WO2016074503A1 - 一种y染色体修饰方法及其应用 - Google Patents

一种y染色体修饰方法及其应用 Download PDF

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WO2016074503A1
WO2016074503A1 PCT/CN2015/085703 CN2015085703W WO2016074503A1 WO 2016074503 A1 WO2016074503 A1 WO 2016074503A1 CN 2015085703 W CN2015085703 W CN 2015085703W WO 2016074503 A1 WO2016074503 A1 WO 2016074503A1
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seq
chromosome
animal
kit
suicide
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French (fr)
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戴蕴平
孙照霖
丁方荣
王海萍
李京
李玲
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中国农业大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos

Definitions

  • the invention relates to a Y chromosome modification method and an application thereof, and belongs to the field of biotechnology.
  • Animal sex control technology is a biotechnology that allows adult females to produce desired offspring of their offspring through human intervention in the normal reproductive process of animals. Gender control technology is of great importance in livestock production.
  • Second, controlling the sex ratio of offspring can increase the selection intensity and speed up the breeding process. By controlling the sex of the embryo, it can also overcome the heterosexual infertility phenomenon in the embryo transfer of cattle, and eliminate the harmful genes.
  • Sex control technology originated from the suggestion of insect "infertility technology", which is mainly used to prevent and control pests.
  • mosquitoes need to release a large number of male sterile mosquitoes into the environment. Therefore, gender separation techniques or sexual control techniques are required during the cultivation process, and a large number of male mosquitoes are obtained.
  • silkworm a high-yield trait insect, also needs sex control technology to obtain high-yield silk and low-consumption male silkworm.
  • the study of fish gender control has important practical significance for aquaculture. Because of many farmed fish, there are differences in biological or economic traits such as growth rate, maturity age, reproductive mode, body color, body size and individual size.
  • livestock animals also urgently need gender control techniques to improve production traits and improve economic efficiency, such as cattle, pigs, sheep and chickens.
  • Practices at home and abroad have shown that in the development of animal husbandry, the contribution rate of varieties reaches more than 40%. Therefore, animal breeding technology plays a vital or even irreplaceable role in promoting animal husbandry and even agricultural development.
  • the main livestock species are heavily dependent on imports. Among the major livestock breeds, the dependence of dairy breeds is 100%, and the dependence of pigs and chicken breeds is close to 90%!
  • insects mainly include: biological methods, traditional genetic methods and transgenic technology methods.
  • livestock mainly use physics, chemistry and the latest molecular techniques to identify sperm or early-pregnancy embryos and obtain the desired sex offspring.
  • This section mainly introduces research in this area.
  • the common methods for gender control of dairy cows mainly include: separation of X, Y sperm, identification of embryo sex and environmental control.
  • Flow cytometry The main reason is that the content of X and Y sperm DNA is different.
  • X sperm contains more DNA than Y sperm, so when dyed with the fluorescent dye Hoechst33342, X sperm absorbs more dyes and emits more fluorescence, so X and Y sperm can be distinguished, and then computer controlled
  • the fluorescent X-sperm has a positive charge, and the Y sperm has a negative charge. When it passes through a high-voltage electric field, it deflects in different directions to achieve separation. The resolution is up to 90%, but the sperm is separated by a flow cell separator.
  • H-Y antiserum is obtained by immunizing female animals, but H-Y antigen itself is a weak antigen, and the difference in immune response of animal individuals themselves is difficult to achieve better immune effect, and sperm is separated. Decreased vitality also affects the conception rate and litter size. At present, there are extensive studies on H-Y clone antibodies at home and abroad, and it is expected that future immunological methods can be applied to control gender.
  • FISH technique identification method fluorescence in situ hybridization (a technique for detecting in situ hybridization samples using non-radioactive fluorescent signals).
  • FISH is widely used due to its intuitive, fast, sensitive and convenient flexibility.
  • the basic principle is that the single-stranded DNA (probe) labeled with fluorescence is annealed to the DNA complementary thereto, and the position of the fluorescent signal on the chromosome is observed to reflect the condition of the corresponding gene. That is, the Y chromosome specific nucleic acid probe is used to hybridize with a specific sequence on the sperm, and then the fluorescent substance is calibrated, and the X sperm and the Y sperm are directly observed and distinguished under a fluorescence microscope.
  • This method is especially suitable for the case where the difference in DNA content between X sperm and Y sperm is very small, and reanalysis cannot guarantee accuracy.
  • the initial use of FISH on bovine sperm was able to clearly identify 79% of sperm.
  • the drawback of this method is that it takes a long time and the price of the reagent is high.
  • the principle is to detect the female or male in the embryonic stage by karyotyping, immunology and SRY-PCR identification, and then select the target sex embryo for subsequent operation.
  • the sex of the embryo is identified by identifying the sex chromosome types of the embryonic cells as XX and XY.
  • the operation process is as follows: a small amount of embryonic cells are fixed and stained by colchicine, and the sex chromosomes are examined, and the sex is determined according to the different bands of the chromosome in the middle of the cell division and the size and shape of the Y chromosome.
  • the accuracy of this method can reach almost 100. %, but the operation is cumbersome and difficult to apply in production. Currently used to verify the accuracy of other gender identification methods.
  • SRY-PCR method a method for identifying embryo sex using male-specific gene probes and PCR amplification techniques. The principle of this method is to design and synthesize a pair of specific primers on both sides of the SRY gene core sequence, respectively complementary.
  • DNA amplification is carried out in three steps of high temperature denaturation, low temperature annealing and chain extension, and the target sequence is amplified by more than one million times.
  • the SRY sequence can be amplified to be male, and vice versa.
  • the PCR amplification reaction can amplify the embryo of the target fragment into a male embryo, otherwise it is a female embryo.
  • Amplification of Y-chromosome DNA by PCR can greatly increase the sensitivity and improve the accuracy.
  • the embryos sampled by living tissue will not be greatly damaged and will not be easily contaminated by sperm adhered to the surface of the embryo or in the zona pellucida, and it is hoped that further freezing will occur.
  • the living cells to be taken are amplified by PCR, and the amplified products are subjected to agarose electrophoresis, and the specific fragments can be observed by staining.
  • the accuracy of the PCR-amplified embryos is more than 90%, which is the most ideal embryo so far.
  • One of the methods of gender identification Because of this, the method has been widely used in the sex identification of livestock, especially cattle and sheep embryos. However, this method has great damage to the embryo, and this analysis takes a long time, and the time of the embryo in vitro and the time of embryo transfer into the receptor are strictly limited. If the time cannot be synchronized, the transplantation efficiency will be affected. influences.
  • the SRY gene is a sex-control gene in most mammals. During mammalian sex development, the presence of the Y chromosome determines its development into males. The gene that plays a decisive role in this process is the Y-linked Sry, which is the only testis determinant in mammals. This gene is a gene directly related to sex in the mammalian Y chromosome, and the presence or absence of this gene. Mutation or not directly determines the sex phenotype of mammals. Individuals with the SRY gene with genotype XX will have a male phenotype, SRY Mutations in genes can also cause sexual reversal or gender abnormalities to some extent.
  • the SRY protein belongs to a subclass containing the HMG box (High mobiity group) and specifically binds to DNA sequence proteins. This subclass includes a variety of transcription factors that activate the expression of many male related genes downstream, thereby controlling male development.
  • the invention provides a Y chromosome modification method and application thereof.
  • the invention provides an animal Y chromosome modification method, which utilizes the TALEN method to modify a specific suicide element of a Y chromosome of an isolated animal somatic cell;
  • the suicide element can specifically kill the germ cell containing the Y chromosome, and does not cause harm to the body cells of the animal in which it is located;
  • the modification is performed on the sex determining gene SRY of the Y chromosome
  • the TALEN method utilizes a TALE protein to mutate a Y chromosome-specific target sequence while homologously recombining the suicide element to a target sequence position.
  • the method for modifying a suicide element specific to the Y chromosome of an isolated animal somatic cell is: expressing TALE protein-I and TALE protein-II in an isolated somatic cell of male animal A, a somatic cell in which the target sequence is mutated on the Y chromosome; and the suicide element is homologously recombined to the target sequence position, and the targeted integration of the suicide element at the target sequence on the Y chromosome of the isolated human somatic cell of the male animal A is achieved;
  • the amino acid sequence of the TALE protein-I is as shown in SEQ ID No. 3;
  • the amino acid sequence of the TALE protein-II is shown in SEQ ID No. 5;
  • the target sequence is shown in SEQ ID No. 1;
  • the target sequence is located on the SRY gene of the Y chromosome
  • TALE protein-I and TALE protein-II can specifically bind to the nucleotide sequences 4 to 18, 35 to 48 from the 5' end of SEQ ID No. 1, respectively, TALE protein-
  • the Fok I domain in I and TALE protein-II forms a dimer that exerts non-specific endonuclease activity, causing mutations in the sequence between TALE-I and TALE-II, respectively, to the specific binding site of the target sequence.
  • the method for expressing TALE protein-I and TALE protein-II in isolated somatic cells of male animal A is: recombinant expression plasmid containing TALE protein-I encoding gene and containing a recombinant expression plasmid encoding a TALE protein-II encoding gene is introduced into the isolated somatic cells of the male animal A;
  • the coding sequence of the TALE protein-I is as shown in SEQ ID No. 2;
  • the coding gene sequence of the TALE protein-II is shown in SEQ ID No. 4.
  • the homologous recombination of the suicide element to the target sequence position is to integrate the suicide element through the linearized homologous recombination vector at the target sequence position;
  • the linearized homologous recombination vector has a fragment of the target sequence homologous left arm-suicide element-target sequence homologous right arm.
  • sequence of the homologous left arm of the target sequence is from 5' in SEQ ID No. 9. The ends are shown in nucleotides 676 to 1636;
  • sequence of the homologous right arm of the target sequence is shown in nucleotides 5526 to 6449 from the 5' end in SEQ ID No. 9.
  • the suicide element comprises a sperm-specific promoter and a suicide gene
  • the suicide gene is activated by the sperm-specific promoter during sperm maturation, killing the sperm containing the Y chromosome in which the suicide gene is located;
  • the sequence of the suicide element is shown in nucleotides 1948 to 3730 of the SEQ ID No. 9 from the 5' end;
  • the sequence of the target sequence homologous left arm-suicide element-target sequence homologous right arm is shown in nucleotides 676 to 6449 from the 5' end in SEQ ID No. 9.
  • nucleotide sequence of the homologous recombinant vector is as shown in SEQ ID No. 9;
  • the linearization is linearization of the restriction endonuclease AhdI;
  • the animal is a cow.
  • a method for obtaining a female animal is also within the scope of the present invention, and the Y chromosome in the isolated somatic cell of male animal A is modified by any of the methods described above to obtain a transgenic cell with a suicide element;
  • the transgenic cell is a nuclear donor cell, and the somatic cell clone male animal B is obtained by somatic cell cloning technology; the somatic cell clone male animal B is the male parent, and the obtained offspring are female;
  • the animal is specifically a cow, such as a Holstein cow.
  • kits are also within the scope of the invention, and the kit contains at least one of the following 1)-4):
  • SEQ ID No. 3 The gene encoding the protein represented by SEQ ID No. 3 is specifically shown in SEQ ID No. 2;
  • the gene encoding the protein represented by SEQ ID No. 5 is specifically shown in SEQ ID No. 4.
  • the DNA molecule represented by SEQ ID No. 9 is also within the scope of protection of the present invention.
  • DNA molecules represented by nucleotides 676 to 6449 from the 5' end of SEQ ID No. 9 are also within the scope of the present invention.
  • DNA molecule represented by SEQ ID No. 2 is also within the scope of protection of the present invention.
  • the DNA molecule shown by SEQ ID No. 4 is also within the scope of the present invention.
  • the suicide element is specifically a DNA molecule represented by nucleotides 676 to 6449 from the 5' end in SEQ ID No. 9, specifically located on the SRY gene of the Y chromosome;
  • the animal is specifically a cow, such as a Holstein cow.
  • kit is also within the scope of the present invention, which kit contains at least one of the following 5)-6):
  • kits in the preparation of products for the selection and/or control of animal sex is also within the scope of the invention.
  • the animal is specifically a cow, such as a Holstein cow.
  • the principle of the present invention is to first modify the sex-determining gene SRY of the Y chromosome in the male animal A somatic cell to "self-kill the Y chromosome-containing sperm" element without destroying the SRY gene, and use it as a nucleus.
  • the donor cell, and then the somatic cell clone male animal B is obtained by somatic cell cloning technology. Since the sperm of the somatic cell clone male B matures, the DTA gene on the Y stain is specifically expressed, killing the sperm containing the Y chromosome, and thus containing Y The sperm of the chromosome die, and the sperm containing the X chromosome survives normally.
  • the sperm obtained by somatic cell cloning of the sperm of male B is all female, thereby achieving the purpose of gender control.
  • Figure 1 is a map of the pSRY-TALEN-F and pSRY-TALEN-R vectors.
  • Figure 2 is a linear map of the homologous recombination donor vector pPRM1-DTA after single digestion.
  • Figure 3 shows the results of PCR identification of cell clones.
  • Figure 4 shows the results of PCR identification of cloned cattle.
  • Figure 5 shows the results of semen PCR identification of cloned cattle.
  • Figure 6 is a PCR result of sex identification of progeny of cloned cattle.
  • DMEM/F12+10% FBS medium was prepared as follows: The medium was mixed with DMEM/F12 and fetal bovine serum (FBS), and the volume ratio of FBS to DMEM/F12 was 1:9.
  • FBS fetal bovine serum
  • pPGKloxPneo2 was purchased from addegen with catalog number 13443.
  • the preparation method of the mature liquid is as follows: M199 medium and fetal bovine serum (FBS) are mixed according to a volume ratio of 9:1 to obtain a mixed solution, and 0.01 U/mL bFSH (follicle-stimulating growth hormone), 0.01 U is added to the mixed solution. /mLbLH (luteinizing hormone) and 1 ⁇ g/mL estradiol.
  • FBS fetal bovine serum
  • the preparation method of the operation liquid was as follows: M199 medium and fetal bovine serum (FBS) were mixed at a volume ratio of 9:1 to obtain a mixed solution, and 7.5 ⁇ g/mL of cell relaxin B was added to the mixture.
  • FBS fetal bovine serum
  • the Zimmerman solution was prepared as follows: an aqueous solution containing 0.3 M mannitol, 0.1 M MgSO 4 , 0.05 M CaCl 2 , 0.5 mM HEPES, 0.05 g/100 mL BSA, pH 7.2, filtered through a 0.22 ⁇ m filter.
  • A23187 liquid was purchased from sigma and the article number was C9275.
  • the CR1aa broth was prepared as follows: 114 mM sodium chloride, 3.1 mM potassium chloride, 26.2 mM sodium hydrogencarbonate, 20.4 mM sodium pyruvate aqueous solution, pH 7.2, filtered through a 0.22 um filter.
  • Both A and B liquids are prepared after high temperature sterilization.
  • Example 1 Preparation of somatic cells of a bull of the Y-chromosome sex control gene SRY by "self-killing the sperm containing Y chromosome"
  • DMEM/F12+10% FBS was added to 6 mL at 37 °C.
  • the cells were cultured for 6-7 days in a 5% CO 2 incubator, and changed every 2 days. After the cells were confluent, they were digested with 0.25% trypsin for 2-3 times, and frozen in a batch of cell frozen solution.
  • a bull cell fibroblast cell line was established by in vitro culture operations such as primary culture, subculture, and freezing.
  • the selected target sequence is internal to the SRY gene and the sequence is as follows:
  • the nucleotide sequence from the 4th to the 18th and the 35th to the 48th nucleotide in the SEQ ID No. 1 from the 5' end is a portion which can specifically bind to the binding domain in the TALENs protein, and the middle portion of the binding site A recognition site for the FokI endonuclease of the TALENs protein.
  • FIG. 1 A schematic of the pSRY-TALEN-F and pSRY-TALEN-R vectors is shown in Figure 1.
  • SRY-F The coding gene sequence of SRY-F in pSRY-TALEN-F is shown in SEQ ID No. 2, the amino acid sequence of SRY-F is shown in SEQ ID No. 3, and SRY-F is TALE protein-I.
  • SRY-R The coding gene sequence of SRY-R in pSRY-TALEN-R is shown in SEQ ID No. 4, the amino acid sequence of SRY-R is shown in SEQ ID No. 5, and SRY-R is TALE protein-II.
  • TALE protein-I and TALE protein-II can specifically bind to nucleotide sequences 4 to 18, 35 to 48 from the 5' end of SEQ ID No. 1, respectively, TALE protein-I and TALE protein
  • the Fok I domain in -II forms a dimer that exerts non-specific endonuclease activity, causing mutations in the sequence between the TALE-I and TALE-II-specific binding sites, respectively; if TALENs play During the cleavage, the cell initiates a self-repair mechanism, and deletion or insertion of small fragments occurs at the cleavage site, and the peak image of the sequencing result is a heterozygous peak map.
  • the genomic DNA of the bovine fibroblasts of step one is extracted, and the PCR amplification products are obtained by using PCR as primers and primers B171 and B172 as primers.
  • the PCR amplification product was 943 bp, which was used as the 3'-end homology arm of the homologous recombination donor vector.
  • the PCR amplification product was ligated into the pMD-19T vector to obtain a recombinant plasmid, which was named pMD19T-3HR, and the pMD19T-3HR was sent for sequencing, and the result was correct.
  • the target sequence is homologous to the left arm, and the 1948th to the 3730th position is the "self-killing Y chromosome-containing sperm" element, the 5526th to the Position 6449 is the target sequence homologous to the right arm.
  • the "self-killing Y-containing sperm” component contains a sperm-specific promoter and a suicide gene.
  • the pPRM1-DTA vector was digested with AhdI to obtain a linearized fragment, and the linearized fragment was purified by absolute ethanol method for transfection of the bull cell fibroblast cell line.
  • the linearized carrier structure is shown in Figure 2.
  • PRM1 represents the sperm-specific promoter of bovine
  • DTA is the diphtheria toxin gene, ie suicide gene
  • PGK is a strong promoter of phosphoglycerin kinase
  • Neo r represents the neomycin resistance gene
  • polyA denotes a transcription termination signal.
  • the neomycin resistance gene is set to facilitate subsequent screening of transgenic cells.
  • telomeres plasmid 3 ug AhdI linearized pPRM1-DTA and 3 ug TALENs plasmid (pSRY-TALEN-F and pSRY-TALEN-R 1.5 ug each co-transfection step 1)
  • Transgenic cells are obtained from bull fibroblasts (cell number approximately 2 x 10 6 ).
  • the TALE protein-I and TALE protein-II encoded by pSRY-TALEN-F and pSRY-TALEN-R can respectively reach the 4th position from the 5' end in SEQ ID No. 1 on the Y chromosome of the bull cell fibroblast.
  • the 18th, 35th, and 18th nucleotide sequences specifically bind, and the Fok I domain in TALE protein-I and TALE protein-II forms a dimer that exerts non-specific endonuclease activity, resulting in TALE protein- Mutation of the sequence between the I and TALE protein-II and the specific binding site of the target sequence, respectively, and simultaneous introduction of the linearized pPRM1-DTA with the AhdI digestion of the foreign gene, "self-killing the sperm containing the Y chromosome”
  • the homologous recombination of the elements to the target sequence position and the realization of the Y chromosome sex control gene SRY in the bovine fibroblasts are precisely modified by the "self-killing Y-containing sperm" elements.
  • the genomic DNA of the transgenic cells was used as a template, and KOD2-F and KOD2-R were used as primers to carry out PCR amplification to obtain a PCR amplification product. If the PCR amplification product was a 2.1 kb fragment, the transgenic cells were positive transgenic cells. At the same time, the above experiment was carried out using ddH 2 O as a template as a control.
  • KOD2-F 5'-tgctcctgccgagaaagtat-3'; (SEQ ID No. 10)
  • Figure 3 shows that the transgenic cell clones numbered 1, 1 were identified as positive transgenic cell clones, and the PCR amplification products were sequenced and the results were correct.
  • the positive transgenic cells were used as somatic cells to clone nuclear donor cells, and the transgenic cells in the following examples were all positive transgenic cells.
  • the ovaries of adult Holstein cows were collected from the slaughterhouse, placed in physiological saline at 30 ° C, sent to the laboratory within 4 h, and the ovaries were washed three times in PBS at 37 ° C, and the diameter was extracted with a needle of 0.7 mm diameter.
  • the aqueous solution of the acid enzyme is shaken in the tube for 2-3 min, then gently blown with a glass tube to completely detach the cumulus cells from the oocyte, and the oocyte with uniform morphology and uniform cytoplasm and the first polar body is selected as the nucleus. Recipient cells.
  • Transgenic cells prepared in Example 1 with serum starvation for 2-4 days were digested with 0.25% trypsin for 2-4 min, and transgenic cells with a diameter of 10-12 ⁇ m were transferred into a 20 ⁇ m diameter glass tube.
  • the enucleated oocyte prepared in step 1 is placed in a zona pellucida, and then placed in a Zimmerman solution for equilibration (currently used) for 3-5 minutes, then placed in a fusion tank to rotate the egg cells to make the nuclear donor cells and enucleated.
  • the oocyte is in contact with the electric field, and is fused in a DC pulse field with a field strength of 2.5 kV/cm, with a pulse time of 10 ⁇ s, a pulse number of 2, and a pulse interval of 1 s (the fusion device is BTX).
  • the fused embryos were obtained and rapidly transferred to M199 containing 10% FBS in volume for several hours, and the fused embryos were selected (specifically, the recombination of donor cells and oocytes completely recombined) The embryo) is activated.
  • the fused embryos were placed in 5 mM A23187 solution for 5 minutes and then transferred to embryonic activator (M199 medium containing 5 ug/ml cytochalasin B, 10 ug/ml cycloheximide) for 5 hours.
  • embryonic activator M199 medium containing 5 ug/ml cytochalasin B, 10 ug/ml cycloheximide
  • the CR1aa containing FBS containing 5% by volume was exchanged, and the blastocyst development rate of the fusion embryo was observed after 7 days of culture in a culture medium at 38.5 ° C in a 5% CO 2 incubator. The results showed that the blastocyst development rate was 20 %-60%.
  • the cloning blastocyst of the 7th day which is excellent in morphology, was transferred into the uterine horn of the recipient cow (Holstein cow) of the same estrus. B-ultrasound was performed on recipient cows on the 30th day after transplantation to determine the conception of the fetus, and rectal examination was performed on the 60th and 90th day after transplantation to determine the pregnancy rate, and the pregnancy rate was 40%.
  • the pregnant cows are kept according to the conventional feeding method. After 280 days, the pregnant cows are normally delivered, and the somatic cloned bulls are obtained.
  • Somatic cell clones were collected from the ears of the bulls, and the genomic DNA was extracted.
  • the genomic DNA was used as a template, and KOD2-F and KOD2-R were used as primers to carry out PCR amplification to obtain PCR amplification products, and ddH 2 O was used as the primer.
  • the template was subjected to the above experiment as a control.
  • the gene knock-in is successful.
  • the somatic cell cloned bulls obtained in Example 2 were subjected to sperm collection, and then identified by PCR to determine whether the spermatozoa were all X-chromosome-containing sperm.
  • the specific steps are as follows:
  • the genomic DNA of the somatic cloned bull sperm was used as a template, and the SRY gene-specific PCR primers SRY-F and SRY-R were used as primers to carry out PCR amplification to obtain a PCR amplification product.
  • the above experiment was carried out using ddH 2 O as a template, and the above experiment was carried out using the genomic DNA of the sperm of the wild type Holstein cow breed bull as a template, and used as a control.
  • the primer sequences are as follows:
  • the sperm containing the Y chromosome specifically contains the SRY gene, so PCR amplification can amplify a 340 bp fragment, while a sperm containing only the X chromosome cannot amplify a band.
  • Figure 5 shows that the semen of the somatic cloned bull did not amplify the target band, indicating that the sperm of the somatic cell clone bull containing the Y chromosome automatically died, leaving only the normal sperm containing the X chromosome.
  • BSA (3.0mg / ml) in BO solution diluted and washed twice (at 1800 rev / min, centrifuged for 8 minutes), after removing the supernatant and then add 1ml of the same containing heparin (50ug / ml), caffeine ( After mixing 0.01 ⁇ mol/ml) and BSA (3.0mg/ml) BO solution, add 50ul to 50ul of fertilization solution containing 20-30 oocytes (containing 10mg/ml BSA, BO solution without fatty acid) After 5 hours of cultivating in the CCP, it is transferred to the in vitro culture medium CR1aa for 5 to 7 days. After the fertilized egg develops to the morula to the blastocyst stage and then transplanted to the pregnant mother, the offspring are subjected to PCR and morphology for sex. Identification.
  • PCR amplification was carried out by using genomic DNA of bovine ear cells as a template and SRY-F and SRY-R as primers to obtain PCR amplification products.
  • the above experiment was carried out using ddH 2 O as a template, and the above experiment was carried out using the genomic DNA of the sperm of the wild type Holstein cow breed bull as a template, and used as a control.
  • Figure 6 shows that the sperm of the wild-type Holstein dairy bull can amplify a 340 bp target fragment, and the progeny labeled 1 and 2 do not amplify the target band, and all the offspring are morphologically identified. Shown as a female cow.
  • the method provided by the present invention is a method for identifying and/or selecting the purpose of a specific sex animal by precisely genetically modifying male mammalian somatic cells.
  • the invention lays a foundation for selectively cultivating animals of specific gender and improving breeding efficiency.

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Abstract

提供了一种Y染色体修饰方法及其应用。该方法是利用TALEN方法对离体的动物体细胞的Y染色体进行特异性的自杀元件修饰。

Description

一种Y染色体修饰方法及其应用 技术领域
本发明涉及一种Y染色体修饰方法及其应用，属于生物技术领域。
背景技术
动物的性别控制(sex control)技术是通过对动物的正常生殖过程进行人为干预，使成年雌性动物产出人们期望性别后代的一门生物技术。性别控制技术在畜牧生产中意义重大。首先，通过控制后代的性别比例，可充分发挥受性别限制的生产性状(如泌乳)和受性别影响的生产性状(如生长速度、肉质等)的最大经济效益。其次，控制后代的性别比例可增加选种强度，加快育种进程。通过控制胚胎性别还可克服牛胚胎移植中出现的异性孪生不育现象，排除伴性有害基因的危害。
性别控制技术最早起源于昆虫“不育技术”的提出，昆虫不育技术主要用于防止和控制害虫。特别是蚊子，需要释放大量的雄性不育蚊子进入环境，因此，培养过程中需要性别分离技术或者性控技术，获得大量的雄性蚊子。同时，家蚕这种高经济性状昆虫，也需要性控技术获得高产丝、低消耗的雄性家蚕。鱼类性别控制的研究,对水产养殖来说,具有重要的实用意义。因为许多养殖鱼类,其生物学或经济性状诸如生长率、成熟年龄、繁殖方式、体色、体型和个体大小等雌、雄鱼之间存在差异。因此,人们可以根据需要专门生产全雌或全雄苗种进行单性养殖以提高经济效益。除了上述物种，家畜动物也迫切需要性别控制技术，从而提高生产性状，提高经济效益，例如牛、猪、羊和鸡等等。国内外实践表明，在畜牧业发展中，品种贡献率达到40％以上，因此动物育种技术在促进畜牧业乃至农业发展中发挥至关重要甚至是不可替代的作用。然而在我国，主要畜牧类品种则严重依赖于进口，在主要的畜牧类品种中，奶牛品种依赖程度达100％，猪、鸡品种依赖程度接近90％！这意味着动物农业整个产业链的源头——品种严重依赖于国际市场。因此如果我们国家要进一步提高畜牧业的效率、进一步保证畜牧业产业链的安全，动物育种则是重中之重。因此，发展性别控制技术可以大大促进动物育种的发展，大大提高经济效益。
目前，从昆虫到家畜发展了很多性别控制方法，其中昆虫主要包括：生物学法、传统遗传学法和转基因技术法。然而，家畜主要利用物理、化学和最新的分子技术的方法，对精子或早期受孕胚胎进行鉴定，获得想要的性别后代。这里主要介绍这方面研究。目前奶牛性别控制的常用方法主要包括：X，Y精子的分离、胚胎性别的鉴定和环境控制等方法。
第一，X，Y精子的分离。
原理根据X，Y精子在物理(体积，密度，电荷，运动性)和化学(DNA含量，表面抗原)等方面的差异，建立的对精子进行分选的各种方法，包括沉降法、电泳法、离心法及其目前应用比较多的流式细胞分离法、免疫学和FISH技术鉴定 方法。
流式细胞分离法：主要依据是X、Y精子DNA的含量不同。一般来说，X精子比Y精子含有较多DNA，所以用荧光染料Hoechst33342染色时，X精子吸收的染料多，发出的荧光也强，就此可以分辨出X与Y精子，然后再利用计算机控制使荧光强的X精子带上正电荷，Y精子带上负电荷，在通过高压电场时便向不同的方向偏转，从而达到分离目的，分辨率可达90％，但用流式细胞分离器分离精子时，精子需要一个个通过，这样就必须稀释精液，这就会造成精子的运动能力下降，而且荧光染料对精子有毒害作用，加之效率太低仪器价格昂贵，在授精后还有产仔数和妊娠率下降的情况，还无法用于生产实践。
免疫学方法：随着免疫学的发展人们发现在雄性组织包括Y精子，存在有H—Y抗原。雄性组织免疫雌性动物产生H—Y抗体，且只有Y精子才能表达H—Y抗原，因而利用H—Y抗体检测精子质膜上存在的H—Y抗原，再通过免疫或分离方法获得X、Y精子。1982年，研究者Zavos把H—Y抗血清注入母兔阴道内，15分钟后输精，所产生的雌性兔占74.2％。H—Y抗血清是利用免疫雌性动物后而获取的，但H—Y抗原自身是一种弱抗原，加之动物个体自身对免疫反应的差异，很难起到较好的免疫效果，而且分离精子活力下降也会影响受胎率和产仔数。目前国内外对H—Y克隆抗体都有广泛的研究，可以期望未来免疫学方法能在控制性别上得到应用。
FISH技术鉴定法：荧光原位杂交技术(是一种利用非放射性的荧光信号对原位杂交样本进行检测的技术)。FISH由于其直观，快速，敏感性高和方便灵活越来越得到广泛的应用。基本原理是：将标记了荧光的单链DNA(探针)和与其互补的DNA退火杂交，通过观察荧光信号在染色体上的位置来反映相应基因的情况。即利用Y染色体特异核酸探针与精子上的特定的序列杂交，而后标定荧光物质，在荧光显微镜下直接观察并区分X精子和Y精子。该方法特别适用于X精子和Y精子DNA含量差别很微小，重分析不能保证准确性的情况。最初运用FISH在牛精子上的结果是可清楚鉴定79％的精子。但是该方法的缺陷是耗费时间长，且试剂的价格较高。
第二：胚胎性别的鉴定
原理是在胚胎期，利用核型分析法、免疫学法和SRY-PCR鉴定法检测其雌性还是雄性，从而选着目的性别胚胎进行后续操作。
核型分析法：通过查明胚胎细胞的性染色体类型为XX型和XY型来鉴定胚胎的性别。操作过程为：取少量的胚胎细胞经秋水仙素处理固定染色，检查性染色体，根据染色体在细胞分裂中期不同的谱带和Y染色体的大小形态来判定性别，这种方法准确率几乎可达到100％，但操作繁琐，难以在生产中应用。目前主要用来验证其他性别鉴定方法的准确率。
免疫学法：先将8细胞桑葚胚期的胚胎与H—Y抗体反应30 min，再与异硫氰酸盐荧光素(FITC)标记的免疫球蛋白IgM抗体反应，然后在荧光显微镜下检查 胚胎是否带有荧光素，若有则判定为H—Y+胚胎，不显荧光则为H—Y-胚胎。在牛有89％的雌性鉴定准确率，猪有81％的鉴定准确率，绵羊有85％，的鉴定准确率。
SRY-PCR法：是一种利用雄性特异性基因探针和PCR扩增技术鉴定胚胎性别的方法，该方法的原理：在SRY基因核心序列的两侧设计并合成一对特异性引物，分别互补于扩增序列的两条链，在TaqDNA聚合酶和胚胎细胞DNA存在的条件下，经高温变性，低温退火和链延伸三个步骤进行DNA扩增，将靶序列扩增至上百万倍以上，经电泳检测扩增结果，能扩增出SRY序列的为雄性，反之为雌性。Herr等在1990年首先成功的建立了牛胚胎性别鉴定的PCR法。即通过合成SRY基因或其他Y染色体上特异性片段的部分序列作为引物，在一定条件下PCR扩增反应能扩增出目标片段的胚胎为雄性胚胎，否则为雌性胚胎。通过PCR扩增Y染色体DNA可大大增加灵敏度，改善准确率，经活组织取样的胚胎不会有很大的损伤且不易被黏附在胚胎表面或透明带里的精子污染，还有希望进一步冷冻，将采取的活细胞经PCR扩增，扩增产物经琼脂糖电泳，染色即可观察有无特异性片段，用PCR扩增的胚胎鉴定的准确率可达90％以上是目前为止最为理想的胚胎性别鉴定方法之一。正因为如此该方法已被广泛应用在家畜，特别是牛，羊胚胎的性别鉴定。但这种方法对胚胎具有很大的损伤，且这种分析需要较长时间，而胚胎在体外的时间和胚胎移入受体时间受到严格的限制，如果在时间上不能同步，将使移植效率受到影响。为了解决这个问题，人们不得不将进行PCR分析的胚胎暂时冷冻保存起来，待PCR结果出来后再进行解冻，这样大大增加了操作的难度，并对胚胎产生进一步的伤害。总之，目前的这些方法不能满足生产应用，急需新的方法。
动物转基因技术是生物技术领域的研究热点，特别是目前最新发展的人造核酸酶介导的精确地基因组编辑技术，它的应用范围已经渗透到基础研究、农业、医药等诸多领域。1997年，首例体细胞克隆绵羊“Dol ly”诞生的报道引起了社会各界的强烈关注，同时也标志着生物领域的一种新的技术—哺乳动物体细胞核移植技术的成功建立。同年报道的首例利用体细胞核移植技术培育出的转基因绵羊“Pol ly”的诞生才是动物转基因研究领域真正的里程碑，它掀开了体细胞核移植技术用于生产转基因大家畜的新篇章,同时伴随着“人造核酸酶技术”ZFN/TALEN和Cas9等精确基因组编辑技术的出现和发展，与传统的大动物体细胞核移植技术相结合，可以实现大动物精确基因组修饰及其多基因修饰，大大加快了大动物转基因技术的发展。尽管，转基因技术不断进展，但是利用此技术进行大动物性别控制没有研究。
SRY基因是大多数哺乳动物的性别控制基因，在哺乳动物的性别发育过程中，Y染色体的存在决定其向雄性发育。在这一过程中起决定作用的基因是Y染色体连锁的Sry，它是哺乳动物唯一的睾丸决定因子，该基因是位于哺乳动物Y染色体上与性别发生直接相关的基因,该基因的有无和突变与否直接决定了哺乳动物的性别表型。基因型为XX的带有SRY基因的个体会以雄性表型存在，SRY 基因的突变也会在一定程度上引起性反转或性别异常，2013年研究人员报道SRY基因敲除小鼠，丧失雄性特征，变为雌性。SRY蛋白属于含有HMG盒(High mobi l itygroup)并特异结合于DNA序列蛋白的一个亚类,该亚类包括多种转录因子,可以激活下游很多雄性相关基因的表达，从而控制雄性发育。
发明公开
本发明提供了一种Y染色体修饰方法及其应用。
本发明提供一种动物Y染色体修饰方法，是利用TALEN方法对离体的动物体细胞的Y染色体进行特异性的自杀元件修饰；
所述自杀元件能特异性杀死其所在的含有Y染色体的生殖细胞，同时对其所在的动物的体细胞不产生危害；
所述修饰是在Y染色体的性别决定基因SRY上进行修饰；
所述TALEN方法是利用TALE蛋白对Y染色体特异靶序列进行突变，同时将自杀元件同源重组到靶序列位置。
上述方法中，所述对离体的动物体细胞的Y染色体进行特异性的自杀元件修饰的方法为：使TALE蛋白-Ⅰ和TALE蛋白-Ⅱ在雄性动物A的离体体细胞中表达，得到Y染色体上靶序列发生突变的体细胞；同时将自杀元件同源重组到靶序列位置，实现自杀元件在雄性动物A的离体体细胞Y染色体上靶序列处的定点整合；
所述TALE蛋白-Ⅰ的氨基酸序列如SEQ ID No.3所示；
所述TALE蛋白-Ⅱ的氨基酸序列如SEQ ID No.5所示；
所述靶序列如SEQ ID No.1所示；
所述靶序列位于Y染色体的SRY基因上；
所述TALE蛋白-Ⅰ和TALE蛋白-Ⅱ能分别与SEQ ID No.1中自5’末端起第4位至第18位、第35位至第48位核苷酸序列特异结合，TALE蛋白-Ⅰ和TALE蛋白-Ⅱ中的Fok I功能域形成二聚体，发挥非特异性内切酶活性，使得TALE蛋白-Ⅰ和TALE蛋白-Ⅱ分别与靶序列特异结合位点之间的序列发生突变。
上述任一所述的方法中，所述使TALE蛋白-Ⅰ和TALE蛋白-Ⅱ在雄性动物A的离体体细胞中表达的方法为：将含有TALE蛋白-Ⅰ编码基因的重组表达质粒和含有TALE蛋白-Ⅱ编码基因的重组表达质粒导入所述雄性动物A的离体体细胞中；
所述TALE蛋白-Ⅰ的编码基因序列如SEQ ID No.2所示；
所述TALE蛋白-Ⅱ的编码基因序列如SEQ ID No.4所示；
所述将自杀元件同源重组到靶序列位置为将所述自杀元件通过线性化的同源重组载体整合在所述靶序列位置上；
所述线性化的同源重组载体上具有靶序列同源左臂-自杀元件-靶序列同源右臂的片段。
上述任一所述的方法中，所述靶序列同源左臂的序列如SEQ ID No.9中自5’ 末端起第676位至第1636位核苷酸所示；
所述靶序列同源右臂的序列如SEQ ID No.9中自5’末端起第5526位至第6449位核苷酸所示。
上述任一所述的方法中，所述自杀元件包含精子特异性启动子和自杀基因；
所述自杀基因在精子成熟过程中被所述精子特异性启动子启动表达，杀死所述自杀基因所在的含有Y染色体的精子；
所述自杀元件的序列如SEQ ID No.9中自5’末端起第1948位至第3730位核苷酸所示；
所述靶序列同源左臂-自杀元件-靶序列同源右臂的的序列如SEQ ID No.9中自5’末端起第676位至第6449位核苷酸所示。
上述任一所述的方法中，所述同源重组载体的核苷酸序列如SEQ ID No.9所示；
所述线性化为限制性内切酶AhdI线性化；
所述动物为牛。
一种获得雌性动物的方法也属于本发明的保护范围，是利用上述任一所述的方法对雄性动物A的离体体细胞中的Y染色体进行修饰，得到带有自杀元件的转基因细胞；以转基因细胞为核供体细胞，通过体细胞克隆技术得到体细胞克隆雄性动物B；以体细胞克隆雄性动物B为父本，获得的后代均为雌性；
所述动物具体为牛,例如荷斯坦奶牛。
一种试剂盒也属于本发明的保护范围，该试剂盒含有如下1)-4)的至少一种物质：
1)含有SEQ ID No.3所示蛋白的编码基因的DNA分子、重组载体、表达盒、转基因细胞系或重组菌；
2)含有SEQ ID No.5所示蛋白的编码基因的DNA分子、重组载体、表达盒、转基因细胞系或重组菌；
3)SEQ ID No.9所示的DNA分子或含有该分子的转基因细胞系或重组菌；
4)含有SEQ ID No.9中自5’末端起第676位至第6449位核苷酸所示的DNA分子、重组载体、表达盒、转基因细胞系或重组菌；
所述SEQ ID No.3所示的蛋白的编码基因具体如SEQ ID No.2所示；
所述SEQ ID No.5所示的蛋白的编码基因具体如SEQ ID No.4所示。
SEQ ID No.9所示的DNA分子也属于本发明的保护范围；
和/或，
含有SEQ ID No.9中自5’末端起第676位至第6449位核苷酸所示的DNA分子也属于本发明的保护范围；
和/或,
SEQ ID No.2所示的DNA分子也属于本发明的保护范围；
和/或,
SEQ ID No.4所示的DNA分子也属于本发明的保护范围。
上述试剂盒或上述DNA分子在制备自杀元件修饰动物离体体细胞Y染色体的产品中的应用也属于本发明的保护范围；
或，
上述试剂盒或上述DNA分子在制备Y染色体上带有自杀元件的雄性动物的产品中的应用也属于本发明的保护范围；
或，
上述试剂盒或上述DNA分子在制备Y染色体上带有自杀元件的雄性动物细胞的产品中的应用也属于本发明的保护范围；
所述自杀元件具体如SEQ ID No.9中自5’末端起第676位至第6449位核苷酸所示的DNA分子，具体位于Y染色体的SRY基因上；
或，
上述试剂盒或上述DNA分子在制备用于动物性别的选择和/或控制的产品中的应用也属于本发明的保护范围；
或，
上述试剂盒或上述DNA分子在动物繁育中的应用也属于本发明的保护范围；
所述动物具体为牛,例如荷斯坦奶牛。
另一种试剂盒也属于本发明的保护范围，该试剂盒含有如下5)-6)的至少一种物质：
5)SEQ ID No.3所示蛋白；
6)SEQ ID No.5所示蛋白。
上述试剂盒在制备自杀元件修饰动物离体体细胞Y染色体的产品中的应用也属于本发明的保护范围；
或，
上述试剂盒在制备Y染色体上带有自杀元件的雄性动物的产品中的应用也属于本发明的保护范围；
或，
上述试剂盒在制备Y染色体上带有自杀元件的雄性动物细胞的产品中的应用也属于本发明的保护范围；
或，
上述试剂盒在制备用于动物性别的选择和/或控制的产品中的应用也属于本发明的保护范围；
或，
上述的试剂盒在动物繁育中的应用也属于本发明的保护范围。
所述动物具体为牛,例如荷斯坦奶牛。
本发明的原理是首先对雄性动物A体细胞中的Y染色体的性别决定基因SRY进行“自我杀死含Y染色体精子”元件修饰，同时不破坏SRY基因，将其作为核 供体细胞，再通过体细胞克隆技术得到体细胞克隆雄性动物B，由于体细胞克隆雄性动物B的精子成熟后，Y染色上的DTA基因特异表达，杀死含有Y染色体的精子，因此含Y染色体的精子死亡，而含X染色体的精子正常存活，体细胞克隆雄性动物B的精子进行受精获得的个体全部是雌性，从而达到性别控制的目的。
附图说明
图1为pSRY-TALEN-F和pSRY-TALEN-R载体图谱。
图2为同源重组供体载体pPRM1-DTA单酶切后的线性图谱。
图3为细胞克隆的PCR鉴定结果。
图4为克隆牛的PCR鉴定结果。
图5为克隆牛的精液PCR鉴定结果。
图6为克隆牛的后代性别鉴定的PCR结果。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
DMEM/F12+10％FBS培养基按照如下方法配制：该培养基由DMEM/F12和胎牛血清(FBS)混匀而成，FBS与DMEM/F12的体积比为1：9。
pPGKloxPneo2购自addegen，产品目录号为13443。
成熟液的制备方法如下：将M199培养基和胎牛血清(FBS)按照体积比9:1混匀，得到混合液，在混合液中加入0.01U/mL bFSH(促卵泡生长激素)、0.01U/mLbLH(促黄体生成素)和1μg/mL雌二醇。
操作液的制备方法如下：将M199培养基和胎牛血清(FBS)按照体积比9:1混匀，得到混合液，在混合液中加入7.5μg/mL细胞松驰素B。
Zimmerman液按照如下方法配制：含有0.3M甘露醇、0.1M MgSO4、0.05M CaCl2、0.5mM HEPES、0.05g/100mL BSA的水溶液，pH7.2，用0.22μm滤膜过滤
A23187液购自sigma，货号为C9275。
CR1aa培养液按照如下方法配制：114mM氯化钠、3.1mM氯化钾、26.2mM碳酸氢钠、20.4mM丙酮酸钠的水溶液，PH7.2，用0.22um滤膜过滤。
BO液的配制：
(1)A液(100ml)
Figure PCTCN2015085703-appb-000001
(2)B液(100ml)
NaHCO3    1.1552g
超纯水  100ml定容。
A、B液均经高温消毒后备用。
(3)BO液(100ml)
A液 80ml
B液 20ml
丙酮酸钠 0.0138g
青霉素 3.1mg
链霉素 3.1mg
肝素钠 3mg。
实施例1、“自我杀死含Y染色体精子”元件精确修饰Y染色体性别控制基因SRY的种公牛的体细胞的制备
一、种公牛成纤维细胞系的建立
取荷斯坦奶牛种公牛的耳部皮肤组织，将耳部下缘背侧去毛后用体积百分含量为70％的乙醇水溶液清洗干净，再用刀片从耳部下缘背侧剔取面积为1cm2左右的皮肤，置于0℃的DMEM/F12培养基中尽快运回实验室，以PBS和体积百分含量为70％的乙醇水溶液清洗数遍后剪碎成1mm3左右的小块，DMEM/F12清洗2遍后分批植块于含1mL DMEM/F12+10％FBS的25cm2的培养瓶中，待组织块贴壁牢固后再补加DMEM/F12+10％FBS至6mL，于37℃、5％CO2培养箱培养6-7d，每2d换液1次，待细胞生长汇合后，以0.25％胰蛋白酶消化传代2-3次，分批以细胞冻存液冻存。这样，经原代培养、传代培养、冷冻等体外培养操作，建立了种公牛成纤维细胞系。
二、靶点序列的确定
选定的靶序列在SRY基因内部，序列如下：
5’-ctTTCTTGTGCTTATTTTCAATATTGACTTCCTTACTCTCGCTAACAAag-3’(SEQ ID No.1)
SEQ ID No.1中自5’末端起第4位至第18位、第35位至第48位核苷酸序列为可被TALENs蛋白中的结合功能域特异结合的部分，结合位点中间部分为TALENs蛋白的FokⅠ内切核酸酶切割识别位点。
三、构建作用于SRY基因的TALEN
pSRY-TALEN-F和pSRY-TALEN-R载体的示意图如图1所示。
pSRY-TALEN-F中SRY-F的编码基因序列如SEQ ID No.2所示，SRY-F的氨基酸序列如SEQ ID No.3所示，SRY-F即为TALE蛋白-Ⅰ。
pSRY-TALEN-R中SRY-R的编码基因序列如SEQ ID No.4所示，SRY-R的氨基酸序列如SEQ ID No.5所示，SRY-R即为TALE蛋白-Ⅱ。
TALE蛋白-Ⅰ和TALE蛋白-Ⅱ能分别与SEQ ID No.1中自5’末端起第4位至第18位、第35位至第48位核苷酸序列特异结合，TALE蛋白-Ⅰ和TALE蛋白 -Ⅱ中的Fok I功能域形成二聚体，发挥非特异性内切酶活性，使得TALE蛋白-Ⅰ和TALE蛋白-Ⅱ分别与靶序列特异结合位点之间的序列发生突变；如果TALENs蛋白发挥切割作用，细胞会启动自身修复机制，在切割位点会出现小片段的删除或者插入，测序结果峰图为杂合峰图。
四、构建同源重组供体载体pPRM1-DTA
1、NotI单酶切p2014Gene-1，得到3070bp的基因片段(如SEQ ID No.6所示)，该基因片段含有同源供体载体的5’端同源臂及“自我杀死含Y染色体精子”元件；NotI单酶切pPGKloxPneo2，得到6307bp的载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为pDonor-5HR-DTA，将pDonor-5HR-DTA送测序，结果正确。
2、提取步骤一的种公牛成纤维细胞的基因组DNA，以其为模板，以引物B171和B172为引物，进行PCR扩增，得到PCR扩增产物。
B171：5’-AAGGATGCAAGCTAGCCTTCCTTACTCTCGCTAACAA-3’；(SEQ ID No.7)
(下划线所示序列为NheI酶切识别位点)
B172：5’-ATGCAAGTGCGTCGACATCAGATTAATCAGACAGGAT-3’。(SEQ ID No.8)
(下划线所示序列为SalI酶切识别位点)
该PCR扩增产物为943bp，将其作为同源重组供体载体的3’端同源臂。
将该PCR扩增产物连接pMD-19T载体，得到重组质粒，将其命名为pMD19T-3HR，将pMD19T-3HR送测序，结果正确。
3、NheI和SalI双酶切pMD19T-3HR，得到943bp的基因片段；NheI和SalI双酶切pDonor-5HR-DTA，得到9.3kb的载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为pPRM1-DTA，将pPRM1-DTA送测序，结果正确。
pPRM1-DTA的序列如SEQ ID No.9所示。
SEQ ID No.9中自5’末端起第676位至第1636位为靶序列同源左臂、第1948位至第3730位为“自我杀死含Y染色体精子”元件、第5526位至第6449位为靶序列同源右臂。
“自我杀死含Y染色体精子”元件包含精子特异性启动子和自杀基因。
五、pPRM1-DTA载体线性化
用AhdI单酶切pPRM1-DTA载体，得到线性化片段，并用无水乙醇法纯化回收线性化片段，用于转染种公牛成纤维细胞系。
线性化载体结构如图2所示。
图2中，5HR和3HR代表同源重组的位置，PRM1表示牛的精子特异启动子；DTA为白喉毒素基因即自杀基因；PGK为磷酸甘油激酶强启动子；Neor表示新霉素抗性基因；polyA表示转录终止信号。
新霉素抗性基因是为了便于后续的转基因细胞的筛选所设置。
六、基因转染
pPRM1-DTA与TALENs质粒的细胞共转染：将3ug AhdI酶切线性化的pPRM1-DTA及3ug TALENs质粒(pSRY-TALEN-F和pSRY-TALEN-R各1.5ug共转 染步骤一制备的种公牛成纤维细胞(细胞数约2 X 106)得到转基因细胞。
pSRY-TALEN-F和pSRY-TALEN-R分别编码的TALE蛋白-Ⅰ和TALE蛋白-Ⅱ能分别与种公牛成纤维细胞Y染色体上的SEQ ID No.1中自5’末端起第4位至第18位、第35位至第18位核苷酸序列特异结合，TALE蛋白-Ⅰ和TALE蛋白-Ⅱ中的Fok I功能域形成二聚体，发挥非特异性内切酶活性，使得TALE蛋白-Ⅰ和TALE蛋白-Ⅱ分别与靶序列特异结合位点之间的序列发生突变；同时将带有外源基因的AhdI酶切的线性化pPRM1-DTA导入后，“自我杀死含Y染色体精子”元件同源重组到靶序列位置，实现种公牛成纤维细胞中的Y染色体性别控制基因SRY被“自我杀死含Y染色体精子”元件精确修饰。
七、PCR鉴定阳性转基因细胞
以转基因细胞的基因组DNA为模板，以KOD2-F和KOD2-R为引物，进行PCR扩增，得到PCR扩增产物，如果PCR扩增产物为2.1kb的片段，表明转基因细胞为阳性转基因细胞，同时以ddH2O为模板，进行上述实验，作为对照。
KOD2-F：5’-tgctcctgccgagaaagtat-3’；(SEQ ID No.10)
KOD2-R：5’-AAACAGTCTGTGAAGTTACCT-3’。(SEQ ID No.11)
结果如图3所示。
图3表明，标号1、6的转基因细胞克隆鉴定为阳性转基因细胞克隆，并对PCR扩增产物进行测序，结果正确。将阳性转基因细胞作为体细胞克隆核供体细胞，下述实施例中的转基因细胞均为阳性转基因细胞。
实施例2、利用“自我杀死含Y染色体精子”元件精确修饰Y染色体性别控制基因SRY的种公牛的体细胞培育体细胞克隆种公牛
一、卵母细胞的成熟培养
从屠宰厂收集成年荷斯坦奶牛的卵巢，置于30℃的生理盐水，在4h内送到实验室，将卵巢在37℃的PBS液中清洗三遍后，以直径为0.7mm针头抽取直径为2-8mm的卵泡，回收形态均匀、结构致密的卵丘-卵母细胞复合体(COCs)，以成熟液洗涤两遍，然后将卵丘-卵母细胞复合体以50-60枚/孔放入含成熟液的4孔板，在38.5℃、5％CO2培养箱中成熟培养18-20h后，得到成熟的细胞，将成熟的细胞放入盛有含体积百分含量0.1％的透明质酸酶的水溶液的管内振荡2-3min后，再用玻璃管轻轻吹打，使卵丘细胞与卵母细胞完全脱离，选择形态完整，细胞质均匀并带有第一极体的卵母细胞为核受体细胞。
二、体细胞克隆种公牛的获得
1、将带有第一极体的卵母细胞移入操作液中，在200倍显微镜下用玻璃针于极体上方将透明带切一小口，再用内径为20μm的玻璃管将第一极体以及其下方的卵母细胞内的染色体一并吸除，再放入含有体积百分含量20％FBS的M199溶液中洗三遍后，得到去核的卵母细胞，将其置于培养箱中备用。
2、将血清饥饿2-4d的实施例1制备的转基因细胞(核供体细胞)用0.25％胰蛋白酶(trypsin)消化2-4min，用20μm直径玻璃管将直径为10-12μm的转基因细 胞移入步骤1制备的去核的卵母细胞的透明带内，然后将其放入Zimmerman液中平衡(现用现配)3-5分钟后放入融合槽内转动卵细胞使核供体细胞与去核的卵母细胞接触而与电场垂直，同时在场强为2.5kV/cm的直流脉冲场中，在脉冲时间为10μs、脉冲次数为2次、脉冲间隔为1s的条件下融合(融合仪为BTX公司的ECM-2001)后，得到融合胚，将其迅速移入含有体积百分含量10％FBS的M199中培养数小时后，挑选融合胚(具体挑选供体细胞和卵母细胞完全融合的重构胚)进行激活处理。将融合胚放入5mM A23187液中5分钟后换到胚胎激活液(含5ug/ml细胞松驰素B、10ug/ml放线菌酮的M199培养液)中5小时，待融合胚被激活后换入含有体积百分含量5％的FBS的CR1aa中，在38.5℃、5％CO2培养箱中培养液中培养7天后观察融合胚的囊胚发育率，结果表明克隆囊胚发育率为20％-60％。
3、胚胎移植与妊娠检测
将形态优良的第7d的克隆囊胚移入同期发情的受体牛(荷斯坦奶牛)的子宫角内。在移植后的第30d对受体母牛进行B超检测以确定受胎情况，并分别在移植后的第60d和第90d进行直肠检测以确定妊娠率，妊娠率为40％。
4、对妊娠母牛按常规饲养方法进行饲养，经过280天，妊娠母牛正常分娩，得到体细胞克隆种公牛。
三、种公牛及的生物学鉴定
采集体细胞克隆种公牛耳部组织样，提取其基因组DNA，以基因组DNA为模板，以KOD2-F和KOD2-R为引物，进行PCR扩增，得到PCR扩增产物，同时以ddH2O为模板，进行上述实验，作为对照。
PCR反应程序：94℃5 min；94℃30 sec，62℃30 sec，72℃120 sec，30个循环；72℃7 min。
如果PCR扩增产物为2.1kb的片段，表明基因敲入成功。
结果表明共有5头个体，PCR都扩增出2.1kb的阳性片段，如图4所示。进一步将PCR扩增产物进行测序，结果正确。证实步骤二成功获得体细胞克隆种公牛，该体细胞克隆种公牛的Y染色体性别控制基因SRY被“自我杀死含Y染色体精子”元件精确修饰。
实施例3、体细胞克隆种公牛精液的分子生物学鉴定
将实施例2培育得到的体细胞克隆种公牛进行精子采集，然后用PCR方法进行鉴定，确定其精子是否全是含X染色体的精子，具体步骤如下：
以体细胞克隆种公牛精子的基因组DNA为模板，以SRY基因特异的PCR引物SRY-F和SRY-R为引物，进行PCR扩增，得到PCR扩增产物。同时以ddH2O为模板，进行上述实验，以野生型荷斯坦奶牛种公牛的精子的基因组DNA为模板，进行上述实验，作为对照。
引物序列如下：
SRY-F:5’-AACGACGATGTTTACAGTCCA-3’；(SEQ ID No.12)
SRY-R:5’-GCCCGGGTATTTGTCTCGGT-3’。(SEQ ID No.13)
PCR反应程序：94℃5 min；94℃30 sec，62℃30 sec，72℃30 sec，30个循环；72℃7 min。
含有Y染色体的精子特异含有SRY基因，因此PCR扩增可以扩增出340bp片段，而只含有X染色体的精子，不能扩增出条带。
结果如图5所示。
图5表明，体细胞克隆种公牛的精液没有扩增出目的条带，说明体细胞克隆种公牛含Y染色体的精子自动死亡，只剩下正常的含X染色体的精子。
实施例4、体细胞克隆种公牛后代个体的鉴定
取Y染色体性别控制基因SRY被“自我杀死含Y染色体精子”元件精确修饰的实施例2获得的体细胞克隆种公牛的精液，加入到含有肝素(50ug/ml)、咖啡因(0.01nmol/ml)、BSA(3.0mg/ml)的BO液中稀释洗涤两遍(以1800转/分钟，离心8分钟)，去上清后再加入1ml相同的含有肝素(50ug/ml)、咖啡因(0.01nmol/ml)、BSA(3.0mg/ml)的BO液混合均匀后，取50ul加到50ul的含有20-30枚卵母细胞的受精液(含10mg/ml BSA，不含脂肪酸的BO液)中共培养5小时后，将其转入到体外培养液CR1aa中继续培养5到7天，待受精卵发育到桑椹至囊胚期然后移植到受孕母体，得到后代个体进行PCR和形态学进行性别鉴定。
以牛耳细胞的基因组DNA为模板，以SRY-F和为SRY-R为引物，进行PCR扩增，得到PCR扩增产物。同时以ddH2O为模板，进行上述实验，以野生型荷斯坦奶牛种公牛的精子的基因组DNA为模板，进行上述实验，作为对照。
结果如图6所示。
图6表明，野生型荷斯坦奶牛种公牛的精子可以扩增出340bp的目的片段，标号为1和2的后代个体均没有扩增出目的条带，并且所有的后代个体经形态学鉴定后都显示为雌性母牛。
工业应用
本发明提供的方法为一种通过精确基因修饰的雄性哺乳动物体细胞，进而实现鉴定和/或选择特定性别动物的目的的方法。本发明为选择性培育特定性别的动物、提高育种效率奠定基础。

Claims (13)

  1. 一种动物Y染色体修饰方法，是利用TALEN方法对离体的动物体细胞的Y染色体进行特异性的自杀元件修饰；
    所述自杀元件能特异性杀死其所在的含有Y染色体的生殖细胞，同时对其所在的动物的体细胞不产生危害；
    所述修饰是在Y染色体的性别决定基因SRY上进行修饰。
  2. 根据权利要求1所述的方法，其特征在于：所述对离体的动物体细胞的Y染色体进行特异性的自杀元件修饰的方法为：使TALE蛋白-Ⅰ和TALE蛋白-Ⅱ在雄性动物A的离体体细胞中表达，得到Y染色体上靶序列发生突变的体细胞；同时将自杀元件同源重组到靶序列位置，实现自杀元件在雄性动物A的离体体细胞Y染色体上靶序列处的定点整合；
    所述TALE蛋白-Ⅰ的氨基酸序列如SEQ ID No.3所示；
    所述TALE蛋白-Ⅱ的氨基酸序列如SEQ ID No.5所示；
    所述靶序列如SEQ ID No.1所示。
  3. 根据权利要求1或2所述的方法，其特征在于：所述使TALE蛋白-Ⅰ和TALE蛋白-Ⅱ在雄性动物A的离体体细胞中表达的方法为：将含有TALE蛋白-Ⅰ的编码基因的重组表达质粒和含有TALE蛋白-Ⅱ的编码基因的重组表达质粒导入所述雄性动物A的离体体细胞中；
    所述TALE蛋白-Ⅰ的编码基因序列如SEQ ID No.2所示；
    所述TALE蛋白-Ⅱ的编码基因序列如SEQ ID No.4所示；
    所述将自杀元件同源重组到靶序列位置为将所述自杀元件通过线性化的同源重组载体整合在所述靶序列位置上；
    所述线性化的同源重组载体上具有靶序列同源左臂-自杀元件-靶序列同源右臂的片段。
  4. 根据权利要求3所述的方法，其特征在于：所述靶序列同源左臂的序列如SEQ ID No.9中自5’末端起第676位至第1636位核苷酸所示；
    所述靶序列同源右臂的序列如SEQ ID No.9中自5’末端起第5526位至第6449位核苷酸所示。
  5. 根据权利要求4所述的方法，其特征在于：所述自杀元件包含精子特异性启动子和自杀基因；
    所述自杀基因在精子成熟过程中被所述精子特异性启动子启动表达，杀死所述自杀基因所在的含有Y染色体的精子；
    所述自杀元件的序列如SEQ ID No.9中自5’末端起第1948位至第3730位核苷酸所示；
    所述靶序列同源左臂-自杀元件-靶序列同源右臂的的序列如SEQ ID No.9中自5’末端起第676位至第6449位核苷酸所示。
  6. 根据权利要求5所述的方法，其特征在于：所述同源重组载体的核苷酸序列如SEQ ID No.9所示；
    所述线性化为限制性内切酶AhdI线性化。
  7. 一种获得雌性动物的方法，是利用权利要求1-6中任一所述的方法对雄性动物A的离体体细胞中的Y染色体进行修饰，得到带有自杀元件的转基因细胞；以转基因细胞为核供体细胞，通过体细胞克隆技术得到体细胞克隆雄性动物B；以体细胞克隆雄性动物B为父本，获得的后代均为雌性。
  8. 一种试剂盒，该试剂盒含有如下1)-4)的至少一种物质：
    1)含有SEQ ID No.3所示蛋白的编码基因的DNA分子、重组载体、表达盒、转基因细胞系或重组菌；
    2)含有SEQ ID No.5所示蛋白的编码基因的DNA分子、重组载体、表达盒、转基因细胞系或重组菌；
    3)SEQ ID No.9所示的DNA分子或含有该分子的转基因细胞系或重组菌；
    4)含有SEQ ID No.9中自5’末端起第676位至第6449位核苷酸所示的DNA分子、重组载体、表达盒、转基因细胞系或重组菌。
  9. 根据权利要求8所述的试剂盒，其特征在于：
    所述SEQ ID No.3所示的蛋白的编码基因如SEQ ID No.2所示；
    所述SEQ ID No.5所示的蛋白的编码基因如SEQ ID No.4所示。
  10. SEQ ID No.9所示的DNA分子；
    和/或，
    含有SEQ ID No.9中自5’末端起第676位至第6449位核苷酸所示的DNA分子；
    和/或,
    SEQ ID No.2所示的DNA分子；
    和/或,
    SEQ ID No.4所示的DNA分子。
  11. 权利要求8所述的试剂盒、权利要求9所述的试剂盒或权利要求10所述的DNA分子在制备自杀元件修饰动物离体体细胞Y染色体的产品中的应用；
    或，
    权利要求8所述的试剂盒、权利要求9所述的试剂盒或权利要求10所述的DNA分子在制备Y染色体上带有自杀元件的雄性动物的产品中的应用；
    或，
    权利要求8所述的试剂盒、权利要求9所述的试剂盒或权利要求10所述的DNA分子在制备Y染色体上带有自杀元件的雄性动物细胞的产品中的应用；
    或，
    权利要求8所述的试剂盒、权利要求9所述的试剂盒或权利要求10所述的DNA分子在制备用于动物性别的选择和/或控制的产品中的应用；
    或，
    权利要求8所述的试剂盒、权利要求9所述的试剂盒或权利要求10所述的DNA分子在动物繁育中的应用。
  12. 一种试剂盒，该试剂盒含有如下5)-6)的至少一种物质：
    5)SEQ ID No.3所示蛋白；
    6)SEQ ID No.5所示蛋白。
  13. 权利要求12所述的试剂盒在制备自杀元件修饰动物离体体细胞Y染色体的产品中的应用；
    或，
    权利要求12所述的试剂盒在制备Y染色体上带有自杀元件的雄性动物的产品中的应用；
    或，
    权利要求12所述的试剂盒在制备Y染色体上带有自杀元件的雄性动物细胞的产品中的应用；
    或，
    权利要求12所述的试剂盒在制备用于动物性别的选择和/或控制的产品中的应用；
    或，
    权利要求12所述的试剂盒在动物繁育中的应用。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1434678A (zh) * 1999-12-27 2003-08-06 刘成宇 通过将转基因靶向至性染色体上控制子代性别比例
CN103352082A (zh) * 2013-07-18 2013-10-16 无锡中德美联生物技术有限公司 一种具有辅助性别鉴定的法医dna检测试剂盒及其鉴定方法
CN104450673A (zh) * 2014-11-14 2015-03-25 中国农业大学 一种y染色体修饰方法及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1745626B (zh) * 2004-09-06 2010-10-06 戴蕴平 一种经修饰的雄性哺乳动物体细胞系及其制备方法与应用
WO2012116274A2 (en) * 2011-02-25 2012-08-30 Recombinetics, Inc. Genetically modified animals and methods for making the same
CN103865897A (zh) * 2014-04-15 2014-06-18 西南大学 芥菜基因组定点突变系统及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1434678A (zh) * 1999-12-27 2003-08-06 刘成宇 通过将转基因靶向至性染色体上控制子代性别比例
CN103352082A (zh) * 2013-07-18 2013-10-16 无锡中德美联生物技术有限公司 一种具有辅助性别鉴定的法医dna检测试剂盒及其鉴定方法
CN104450673A (zh) * 2014-11-14 2015-03-25 中国农业大学 一种y染色体修饰方法及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAOYI WANG ET AL.: "TALEN-mediated editing of the mouse Y chromosome", NATURE BIOTECHNOLOGY, 12 May 2013 (2013-05-12), pages 1 - 3, ISSN: 1546-1696 *
XU, CHAO.: "Bovine SOX9 Cloning and Analysis of Sex-related Gene Expression in early Fetus", CHINA MASTER'S THESES FULL-TEXT DATABASE, AGRICULTURAL SCIENCE AND TECHNOLOGY, 15 February 2011 (2011-02-15), pages D050 - 28 *

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