Classifications

• • 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0603—Embryonic cells ; Embryoid bodies

Definitions

• the present invention is in the field of biotechnology, and in particular, the present invention relates to a lone male haploid stem cell line and methods of manufacture and use thereof. Background technique
• Mammals are all diploid, that is, there are two sets of chromosomes in the cell, one from the father and one from the mother. In sexually reproducing individuals, haploid gametes (eggs and sperm) are able to mediate gene transfer to the next generation. However, because sperm cannot be cultured in vitro, it greatly limits its genetic manipulation; in addition, it is difficult to identify animals. Which set of chromosomes is determined by a certain set of chromosomes, so diploid cells are a huge difficulty for genetic research.
• Haploid cells facilitate gene research because they contain only one set of chromosomes. Although mouse haploid embryos have been obtained, the embryonic stem cells established with these haploid embryos will later exhibit a diploid karyotype. Parthenogenetic mouse haploid embryonic stem cell line can be established by parthenogenetic activation
• haESCs have been applied to the field of gene screening at the mammalian cell level.
• the ability of these haploid embryonic stem cells to acquire an individual remains to be proven. Since the diploid parthenogenetic embryos formed by the haploid embryonic stem cells and egg cells of parthenogenetic mice cannot develop into individual animals, the study of parthenogenetic haploid stem cells is still at the genetic level.
• the object of the present invention is to provide a method for establishing a lone male haploid stem cell line and an application thereof.
• a orphan male haploid cell line wherein the cell nucleus contains only a single autosomal and sex chromosome, and the sex chromosome is an X chromosome.
• the orphan male haploid cell line not only expresses a traditional embryonic stem cell marker, but also proliferates and maintains a haploid karyotype in vitro, and has pluripotency, which can be doubled in injection.
• the blastocysts are post-differentiated into various types of tissue cells including germ cells.
• the orphaned haploid cell line is capable of passage for more than 30 passages in vitro.
• the cells are derived from a vertebrate, preferably from a human or non-human mammal.
• the non-human mammal is selected from the group consisting of rabbit, mouse, cow, monkey, or sheep.
• the orphan male haploid cell line does not contain the Y chromosome.
• the nuclear genetic material of the orphan male haploid cell line is derived from a male parent or sperm. In another preferred embodiment, the nuclear genetic material is DNA.
• the orphan male haploid cell line is capable of maintaining a paternal genomic imprint; preferably, the maintaining the paternal genomic imprinting means: the paternal expression gene is up-regulated or the maternal expression gene is down-regulated; Or, the parental imprinting gene is normal in methylation level, or the maternal imprinting gene methylation level is decreased.
• the paternal expression gene or the maternal imprinting gene is selected from the group consisting of: Snrpn,
• the maternal expression gene or paternal imprinting gene is selected from the group consisting of Phlda2, Rian, Grbl0, Meg3, Ube3a, Gtl2, or a combination thereof.
• the orphan male haploid cell line is a lone male haploid embryonic stem cell line.
• a orphan male blastocyst the nuclear genetic material of the blastocyst comprising only a single autosomal and sex chromosome, the sex chromosome being an X chromosome, the nuclear inheritance The substance comes from the father or the sperm.
• the blastocyst is derived from a human or a non-human mammal.
• the non-human mammal is selected from the group consisting of rabbit, mouse, cow, monkey, or sheep.
• the nuclear genetic material is DNA.
• the orphaned blastocyst contains no Y chromosome.
• the blastocyst is a haploid lone male blastocyst.
• a method of preparing a lone male blastocyst comprising the steps of: (i) obtaining a zygotic cell free of a prokaryotic nucleus;
• the zygotic cells containing no prokaryotic cells in step (i) are prepared by a method selected from the group consisting of:
• the zygotic cell described in (2) is a zygote cell at the PN3 stage.
• a method for preparing a lone male haploid cell comprising the steps of: culturing the orphan male blastocyst of the second aspect of the invention, obtaining a lone male haploid cell.
• a lone male haploid cell is obtained from a lone male blastocyst according to the DNA content using a flow cytometric sorting method.
• the cells are further enriched from the obtained lone male monoploid cells according to the DNA content by a method of flow cytometric sorting.
• the orphan male haploid cell line is a lone male haploid embryonic stem cell line.
• the orphan male haploid cell line of the first aspect for:
• the gametes are sperm.
• the orphan male haploid cell line is a lone male haploid embryonic stem cell line.
• a method of preparing an animal comprising the steps of:
• the orphan male haploid cells are genetically transformed.
• the genetic transformation comprises gene knockout, gene mutation, introduction of a foreign gene.
• the orphan male haploid cell line is a lone male haploid embryonic stem cell line.
• a method of preparing a transgenic animal comprising the steps of:
• the transgenic animal is a knockout transgenic animal.
• the genetic transformation comprises gene knockout, gene mutation, introduction of a foreign gene.
• the orphan male haploid cell line is a lone male haploid embryonic stem cell line.
• Figure 1 shows the establishment process and identification results of AG-haESCs.
• Figure 1A shows the schematic diagram of establishing AG-haESCs from the isolated blastocysts. The sperm carries the Oct4-EGFP transgene.
• Figure 1B shows the reconstructed embryos activated for 1 h and then passed Hoechst. Staining can be seen that the sperm head begins to depolymerize;
• Figure 1C shows the results of epigenetic modification of the male pronucleus formed by sperm injection into the enucleated oocyte. The left panel shows that 5hmc and 5mc are enriched in the male pronucleus in the fertilized egg, respectively.
• the right panel shows that the male pronucleus formed by sperm injection into the enucleated oocyte also has 5hmc enrichment;
• Figure 1D shows the formation of the orphan after the spermatozoon carrying the Oct4-EGFP transgene is injected into the enucleated oocyte.
• FIG. 1 shows the fluorescence and phase contrast maps of mulberry and blastocysts, the scale is ⁇ ;
• Figure IE shows the haES cell line (using AGH-OG-3 as an example) after enrichment of haploid cells by several rounds of flow sorting, sorting by DAPI channel Hoechst-stained DNA, congtrol is a flow diagram of the double-body ES;
• Figure 1F shows the cloned form of the AG-haESC cell line (AGH-OG-1) with a scale of 50 ⁇ ;
• Figure 1G shows the PCR junction for sex chromosomes.
• the AG-haESC cell line has only the X chromosome and no Y chromosome;
• Figure 1H shows the results of the karyotype analysis, showing that AGH-OG-3 has 20 chromosomes;
• Figure II shows haESCs (AGH-OG-3) and male mouse kidney.
• the CGH analysis of the cells (C57BL/6) showed that the upper row was haESCs vs. kidney cells and the lower row was kidney cells vs. kidney cells.
• Figure 2 shows the pluripotency of AG-haESCs
• Figure 2A shows the expression of ES marker in AG-haESCs with a scale of 50 ⁇
• Figure 2 ⁇ shows the gene expression profile of AG-haESCs, Pearson correlation. Diploid indicates diploid
• Figure 2C The above figure shows the chimeric mouse produced by the injection of Actin-EGFP-labeled AGH-EG-1 ES cells into normal diploid blastocysts, and the extra-embryonic tissue (left) is derived from the mother.
• the lower panel shows the ovary from the chimera (right) compared to the ovary from the control group (left);
• Figure 2D shows the injection of AGH-OG-2 haESCs (C57BL/6, black) into normal ICR (white) 7-week-old chimeric mice produced after blastocysts;
• Figure 2E shows the distribution of GFP-positive and GFP-negative cells in the skin of neonatal chimeric mice by flow cytometry. E-Texas-Red and FITC channels were used to detect PI and GFP signals.
• Figure 3 shows the paternal imprinting status of AG-haESCs, wherein Figure 3A shows the expression of imprinted genes by quantitative reverse transcription PCR (qPCR), * : 0.01 ⁇ p ⁇ 0.05; **: 0.001 ⁇ p ⁇ 0.01; Figure 3B is small Methylation of Gtl2, H19, and Snrpn DMRs in rat tails, sperm and AG-haESCs (AGH-OG-3, pl5), empty circles and filled circles represent non-methylation and methylation, respectively.
• qPCR quantitative reverse transcription PCR
• Figure 4 shows the results of embryo development after injection of AG-haESCs into egg cells; wherein, Figure 4A is a schematic diagram of MCHC-producing SC mice, after activation of embryos, the second polar body (PB) produced by egg cells and the M phase produced by MES After the pseudo-polar bodies (PPB) are discharged, a diploid embryo is formed, AG-haESCs carry the EGFP transgene, PPN is the pseudo-nucleus from haESC; Figure 4B shows the reprogramming of AG-haESC, and the left and middle are reconstructed embryos. The Hoechst staining map of Oh and lh was activated, and the staining map of the reconstructed embryo was activated for 6 h.
• PB second polar body
• PPN pseudo-polar bodies
• FIG. 4C shows the blastocyst map generated by ICAMCI and ICSI, donor haESCs. And the sperm carrying the Oct4-EGFP transgene, the scale is ⁇ ;
• Figure 4D shows the IHCCI-derived semi-clone (SC) mice from AGH-OG-3 haESCs, showing the caesarean section at the 19.5 days of gestation of the pseudopregnant mother.
• Figure 4F shows control ICSI mice (top), normal SC mice (Middle) and blocking methylation status of H19DMR in SC mice (small);
• Figure 4G shows two eight-week-old SC mice obtained by the AGH-OG-2 manipulation by the ICAMCI method.
• Figure 5 shows the transfer of genetic traits from AG-haESCs to SC mouse progeny; wherein, Figure 5A shows that isolated from newborn SC mice and peripheral large SC mice bearing the Oct4-EGFP transgene AGH-OG-1 The ovary (top) and GV egg cells (bottom), the green fluorescence indicates the germ cells expressing Oct4-EGFP, the scale is 200 ⁇ m (top) and 100 ⁇ m (bottom); Figure 5B shows the AGH-OG-1 Obtained SC mother Figure 5C shows the genotypic analysis of the offspring.
• FIG. 5D shows that the progeny of the SC mother and the offspring of both sexes have AG -haESC's germline inheritance
• the left two images show the expression of Oct4-EGFP transgene in the vas deferens of one week old male F2 pups, and Oct4 will have Oct4 expression in the entire germ cell population of this newborn male.
• the two images on the right show the expression of Oct4-EGFP reporter gene in GV eggs isolated from two-week old female F2 pups.
• the EGFP signal indicates the presence of Oct4 expression in the developing oocytes, with a scale of 100 ⁇ m.
• Figure 6 shows the genetic manipulation in AG-haESCs, wherein Figure 6A is a homologous recombination targeting strategy for the Vwce gene, the coding exon is indicated by a black box, and the 5' non-coding region of exon 1 is partially blank.
• the box indicates that the Frt site on the side of the neo screening marker is indicated by a blank triangle, and the ⁇ site next to the targeting region is indicated by a gray triangle.
• the primers for genotyping of AG-haESCs are indicated by horizontal arrows in the figure.
• the locus (first column), an untargeted AG-haESC cell line was used as a negative control (second column), in which targeted diploid represents twice the target being targeted and untargeted as untargeted;
• Figure 6C shows the amplification of the successfully targeted AG-haESC-Vwce cell line to obtain a stable haploid cell population.
• Figure 7 shows that the orphan-bearing haploid stem cells of the present invention are capable of producing genetically modified animals; wherein, Figure 7A shows that injection of AG-haESC-Vwce cells with targeted alleles into oocytes can be used for gene knockout.
• Figure 7A shows that injection of AG-haESC-Vwce cells with targeted alleles into oocytes can be used for gene knockout.
• the placenta and fetus of the semi-cloned mice were all EGFP-positive, indicating that the injected AG-haESC-Vwce cell line carries the EGFP gene;
• Figure 7B shows the different parts of the mouse with primers (P1-P6). (Tail (tail), ovary (ovary), placenta (placenta)) genotype identification results. detailed description
• the present inventors have for the first time established a stable orphan male haploid cell line through extensive and intensive research, and provided a method for producing the cell line and its application.
• the present inventors provide a lone male haploid cell line and a lone male blastocyst, wherein the cell nucleus of the cell line or blastocyst contains only a single autosomal and sex chromosome, and the sex chromosome is X. Chromosome, no Y chromosome.
• the orphan male haploid cells of the present invention can replace sperm cells as ligands to produce fertile animal individuals; the orphan male haploid cells of the present invention are advantageous for genetic manipulation and can transmit genetic information to offspring.
• the invention also provides a preparation method for establishing a solitary haploid cell and an application thereof. The present invention has been completed on the basis of this. the term
• Haploid cells and diploid cells refers to a cell in which the number of chromosomes in a somatic cell is the number of chromosomes of a gamete of this species. Gamete cells are a haploid cell.
• diploid cell refers to a cell containing two sets of chromosomes. Female and male gametes usually develop into diploid organisms after binding. Mulberry embryo, blastocyst and lone male blastocyst
• mulberry embryo refers to the early stage of embryonic development of an animal.
• a fertilized egg undergoes multiple divisions to form tens of to hundreds of cells.
• the early embryo composed of this cell mass is a morula.
• the morula is further developed, and the cells begin to differentiate and accumulate on the side of the embryo.
• the larger cells of the individual are the inner cell masses of various tissues that will develop into the fetus in the future, and the smaller cells that expand and align along the inner wall of the zona pellucida are the future.
• Trophoblast cells that develop into the membrane and placenta. As the embryo develops further, blastocysts containing fluid begin to appear inside the embryo.
• the invention provides a lone male blastocyst, wherein the nuclear genetic material of the blastocyst contains only a single autosomal and sex chromosome, the sex chromosome is an X chromosome, the nuclear genetic material is derived from a male parent or a sperm, and the isolated male blastocyst
• the Y chromosome is not included; preferably, the blastocyst is derived from a human or a non-human mammal, and the nuclear genetic material is DNA.
• the present invention provides a lone male haploid cell obtained from a lone male blastocyst, which is named a lone male haploid stem cell (AG-haESCs), as used herein, the terms “AG-haESCs”, “orphan Male haploid stem cells, or “loose male haploid embryonic stem cells” can be used interchangeably.
• the cells not only have the typical morphology of traditional embryonic stem cells, but also express traditional embryonic stem cell markers. They also have pluripotency and can be differentiated into various tissue cells including germ cells after being injected into twice as many blastocysts.
• a solitary mouse haplocyte of a mouse is injected into a mouse oocyte to obtain a surviving mouse individual.
• These mice not only carry the genetic traits of haploid stem cells themselves, but also grow into fertile adults.
• the solitary male haploid cells provided by the present invention have a typical imprint of a male reproductive line, can replace the sperm to complement the development of the embryo, and maintain the male imprint; and can also perform gene targeting operations by homologous recombination.
• the present invention provides two methods for establishing a solitary haploid cell, comprising the steps of: ⁇ obtaining a zygotic cell free of estrogen; and (ii) culturing the zygote cell to obtain a lone male blastocyst; preferably,
• the zygotic cells containing no prokaryotic nucleus in step (i) are prepared by a method selected from the group consisting of: (1) combining sperm cells with enucleated oocytes to obtain reconstituted zygotic cells free of estrogen; Or (2) removing the pronucleus in the zygote cells to obtain zygotic cells without the prokaryotic nucleus.
• a universal nuclear transfer method is used, and a sperm head is used instead of a somatic cell as a donor of genetic material.
• the mouse is subjected to chorionic gonadotropin injection treatment, and the egg cells are obtained and cultured; the piezoelectric-driven blunt-head habit is used for enucleation;
• the head of a sperm is injected into the cytoplasm of the egg cell to form a reconstituted egg cell; the reconstructed egg cell is cultured, and activation treatment is performed in the activated culture medium to obtain a lone male haploid cell line.
• the method comprises the steps of: mating a female mouse with a male mouse to obtain a zygote, collecting a zygote during the PN3 period; and driving the transparent band on the female pronucleus by a pulse during the PN3-4 period Perforation, the male pronucleus has a male pronucleus in the size difference, and away from the polar body, the female pronucleus is removed by a micromanipulator to obtain a zygote containing only the male pronucleus; the zygote is cultured to obtain a lone male haploid cell line. .
• a lone male haploid cell is obtained from a lone male blastocyst according to the DNA content using a flow cytometric sorting method.
• the cells are further enriched from the obtained lone male monoploid cells according to the DNA content by a method of flow cytometric sorting.
• Method for establishing non-human primate solitary male haploid embryonic stem cell line
• the invention also provides a method for establishing a non-human primate orphan male haploid embryonic stem cell line.
• Standard procedure for monkey nuclear transfer (Ref. Nature. 2007 Nov 22;450(7169):497-502. Epub 2007 Nov.
• Blastocysts are used to establish embryonic stem cell lines, see Mitalipov et al. (Stem Cells. 2006 Oct; 24(10): 2177-86. Epub 2006 Jim 1; Nature. 2007 Nov 22; 450(7169): 497-502. Epub 2007 Nov 14 ⁇ ).
• ICM Inner cell mass
• the blastocyst was treated in a standard ESC establishment culture medium containing 0.5% pronase for 50 seconds to remove the zona pellucida.
• Inner cell mass (ICM) was obtained by two methods. The first is mechanical, that is, the trophectoderm cells are removed mechanically; the second is immunosurgery, where the blastocysts are placed in rabbit anti-sera (Axell Labs, Westbury, New York, USA) for 30 minutes, then It was treated in guinea pig complement (sigma) for 30 minutes, and finally ICM was isolated by a slight blow.
• the isolated ICM was transferred to a well of a 4-well plate and cultured in DMEM/F12 medium containing 1% non-essential amino acids, 2 mM L-glutamine, 0.1 mM b-mercaptoethanol, and 15% FBS. After the ICM adheres to the MEF and grows out of the cell mass, the cell pellet is mechanically separated into small cell clusters and transferred to a new MEF. After the first passage, clones of standard monkey embryonic stem cell morphology were grown for further subculture. To screen for haploid cells, ES cells were first trypsinized, washed with DPBS, and then co-cultured with 15 ug/ml Hoechest 33342 in a 37 ° C water bath.
• haploids can be purified by BD FACS Ariall for subsequent culture.
• cells were treated with 20 ug/ml NA enzyme A and stained with 50 ug/ml PI after fixation in 70% ethanol.
• Analytical profiles were recorded using the BD LS II SO P software.
• the invention utilizes Macaca Fascicularis, but rhesus macaques may also be used, as well as monkeys of other species.
• AG-haESCs can obtain F 1 generation (limited to females) faster and more reliably, avoiding the genotype transmission of chimeras obtained from diploid embryonic stem cells, and the traditional method of obtaining chimeras is gene targeting. Speed limit step. For large animals (including pigs, cattle, monkeys, etc.), there are currently no embryonic stem cell lines that can be used for efficient chimera formation and reproductive line delivery. Large animals, even if they have chimeras, are difficult to have enough chimeras to mate for screening for germline transmission. In contrast, the hybrid F1 generation directly obtained by AG-haESCs ensures reliable germline transmission within limited mating, as half of the offspring can inherit genetic modification. AG-haESCs can potentially be obtained from male individuals diagnosed with various mutations. Once these cells have been corrected for mutations, they can be used for in vitro fertilization with healthy individuals.
• the invention also provides a method for preparing a transgenic animal, comprising the steps of:
• the transgenic animal is a knockout transgenic animal.
• the genetic transformation includes gene knockout, gene mutation, and introduction of a foreign gene.
• the orphan male haploid cells of the present invention have a typical imprint of a male reproductive line, which can replace the sperm to complement the development of the embryo and maintain the male imprint;
• the orphan male haploid cells of the present invention can perform gene targeting operations by homologous recombination
• the orphan-bearing haploid stem cells of the present invention are capable of producing genetically modified animals, which are advantageous for genetic manipulation and can transmit genetic information to offspring.
• the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
• the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Materials and Methods
• ES cells embryonic stem cells
• colchicine purchased from Sigma
• trypsinized and then ES cells were resuspended in 0.075 M KC1 hypotonic solution at 37 ° C for 30 min.
• the treated cells were fixed in a mixture of methanol:acetic acid (3:1) for 30 min, and then dropped on prewashed glass slides; then treated with 5 g of HC1, stained with Giemsa dye for 15 min; .
• Immunostaining immunofluorescence analysis
• the cells on the slides were fixed with PBS containing 4% paraformaldehyde for 15 min at room temperature; then permeabilized with PBS containing 0.2% Triton X-100 for 15 min at room temperature; and blocked with PBS containing 1% BSA for 30 min;
• One antibody was: anti-Oct4 (sc-5279, purchased from Santa Cruz), Nanog (CAB002P-F, available from eprocell), SSEA-1 (mab4301, available from Millipore), Sox2 (ab5603, purchased from Millipore), diluted In the same blocking buffer, co-culture with the sample at 4 ° C overnight; these cells were treated with a fluorescently coupled secondary antibody and incubated for 1 h at room temperature; the nuclei were stained with Hoechest 33342 (purchased from sigma) for 5 min at room temperature. . Microscopic observation was performed using an SZX7 Olympus stereoscope. Cell size measurement
• Haploid embryonic stem cells and diploided haploid embryonic stem cells were synchronized in the middle phase and then purified by FACS; the same number of cells were resuspended in 10 wl ES medium, then 10 ⁇ l 0.2% Pan blue mix to distinguish and dead cells. The size of these cells was measured using an Invitrogen Countess Cell Counter instrument. Male haploid embryonic stem cells injected into diploid blastocysts
• Two-dimensional embryos were collected from the uterus of females mated with 3.5d superovulated eggs and cultured in KSOM containing amino acids.
• male haploid embryonic stem cells were trypsinized and then resuspended in Leukemia inhibitory factor (LIF) in DMEM and placed on ice; for ES cell injection is a flat-head microinjection pipette; inhalation of more than 100 ES cells at the end of the injection pipette, about 10-15 ES cells Inject into a blastocyst cavity; cultured in KSOM containing amino acids before embryo transfer; 8-10 post-injection blastocysts are transferred to the uterine horn of ICR female rats for 2.5 days of pregnancy; The recipient undergoes caesarean section after 19.5 days of gestation.
• Quantitative reverse transcription PCR Quantitative reverse transcription PCR
• Embryos injected into the intracytoplasmic male haploid embryonic stem cells are cultured in KSOM medium until two cells or blastocyst stage; 15-20 two-cell embryos or 8-10 blastocysts are transferred into pseudopregnant 0.5d or 2.5d In the fallopian tube or uterus of a female ICR; the recipient female is euthanized at 19.5 days of gestation, and the fetus is removed from the uterus as soon as possible; after removing the fluid in the respiratory tract, the fetus is placed in a hot box of oxygen, the surviving fetus Feeded by a lactating mother. Chip analysis
• RNA was extracted using the RNeasy kit (purchased from Qiagen).
• Gene expression analysis of the Affymetrix Genechip 430 2.0 chip was performed by Imagenes.
• the data was analyzed using Genespring GX software (Agilent Technologies). Labeling and hybridization were performed at Shanghai Biochip Corporation in accordance with the corresponding protocol of the Affymetrix GeneChip 3'ITV Express Kit User Manual.
• the correlation of the transcriptional map was determined by calculating the Pearson correlation coefficient.
• DNA samples (from AGH-OG-2, AGH-OG-3 and AGH-OG-1) used for comparative genomic hybridization were extracted and sent to Capital Biotechnology (Changping District, Beijing) for comparative genomic hybridization analysis.
• the chip used was a NimbleGen 3 X 720K mouse whole genome chip with an average span of 3.5 Kb.
• the kidneys of adult C57BL/6 male rats were used as a reference.
• Egg cells The egg cell donor was from B6D2F1 (C57BL/6 X DBA2) female mice, and the B6D2F1 female mice were purchased from Nanjing Model Animal Research Institute.
• the first method the standard nuclear transfer method (see n et al., 2011; Wakayama et al., 1998), in which the somatic donor is replaced with a sperm head.
• egg cells were obtained after 14 h of injection of chorionic gonadotropin (HCG) in mice, and then cultured in HEPES-CZB culture medium containing 5 wg/ml cytochalasin B; The habit of enucleation; after denuclearization, the head of a single sperm is injected into the cytoplasm of the egg cell to form a reconstructed egg cell; the reconstructed egg cell is cultured in the CZB culture medium for 1 h, and then contains 10 nM Sr2+.
• B6D2F1 female mice were mated with Actin-EGFP transgenic mice (C57BL/6 genetic background X mice were purchased from Nanjing Model Animal Research Institute), and zygotes were collected during PN3 period; female pronuclei during PN3-4 period By punching the transparent belt with a pulse, and then removing it with a micromanipulator; the female pronucleus has a male pronucleus that differs in size, and is far from the polar body. The zygote containing only the male pronucleus is cultured at 37 ° C, 5% CO 2 . KSOM in the environment containing amino acids. Finally, the reconstructed embryos that reached the morula or blastocysts at 3.5 days were transferred to ES culture.
• AGH-EG-1 haploid stem cell
• the inventors obtained a total of five solitary male haploid stem cells in this example, in vitro. It can be cultured for more than 30 generations, and no strain containing Y chromosome (the solitary embryo with single or double Y chromosome can not develop to the blastocyst stage).
• Karyotype analysis found that, regardless of the number of generations, these stem cells have only one genome of 20 chromosomes (Fig. 1H);
• Fig. 1 G is the PCR result for sex chromosomes, AG-haESC cell line has only X chromosome, no Y chromosome
• CGH genomic hybridization experiments
• iJh@3a 2M is a gene expression profile of embryonic fibroblasts (MEFs) and AG-haESCs in normal ES cells and male individuals.
• MEFs embryonic fibroblasts
• samples in the G1/G0 phase were collected by FACS. Cluster analysis of these cells was performed based on microarray expression data. The results showed that AG-haESCs and diploid ES cells had high similarity but no similarity to MEFs (Fig. 2B).
• Actin-EGFP-labeled haploid cells were used.
• Example 4 The injection of orphan male haploid stem cells into oocytes can support the subsequent development of embryos by intracytoplasmic solitary haploid stem cell injection (ICAHCIX Figure 4A) to study the injection of matured haploid stem cells into mature eggs. Whether the mother cell can replace the sperm and support the complete development of the embryo.
• the inventors synchronized the solitary haploid stem cells in the medium term and then selected the small cells for ICAHCI, which are almost all M-phase haploid cells.
• the results show that the injected haploid stem cell nucleus forms a pseudo-nucleus, similar to the male pronucleus, indicating that it is undergoing a focused programming process (Fig. 4B); in addition, the second polar body and the pseudopolar body are also separated from the spindle The chromosome complex and the donor nucleus are excreted, resulting in a reconstructed embryo containing twice the body DNA.
• the efficiency of the injected egg cell to the blastocyst is about 51% (Fig. 4C), and the ICSI experiment with the control. The efficiency is similar.
• mice The developmental potential of ICAHCI embryos. 451 2-cell embryos and 424 blastocysts obtained from all five solitary male haploid stem cells (from the seventh to the 22nd generation), and finally 43 live births after caesarean section on the 19.5th day of pregnancy. Rat (Fig. 4D). All mice were female and were consistent with the expected judgment of injection of these solitary male stem cells with X chromosome. The genotype identification results showed that they all carry the GFP transgene derived from the solitary male haploid stem cells, which is obtained by the combination of the lone male haploid stem cells and the normal egg cells. These mice are therefore referred to as semi-clonal (sc) mice.
• the birth rate of the semi-cloned mice was 4.5% and 5.3%, respectively, according to the number of transplanted blastocysts and the number of transplanted 2 cells, which was comparable to the traditional embryonic stem cell nuclear transfer rate.
• the semi-cloned mice have both a normal phenotype comparable to that of a normal newborn mouse and a developmental arrest type (Fig. 4D, Fig. 4E). All arrested offspring died within one hour of birth, and both parental mice were reconstituted from both fully-growed oocytes and non-growth oocytes, either alone or in a H19 differential methylation region (DMR). It is also a double-knocked differentially methylated region (IG-DMR) between the H19 and Dlkl-Dio3 genes that has this phenotype.
• DMR differential methylation region
• the 5' and 3' homologous DNAs were flanked by BAC clones of mouse genomic DNA by standard genetic recombination engineering techniques.
• the targeting vector contains about 4.9 kb and 5.6 kb of homology arms, a PGK-neo drug screening expression cassette and a 3.5 kb genomic region sequence containing exons 2-4.
• the negative selection gene herpesvirus thymidine kinase (HSV-tk) gene is placed outside the target gene fragment.
• the recombinant transformed cells have dual resistance to G418 and ganciclovir, and the non-homologous recombinant target cells cannot survive in the ganciclovir selection medium because they contain the HSV-tk gene integrated by terminal insertion.
• the gene-targeting cell is the AG-EG-1 (25th generation) haploid ES cell line.
• ES cells Three hours after the replacement of the culture medium, ES cells are trypsinized into single cells, and then Ca2+/Mg2+-free.
• PBS in concentration per 1 x 107 ml was resuspended and electroporated in a 0.4 cm wide sterile trough containing 2.5 ⁇ g of pL253-Vwice targeting vector.
• the condition of single pulse was 260 V, 500 ⁇ These cells were at room temperature. After 5 min of placement, they were plated in 10 cm culture dishes with neomycin-resistant MEF trophoblasts.
• ES selection medium containing approximately 200 ⁇ g/ml of G418 and 2 ⁇ M of ganciclovir, and the selection of the permeate was changed daily.
• Pick ES clones after 10 days. The collected clones were first trypsinized and then transferred to a 24-well plate containing ES-selective perfusate. The selected medium contained 100 ⁇ g/ml of G418 and 2 ⁇ M of ganciclovir for 3-5 days. Thereafter, G418-resistant clones were screened for homologous recombination by extensive PCR using primers (P 1-4) spanning the left and right recombination arms.
• the clones identified to be stable contain a large number of haploid cell populations with targeted alleles, but no wild-type, indicating that homologous recombination can occur in haploid cells after electroporation targeting, indicating that Haploid embryonic stem cells can be used in standard gene targeting operations.
• the AG-haESC-Vwce cells carrying the targeted allele prepared in Example 5 were injected into oocytes, and cultured to obtain gene knockout heterozygous mice (semi-cloned mice).
• Figure 7A shows that the half-cloned mice after birth have EGFP-positive placenta and fetus, indicating that the injected AG-haESC-Vwce cell line carries the EGFP gene;
• Figure 7B shows that the mice are different with the primers (P1-P6). Identification results of the tail (tail), ovary (ovary), placenta (placenta) genotypes.
• Example 7 Establishment of a non-human primate solitary haploid cell line
• HECM-9 containing 2 mM DMAP for 5 hours.
• Reconstituted embryos were cultured in HECM-9 medium containing 10% FBS and 12 mM b-mercaptoethanol (BME) for no more than ten days to obtain blastocysts, and the culture medium was changed daily.
• Blastocysts are used to establish embryonic stem cell lines, see Mitalipov et al. (Stem Cells. 2006 Oct; 24(10): 2177-86. Epub 2006 Jim 1; Nature. 2007 Nov 22; 450(7169): 497-502. Epub 2007 Nov 14 ⁇ ).
• the blastocysts were treated in a standard ESC establishment culture medium containing 0.5% pronase for 50 seconds to remove the zona pellucida.
• Inner cell mass (ICM: blastocysts were first placed in rabbit anti-posterior serum (Axell Labs, Westbury, New York, USA) for 30 minutes by immunosurgery and then treated in guinea pig complement (sigma) 30 Minutes, and finally ICM was isolated by a slight blow. The isolated ICM was transferred to a well of a 4-well plate containing 1% non-essential amino acids, 2 mM L-glutamine, 0.1 mM b-mercaptoethanol, and 15% FBS. The medium is cultured in DMEM/F12.
• the cell pellet After ICM adheres to the MEF and grows out of the cell mass, the cell pellet is mechanically divided into small cell clusters and transferred to a new MEF. After the first passage, the standard is grown. Cloning of monkey embryonic stem cell morphology was used for further subculture. To screen for haploid cells, ES cells were first trypsinized, washed with DPBS, and then co-cultured with 15 ug/ml Hoechest 33342 in a 37 ° C water bath. Subsequently, most of the haploids can be purified by BD FACS Ariall for subsequent culture.

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