAAGACA (SEQ ID no:118)

CCUCCAGAAUAGAGCCAU (SEQ ID no:119)

GGUCAGAGUUACAGACACA (SEQ ID no:120)

CAGUGGAUUUCGAAGCUUU (SEQ ID no:121)

CAACGGUGUUUGUGCAGAU (SEQ ID no:122)

or any of them.

In a particularly preferred embodiment, nucleic acid molecules that can be used to reduce the level of protein from chicken myostatin contain the sequence CAGGUGAGUGUGCGGGUAU (SEQ ID no:87) or its variant.

Vectors and cells-owners

The present invention also relates to a vector, codereuse a nucleic acid molecule containing a double-stranded region or one chain of the present invention. Preferably, the vector is an expression vector that can Express open frame(CI) reading, coding of dsrnas in the cell host and/or a cell-free system. A host cell can be a cell of any type, such as, but not limited to, cells of bacteria, fungi, plants or animals.

Typically, the vector according to the invention contains a promoter functionally linked to an open reading frame that encodes a nucleic acid molecule according to the invention or its chain.

In this document, the term "promoter" means a nucleic acid sequence that is capable of directing the transcription of functionally related nucleic acid molecule and includes, for example, the promoters of RNA polymerase II and RNA-floor is meraz III. This definition also covers such regulatory elements of transcription (e.g., enhancers)that are sufficient for implementation-dependent promoter gene expression, controlled, specific for the cell type-specific tissue or temporary manner, or which are induced by external agents or signals.

In this document, "functionally linked" means a functional relationship between two or more segments of nucleic acid (e.g. DNA). Usually this means the functional relationship of the regulatory element, the transcription with the transcribed sequence. For example, a promoter functionally linked to the coding sequence, such as an open reading frame encoding a double-stranded RNA molecule, described in this document, if it stimulates or modulates the transcription of the coding sequence in the appropriate box. Basically, promoter regulatory elements of transcription, which is functionally linked to the transcribed sequence, physically located next to the transcribed sequence, that is, they are inCIS-position. However, some regulatory elements of transcription, such as enhancers, do not necessarily have to be physically next to or close to about the coding sequences, the transcription of which they reinforce.

By "promoter of RNA polymerase III" or "promoter RNA-Pol III" or "polymerase III promoter" or "promoter Paul III" is understood as any promoter invertebrates, vertebrates or mammals such as chickens, humans, mice, pigs, cattle, primates, monkeys and so on, which in nature in the cell binds or interacts with the RNA polymerase III transcription of its functionally related gene or any option, natural or artificially created, which will interact in the selected cell host with RNA polymerase III for transcription of functionally related nucleic acid sequence. Under the U6 promoter (for example, U6 chickens, U6 human, mouse U6, H1 promoter or 7SK promoter understand any promoter invertebrates, vertebrates or mammals or polymorphic variant or mutant, existing in nature, for interaction with RNA polymerase III transcription product of its cognate RNA, that is, RNA U6, H1 RNA or RNA 7SK, respectively. Examples of suitable promoters include cU6-1 (SEQ ID no:7), cU6-3 (SEQ ID no:8), cU6-4 (SEQ ID no:9) and c7SK (SEQ ID no:10).

If the host cell usedE. colithere are no restrictions, except that the vector should have "ori" to amplification and mass p is oductio vector in E. coli(for example, JM109, DH5α, HB101, or XL1Blue), and a marker gene for selection of transformed cellsE. coli(for example, the gene of resistance to drug taken using drugs, such as ampicillin, tetracycline, kanamycin, or chloramphenicol). For example, you can use the vector series M13 vectors series pUC, pBR322, pBluescript, pCR-Script, and the like. pGEM-T, pDIRECT, pT7, and the like can also be used to sublimirovanny and cut the gene encoding dsrnas, as well as the vectors described above.

With regard to expression vectors for use inE. colisuch vectors include JM109, DH5α, HB101, or XL1Blue, the vector should have a promoter, such as the lacZ promoter, the araB promoter or the T7 promoter, which can effectively contribute to the expression of the desired gene in theE. coli. Other examples of vectors are "QIAexpress system" (Qiagen), pEGFP, and pET (for a given vector as the owner it is preferable to use BL21, a strain expressing the RNA polymerase T7).

In addition to vectors forE. colivector, for example, can be an expression vector mammals (for example, pcDNA3 (Invitrogen), pEGF-BOS, pEF, and pCDM8), expression vector from insect cells (e.g., "baculovirus expression system Bac-to-BAC" (GibcoBRL) and pBacPAK8), expression vector from plants (for example, pMH1 and pMH2), expression vector from which Urusov animals (e.g., pHSV, pMV, and pAdexLcw), the expression vector of retroviruses (for example, pZIPneo), expression vector from yeast (for example, set "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), the expression vector ofBacillus subtilis(for example, pPL608 and pKTH50).

In order to Express the nucleic acid molecules in animal cells such as CHO cells, COS, Vero, and NIH3T3, the vector should have a promoter necessary for expression in such cells, for example, the SV40 promoter, the promoter MMLV-LTR, the EF1α promoter, CMV promoter, and so forth, and, more preferably, it has a marker gene for selecting transformants (for example, the gene of resistance to drug taken using drugs (e.g., neomycin, G418, and so forth).

Examples of vectors with such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

The nucleic acid molecules containing double-stranded region, according to the present invention can be Express in animals, for example, by embedding open frame(s) reading, coding(their) nucleic acid into an appropriate vector and introducing the retroviral vector method, liposomal way, cation-liposomal way, adenovirus way and so on. Used vectors include, but are not limited to, adenoviral vectors (e.g., pAdexlcw) and retroviral vectors (e.g., pZIPneo). Common ways m which nipulate genes, such as integration of nucleic acids according to the invention in a vector, can be performed by standard methods.

The present invention also relates to a cell-master, which is introduced exogenous nucleic acid molecule, typically in the vector of the present invention. Cell host according to this invention can be used as, for example, producing system for producing or expression of the nucleic acid molecule. For productsin vitroyou can use eukaryotic cells or prokaryotic cells.

Useful eukaryotic cells-owners may be the cells of animals, plants or fungi. As animal cells can be used mammalian cells such as CHO cells, COS, 3T3, myeloma, kidney newborn hamster (BHK), HeLa or Vero cells, MDCK cells, DF1 cells, cells of amphibians, such as oocytesXenopus, or insect cells such as Sf9 cells, Sf21 or Tn5. You can use CHO cells lacking DHFR gene (dhfr-CHO, or CHO K-1. The vector can be introduced into the cell of the host, for example, using the calcium-phosphate method, DEAE-dextranomer method, cation-liposomal way DOTAP (Boehringer Mannheim), electroporation, lipofection and so on.

Useful prokaryotic cells include cells of bacteria, such asE. colifor example, JM109, DH5a and HB101, orBacillus subtilis.

Composition

The present invention also relates to compositions containing a nucleic acid molecule containing a double-stranded region that can be entered in the eggs of birds. A composition comprising a nucleic acid molecule containing a double-stranded region may include pharmaceutically acceptable carrier, making the composition suitable for injection.

Suitable pharmaceutical carriers, excipients and/or diluents include, but are not limited to, lactose, sucrose, starch powder, talc powder, cellulose esters alkanovykh acid, magnesium stearate, magnesium oxide, crystalline cellulose, methylcellulose, carboxymethylcellulose, gelatin, glycerin, sodium alginate, antibacterial agents, antifungal agents, gum Arabic, Arabian gum, sodium and calcium salts of phosphoric and sulfuric acids, polyvinylpyrrolidone and/or polyvinyl alcohol, f is Zoologicheskii solution and water. In one embodiment, the implementation of the media, excipient and/or diluent is a phosphate-saline buffer solution or water.

The composition may also contain facilitate transfection agent. Promoting transfection tools used to facilitate uptake of nucleic acids in living cells, are well known in this field. Reagents that enhance the transfection include chemical family type of polycation, dendrimers, DEAE-dextran, block copolymers and cationic lipids. Preferably, facilitating transfection tool is a containing lipid compound (or drug)that creates a positively charged hydrophilic region and the hydrophobic acyl region, making possible the self-Assembly in aqueous solution in vials, known as micelles or liposomes, as well as lipopolymer.

Obviously, you can use any conventional medium or agent, provided that they are not incompatible with the compositions or methods according to the invention.

Introduction

The introduction of a nucleic acid molecule containing a double-stranded region (including composition comprising a nucleic acid molecule containing a double-stranded region), usually carried out by injection into the egg, and, as a rule, is injected into the air bag. The however is that that the air bag is the preferred route of administrationin ovothe introduction of the injection can be carried out in other areas, such as yolk SAC or allatoona liquid chorion. If the target for this introduction is not air bag, indicators of liluplanet can be slightly reduced, although not necessarily, in a commercially unacceptable magnitude. The mechanism is not critical to practice of the present invention, although it is preferable that the needle did not cause excessive damage to the eggs or tissues and organs of the developing embryo or Nazaraliev membranes surrounding the embryo.

If productive characteristic is gender, it is preferable to introduce a nucleic acid molecule within four days from the moment of deposition of the eggs.

As a rule, is appropriate syringe for subcutaneous injection with a needle about 22 caliber. The method according to the present invention is well adapted for use with an automatic device for injection, such as described in US 4903635, US 5056464, US 5136979 and US 20060075973.

The nucleic acid molecule is administered in an effective amount, sufficient to at least to some extent change the planned sign. As for sex, the change can be detected by comparing the corresponding number of samples, the processing is the R by the method according to the present invention, with the same number of unprocessed samples. A statistically significant difference in the gender of birds between the two groups would indicate that the effective number. Other methods of determining the effective amount to change gender or other characteristics, are within the competence of specialists in this field.

Preferably, the egg is administered from about 1 ng to 100 μg, more preferably, from about 100 ng to 1 μg of nucleic acid. Moreover, it is preferable to introduce the nucleic acid in the amount of from about 1 μl to 1 ml, more preferably, from about 10 μl to 500 μl.

EXAMPLES

Example 1 - Identification of molecules MSRC to reduce the production of DMRT1 protein in chickens

Selecting sequences MSRC aimed at DMRT1

Developed using Ambion pointer targets for miRNAs (www.ambion.com/techlib/misc/siRNA finder.html), the authors present invention have selected 30 of the predicted sequences of miRNAs for Dmrt1. Then 30 sequences of the miRNAs were subjected to screening for selection MSRC (table 1). There are several algorithms that are suitable for selection of potential sequences for specific miRNAs target genes. However, it is shown that many of these predicted miRNAs do not operate efficiently when the processing of the expressed MSRC. Taxman et al. (200) have developed an algorithm for predicting the effective molecules MSRC, and the inventors have created our own modification of the algorithm to improve predictions MSRC. The inventors have used a modified algorithm Taxman to 30 selected miRNAs to choose the sequence for testing as msrdc for specific knockdown of Dmrt1 gene expression.

When using the algorithm Taxman there are four criteria. Three of these criteria is calculated from the maximum number of 4 points. These criteria are: 1) C or G at the 5'end of the sequence = 1 point, A or T at the 5'-end = -1 point; 2) A or T at the 3'-end = 1 point, C or G at the 3'-end = -1 points; 3) 5 or more A or T in seven 3'reason = 2 points, 4 A or T in seven 3'reason = 1 point. Preferred sequences are MSRC with the maximum number of points. The fourth criterion is based on the calculation of free energy 6 Central grounds sequence MSRC (base 6-11 semantic chain hybridisable with bases 9-14 antisense chain). Preferred are MSRC with ΔG>-12,9 kcal/mol of the Central duplex. Modification of the algorithm Taxman is the use of other parameters of the free energy to predict the stability of duplex RNA, as described Freier et al. (1986). Based on the algorithm, the inventors chose the 6 sequences of miRNAs proposed by the pointer mishandle miRNAs, as a potentially effective MSRC to test their ability to perform the knockout gene expression of Dmrt1. The selected sequences are shown in bold in table 1 and their 5'- 3' sequence presented in table 2. These 6 sequences used to construct plasmids dd (ddRNAi) for the expression of 6 MSRC.

Construction of plasmids dd for the expression of selected MSRC

Promoters cU6-1 (GenBank incoming number DQ531567) and cU6-4 (DQ531570) chicken polymerase III were used as matrices for constructing expression plasmids dd for the selected MSRC for dmrt1 and control (EGFP or an unrelated protein) by one-step PCR (figure 1). In PCR to construct plasmids MSRC used primer TD175 paired with TH346 (Dmrt1-346), TH461 (Dmrt1-461), TH566 (Dmrt1-566), TH622 (Dmrt1-622), TH697 (Dmrt1-697), TH839 (Dmrt1-839) or TD195 (EGFP) (see table 3 for the sequence of the primer and details regarding specific amplified MSRC). Reverse primers in each PCR were designed so that they contained the last 20 NT (nucleotides) of each promoter sequence, semantic sequence MSRC, the loop and the antisense sequence MSRC, and were HPLC purified. Full amplificatoare products polygenic expression cluster ligated in pGEM-T Easy and then seque Aravali. The final expression plasmid MSRC used in the analyses of knockout genes, called pshDmrt1-346, pshDmrt1-461, pshDmrt1-566, pshDmrt1-622, pshDmrt1-697, pshDmrt1-839 and pshEGFP. Also constructed outside the control plasmid cU6-1 and used it as a test for comparison in the analysis of gene expression (see below). For this test plasmids direct primer TD135 used paired with the reverse primer TD149 containing the last 20 NT of the U6 promoter-1 hens and all other components outside MSRC. The PCR product ligated into pGEM-T Easy and sequenced.

Table 1 The selection algorithm sequences MSRC aimed at Dmrt1
MSRC	the 5' end points	Δ G center	the 3' end points	A+T 3'	Points

Table 2 The sequence MSRC for Dmrt1
MSRC	5'-3' sequence

Table 3 The sequence and details of the primers used
Name	The sequence 5' - 3'	Location/ description

Each plasmid for dd designed so that the beginning of each sequence MSRC at position +1 natural transcripts of mark (snRNA) U6. Site for restriction enzymeXhoI created below in the course of transcription from signal termination, to be able screening on the full-sized products MSRC built into pGEM-T Easy. All final expression vectors MSRC consisted of any of the full-length U6 promoters chickens, semantic sequence MSRC, the loop sequence, the antisense sequence MSRC, consistency termination and customersXhoI. a Sequence of loops used in all MSRC, was a 5' UUCAAGAGA 3'.

Testing the selected msrdc the knockout gene expression

Dmrt1

For testing MSRC off Dmrt1 used analysis of EC on the processes reporter gene. Reporter gene was a transcriptionally fused gene from the gene Dmrt1, embedded below during transcription from the 3' end of the gene enhanced green fluorescent protein (EGFP) in pEGFP-C (Clontech). Reporter plasmid was constructed as follows: Dmrt1 cDNA was back transcribable from total RNA extracted from 4-day embryo, and cloned into the multiple cloning site pCMV-Script (Stratagene). Insert Dmrt1 was removed from the cloning vector in the form of a fragment of NotI - EcoRI and cloned below in the course of transcription from a gene EGFP in pEGFP-C (Clontech). The obtained plasmid was named pEGFP-Dmrt1. This plasmid was transfusional cells DF-1 chicken and the expression of transcriptionally fused gene was confirmed by measuring the fluorescence of EGFP by flow cytometry, as described below. Cells DF-1 represent a stable cell line, chick embryo fibroblasts derived from an embryo EV-0 (ATCC, CRL-12203), and thus represent a model system for the study ofin ovoeffects of Rnci molecules.

Analyses on off Dmrt1 gene was performed by co transfection of cells DF-1 reporter plasmid pEGFP-Dmrt1 and each of the plasmids dd expressing specific for Dmrt1 and control MSRC. Experiments on joint transfection was carried out as follows: cells DF-1 (ATCC CRL-12203, chicken fibroblasts) were supported in the wet and the atmosphere, containing 5% CO₂at 37°C in modified according to the method of Dulbecco environment Needle (DMEM)containing 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 10% fetal calf serum (FCS), 2 mm L-glutamine with added penicillin (100 u/ml) and streptomycin (100 µg/ml). The DF1 cells were perceivable as necessary, using a 0.25% (wt./about.) a mixture of trypsin-ethylenediaminetetraacetic acid (EDTA).

Joint transfection of plasmids pEGFP-Dmrt1 and dd for analyses on off fused gene EGFP-Dmrt1 was performed in cells DF-1, were grown to 80-90% of confluently, 24-hole tablets for cultivation (Nunc) for analysis by flow cytometry. The cells were transfusional a total of 1 μg of plasmid DNA per well using the reagent for transfection with Lipofectamine™2000 (Invitrogen). The expression of EGFP was analyzed in transfected cells DF-1 through 60 hours after transfection using flow-cytometrics analysis transpency, carried out in three repetitions. Cells were treated with trypsin, using 100 ál of 0.25% of a mixture of trypsin-EDTA, besieged at 2000 rpm for 5 minutes, washed once in 1 ml of cold phosphate-saline buffer solution-A (PBSA) (Oxoid), twice in 1 ml FACS wash solution (PBSA + 1% FCS) and resuspendable in 250 ál of wash solution FACS. Flow-cytometrics analysis of samples was performed in a cell sorting device with Akti is the situation fluorescence FACScalibur (Becton Dickinson). Data collection and calculation of the mean values of fluorescence intensity (MFI) of samples for collaborative transfection in three repetitions were performed using CELLQuest software (Becton Dickinson). The results of the analysis to shut down gene are presented in figure 2. pshEGFP was used as a positive control. It is known that MSRC, expressed from this plasmid, now focused on the area of the EGFP fused transcript, and, as shown, reduces reporter fluorescence by about 50%. Compared with negative control representing outside MSRC, expressed with pshIrr found that are specific to Dmrt1 molecules MSRC repress expression of reporter gene in various degrees. Dmrt1-622 induced maximum off gene, which is around 60%.

Example 2 Identification of molecules MSRC to reduce the production of the protein of myostatin in chickens

Selecting sequences MSRC target is myostatin (Gdf8)

Developed using Ambion pointer targets for miRNAs (www.ambion.com/techlib/misc/siRNA finder.html)identified 79 of the predicted sequences of miRNAs for Gdf8 (table 4). Additional sequences of miRNAs that are optimized using the algorithm Taxman, are given in table 5. The inventors chose 3 of these sequences (Gdf8-258, Gdf8-913 Gdf8-1002) to construct the plasmid dd for expression MSRC (in bold in table 4).

Construction of plasmids dd for the expression of selected MSRC

The promoter cU6-1 (GenBank incoming number DQ531567) chicken polymerase III was used as a matrix for constructing expression plasmids dd for the selected MSRC to Gdf8 and cEGFP by one-step PCR (figure 1). In PCR to construct plasmids MSRC used primer TD135 paired with DS304 (Gdf8-253), DS305 (Gdf8-913), DS306 (Gdf8-1002) or TD148 (EGFP) (see table 6 for the sequence of the primer and details regarding specific amplified MSRC). Reverse primers in each PCR were designed so that they contained the last 20 NT of each promoter sequence, semantic sequence MSRC, the loop and the antisense sequence of the siRNA and conducted clearing by HPLC. Full amplificatoare products polygenic expression cluster ligated in pGEM-T Easy and then sequenced. The final expression plasmid MSRC used in the analyses of knockout genes, called pshGdf8-253, pshGdf8-913, pshGdf8-1002 and pshEGFP.

Table 4 The sequence MSRC for Gdf8
MSRC	5'-3' sequence	MSRC		5'-3' sequence

Table 5 The sequence of miRNAs for myostatin optimized using algorithm Taxman
Name	5' - 3' sequence

Each plasmid for dd designed so that the beginning of each sequence MSRC at position +1 natural transcripts of mark (snRNA) U6. Site for restriction enzymeXhoI created below in the course of transcription from signal termination, to be able screening on the full-sized products MSRC built into pGEM-T Easy. All final expression vectors MSRC consisted of full-length U6 promoter chickens, semantic sequence MSRC, consistency pet and, antisense sequences MSRC, consistency termination and customersXhoI. a Sequence of loops used in all MSRC, was a 5' UUCAAGAGA 3'.

Table 6 The sequence and details of the primers used
Name	The sequence 5'-3'	Location/ description

Testing the selected msrdc the knockout gene expression Gdf8

To test the three selected MSRC off Gdf8 gene used the analysis on the expression of reporter gene. Reporter gene was a transcriptionally fused gene of the Gdf8 gene embedded below during transcription from the 3' end of the gene enhanced green fluorescent protein (EGFP) in pEGFP-C (Clontech). Reporter plasmid was constructed as follows: cDNA Gdf8 back transcribable from total RNA isolated from 7-day embryo, and cloned into the multiple cloning site pGEM-T Easy (Promega). Insert Gdf8 removed from the cloning vector in the form of a NotI fragment and cloned below in the course of transcription from a gene EGFP in pEGFP-C (Clontech). The obtained p is amidu was named pEGFP-Gdf8. This plasmid was transfusional cells DF-1 chicken and the expression of transcriptionally fused gene was confirmed by measuring the fluorescence of EGFP by flow cytometry, as described below.

Analyses on off Gdf8 gene was performed by co transfection of cells DF-1 reporter plasmid pEGFP-Gdf8 and each of the plasmids dd expressing specific to Gdf8 or EGFP and control MSRC. Experiments on joint transfection was carried out as follows: cells DF-1 (ATCC CRL-12203, chicken fibroblasts) were maintained in a humid atmosphere containing 5% CO₂at 37°C in modified according to the method of Dulbecco environment Needle (DMEM)containing 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 10% fetal calf serum (FCS), 2 mm L-glutamine with added penicillin (100 u/ml) and streptomycin (100 µg/ml). The DF1 cells were perceivable as necessary, using a 0.25% (wt./about.) a mixture of trypsin-ethylenediaminetetraacetic acid (EDTA).

Joint transfection of plasmids pEGFP-Gdf8 and dd for analyses on off fused gene EGFP-Gdf8 held in the cells of DF-1, were grown to 80-90% of confluently, 8-hole slides (Nunc) for analysis by fluorescence microscopy. The cells were transfusional a total of 1 μg of plasmid DNA per well using the reagent for transfection with Lipofectamine™2000 (Invitrogen). The expression of EGFP was analyzed by the transfected cells DF-1 through 60 hours after transfection as follows: jointly transfetsirovannyh cells in the 8-hole slides were washed in PBSA, the frame slides were removed and the cover glass was placed on the monolayer of cells. Microscopic examination was performed using a fluorescent microscope (Leica DM LB (Leica Microsystems, Germany) and images were recorded at 50× magnification, using a color digital camera Leica DC300F (Leica Microsystems, Germany) and software for image processing Photoshop 7.0 (Adobe®). The results are presented in figure 3. Gdf8-1002 very effectively turn off the expression of the fused transcript and, therefore, is an excellent candidate for off natural transcript Gdf8.

Specialists in this area should recognize that it is possible to make numerous variations and/or modifications of the invention presented in specific embodiments, implementation, without changing the nature and scope of the invention described widely. As a consequence, real options implementation should be considered in all respects as illustrative and not restrictive.

In this application claims the priority of US 60/943708, filed on June 13, 2007, the full contents of which are incorporated herein by reference.

The full contents of all publications discussed or cited as references included in this document.

Any discussion of documents, acts, materials, devices, products and the like, included in this description is intended exclusively for the purpose of creating a context for the present invention. This should not be construed as an admission that any or all of the above is part of the prior art, or is a General knowledge in the field related to the present invention, existing before the priority date of each claim of this application.

LITERATURE

Amarzguioui et al. (2004) Biochem Biophys Res Commun 316: 1050-1058.

Elbashire et al. (2001) Nature 411: 494-498.

Hori et al. (2000) Mol Biol Cell 11: 3645-3660.

Freier et al. (1986) Proc Natl Acad Sci USA 83: 9373-9377.

Needleman and Wunsch (1970) J Mol Biol 48: 443-453.

O'neill et al. (2000) Dev Genes Evol 210: 243-249.

Raymond et al. (1999) Dev Biol. 215: 208-220.

Reynolds et al. (2004) Nat. Biotech., 22: 326-330.

Smith et al. (1999) Nature 402: 601-602.

Smith et al. (2000) Nature 407: 319-320.

Taxman et al. (2006) BMC Biotechnol, Jan 24, 6: 7.

Waterhouse et al. (1998) Proc Natl Acad Sci USA 95: 13959-13964.

1. How to change the sign of the bird, including the introduction in bird egg at least one RNA molecule containing a double-stranded region (dsrnas), where the RNA molecule leads to a reduction in the egg at least one RNA molecule and/or protein involved in sex determination, and where the method does not include electroporation eggs, and where the sign represents the floor.

2. How to change the sign of the bird, including the introduction in bird egg at least one molecule of P Is K, containing double-stranded region (dsrnas), where the RNA molecule leads to a reduction in the egg at least one RNA molecule and/or protein involved in sex determination, and where the RNA molecule is injected into the air SAC, yolk SAC or allantoin liquid chorion, and where the sign represents the floor.

3. The method according to claim 1 or 2, where dsrnas represents miRNAs or MSRC.

4. The method according to claim 1 or 2, where the molecule of nucleic acid reduces the level of protein encoded by gene DMRT1.

5. The method according to claim 4, where the nucleic acid molecule contains at least one nucleotide sequence selected from:
CCAGUUGUCAAGAAGAGCA (SEQ ID no:11)
GGAUGCUCAUUCAGGACAU (SEQ ID no:12)
CCCUGUAUCCUUACUAUAA (SEQ ID no:13)
GCCACUGAGUCUUCCUCAA (SEQ ID no:14)
CCAGCAACAUACAUGUCAA (SEQ ID no:15)
CCUGCGUCACACAGAUACU (SEQ ID no:16)
GGAGUAGUUGUACAGGUUG (SEQ ID no:17)
GACUGGCUUGACAUGUAUG (SEQ ID no:18)
AUGGCGGUUCUCCAUCCCU (SEQ ID no:19),
or any of them.

6. The method according to any one of claims 1, 2 or 5, where the nucleic acid molecule is administered by injection.

7. The method according to any one of claims 1, 2 or 5 where the bird choose from chickens, ducks, turkeys, geese, antamok and quail.