WO2019141052A1 - 非人灵长类的体细胞克隆动物的制备方法 - Google Patents

非人灵长类的体细胞克隆动物的制备方法 Download PDF

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WO2019141052A1
WO2019141052A1 PCT/CN2018/123507 CN2018123507W WO2019141052A1 WO 2019141052 A1 WO2019141052 A1 WO 2019141052A1 CN 2018123507 W CN2018123507 W CN 2018123507W WO 2019141052 A1 WO2019141052 A1 WO 2019141052A1
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reconstructed
embryo
monkey
egg
reprogramming
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PCT/CN2018/123507
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French (fr)
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孙强
刘真
张洪钧
蔡毅君
廖兆蒂
许玉婷
王燕
聂艳红
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中国科学院上海生命科学研究院
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Priority to EP18901398.0A priority Critical patent/EP3760727A4/en
Priority to US16/963,074 priority patent/US20210017543A1/en
Priority to JP2020560530A priority patent/JP7199741B2/ja
Publication of WO2019141052A1 publication Critical patent/WO2019141052A1/zh

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Definitions

  • the present invention relates to the field of mammalian propagation technology, and in particular to a method for preparing a somatic cell cloned animal of a non-human primate.
  • non-human primates Because of its evolutionary proximity to humans, many aspects of brain structure and functional activities of non-human primates are highly similar to humans. Relative to other experimental animals, non-human primates have the unique advantage of solving human problems, especially brain-related problems: in addition to being a good experimental animal for studying the advanced functions of human brain normal brain, they also study the mechanism of brain diseases. Good model animals for treatment and treatment. China has a very rich non-human primate resource. In order to make full use of non-human primates to study brain diseases and normal high-level functions of human brain, it is necessary to use transgenic technology to construct a variety of non-human primate brain disease models and optogenetics. And other tool models.
  • CRISPR-Cas9 The most commonly used methods for obtaining non-human primate genetically modified animal models are the lentivirus-infected oocyte method and the editable nuclease method (CRISPR-Cas9, etc.).
  • CRISPR-Cas9 the editable nuclease method
  • the rapid development of gene editing technology represented by CRISPR-Cas9 has made it easy for scientists to edit genes for non-human primates. Even so, there are still many key issues in the construction of non-human primate transgenic animal models using existing transgenic techniques, such as 1, whether using lentivirus-infected oocyte methods to obtain transgenic animals or using editable nucleases.
  • the first generation (F0) animal model is ubiquitous in chimerism, which leads to the first animal model obtained by such methods cannot be used for scientific research.
  • Traditional rodents such as large mice can obtain F1 animal models without chimerism by passage, while for non-human primate models, it takes about 5 years to obtain F1 animal models.
  • 2, for carrying a complex genetically modified non-human primate model (conditional knockout or gene knock-in) the introduction of site-specific DNA double-strand breaks and simultaneous injection of homologous fragments by embryo injection of editable nucleases
  • the method of spot-spot insertion of source fragments has not been reported successfully at the level of mammalian levels, and has not been successfully reported at the level of non-human primates.
  • mice can achieve the same genetic background through continuous self-crossing, and for non-human primates with a long maturity cycle, it is unrealistic to achieve the genetic background through this self-crossing. It can be said that due to the existence of the above three key problems, although the animal model of non-human primate gene modification needs to be urgent, the current animal model of non-human primate gene modification has not been widely used in scientific research.
  • somatic cell-derived cloned monkeys have not been successfully reported.
  • the main reason why the subject has not been successful is: 1. High technical requirements, and primate nuclear transfer technology is much more difficult to operate than other mammals that have been successful. 2, the development efficiency of cloned embryos is low, and the development efficiency of cloned embryos of monkey somatic cells is far lower than that of other mammals.
  • Another object of the present invention is to provide a process for optimizing the operation of a non-human primate somatic cell nuclear transfer and a method for improving the efficiency of non-human primate somatic cell nuclear transfer.
  • the cloned monkeys derived from healthy and healthy somatic cell nuclear transfer were obtained by the optimized operation and method.
  • a method for preparing a non-human primate somatic cell clone animal comprising the steps of:
  • the reprogramming process is performed with a reprogramming activator.
  • the reprogramming activator is selected from the group consisting of:
  • the reprogramming activator comprises a reprogramming activator selected from the group consisting of:
  • the reprogramming activator further comprises a reprogramming activator selected from the group consisting of:
  • the reprogramming activator further comprises a reprogramming activator selected from the group consisting of:
  • the reconstituted or reconstituted embryo is a reconstituted egg or a reconstituted embryo that has not been genetically engineered or genetically engineered.
  • the genetic engineering operation comprises ZFN (zinc-finger nucleases), TALEN or Crispr technology.
  • the genetically engineered reconstituted or reconstituted embryo comprises a knockout of a reconstructed egg of the BMAL1 gene or a reconstituted embryo.
  • the reprogramming process comprises: injecting mRNA encoding a Kdm4d protein into the reconstructed egg or embryonic cells of the reconstructed embryo.
  • the reprogramming process comprises: injecting mRNA encoding one or more of a Kdm4a, Kdm4b or Kdm4d protein into the reconstructed egg or embryonic cells of the reconstructed embryo.
  • 10 2 -10 8 (preferably 10 4 -10 6 ) copies/cell of mRNA encoding the Kdm4d protein are injected into the reconstituted egg or the embryonic cell.
  • 10 2 -10 8 (preferably 10 4 -10 6 ) copies/cell of mRNA encoding one or more of the Kdm4a, Kdm4b or Kdm4d proteins are injected into the reconstituted egg or Embryonic cells.
  • the reconstructed embryo is in a 4-cell phase or an 8-cell phase.
  • the mRNA encoding Kdm4d is in a solution having a concentration of 10-6000 ng/ ⁇ l (preferably, 50-4000 ng/ ⁇ l, more preferably 80-3000 ng/ ⁇ l). .
  • the mRNA encoding Kdm4a or Kdm4b is in a solution having a concentration of 10-6000 ng/ ⁇ l (preferably, 50-4000 ng/ ⁇ l, more preferably 80-3000 ng/ Ll).
  • the regeneration is carried out in the uterus of a surrogate animal of a non-human primate.
  • the species of the surrogate animal is the same as the species of the reconstructed egg.
  • the regeneration is performed in an artificial uterus.
  • the activated reconstructed embryo is obtained; and in the step (iii), the reconstructed embryo is subjected to a reprogramming process.
  • the reprogrammed reconstructed embryo is regenerated to obtain a non-human primate somatic clone animal.
  • step (iv) includes:
  • the transplant is a non-invasive graft (ie, does not cause any trauma).
  • the method is non-therapeutic and non-diagnostic.
  • the reconstituted egg is prepared using an in vitro method comprising the following steps:
  • the somatic cells are selected from the group consisting of fibroblasts, cumulus cells, embryonic stem cells, spermatogonia, testicular support cells, mesenchymal stem cells, skin cells, breast cells, oviduct cells, Ear cells, ovarian cells, epithelial cells, endothelial cells, muscle cells, nerve cells, osteoblasts, or a combination thereof.
  • the activation treatment is performed with an activation treatment agent.
  • the activation conditions of the activation process are as follows:
  • Ionomycin was used for 2-6 minutes at 37 ° C, 5% CO 2 , 6-DMAP for 3-6 hours, and TSA for 5-15 hours.
  • the activating treatment agent is selected from the group consisting of a calcium ion activator, a histone deacetylase inhibitor, 6-DMAP, cycloheximide (CHX), or a combination thereof.
  • the histone deacetylase inhibitor is selected from the group consisting of TSA, scriptaid, or a combination thereof.
  • the concentration of the activating treatment agent is from 0.1 nM to 100 mM; preferably, from 1 nM to 50 mM.
  • the concentration is from 0.5 mM to 30 mM, preferably from 0.8 mM to 15 mM, more preferably from 1 to 10 mM.
  • the concentration of the histone acetylase inhibitor (TSA) in the activation treatment agent is from 0.5 to 80 nM, preferably from 1 to 50 nM, more preferably from 3 to 30 nM.
  • step (ii) comprises one or more characteristics selected from the group consisting of:
  • step (iii) the activated reconstituted egg is activated again for 2-10 hours, preferably 3-7 hours.
  • a kit comprising:
  • a first container containing a reprogramming activator, wherein the reprogramming activator comprises a reprogramming activator selected from the group consisting of:
  • the reprogramming activator further comprises a reprogramming activator selected from the group consisting of:
  • the reprogramming activator further comprises a reprogramming activator selected from the group consisting of:
  • the method of the first aspect of the invention is described in the label or the specification.
  • kits of the second aspect of the invention for the preparation of a kit for the preparation of a somatic clone animal of a non-human primate.
  • a non-human primate somatic cell cloned animal is provided, which is prepared by the method of the first aspect of the invention.
  • Figure 1 shows the optimization of the operation process of cynomolgus monkey somatic cell nuclear transfer
  • A. cynomolgus monkey fetal fibroblasts used as nuclear donors A. cynomolgus monkey fetal fibroblasts used as nuclear donors
  • Figure 2 shows the efficiency statistics using fetal monkey fibroblasts and adult monkey cumulus cells as nuclear donors under different conditions
  • Figure 3 shows the pregnancy rate and fetal development statistics of cynomolgus monkey somatic cell nuclear transfer embryo transfer
  • A B. nuclear transfer pregnancy receptor B-character.
  • the A receptor has only a gestational sac and no fetus.
  • B receptor has gestational sac and fetus
  • Figure 4 shows genetic analysis of somatic cell nuclear transfer cloned monkeys
  • Figure 5 shows nuclear transfer of cynomolgus monkey fetus fibroblasts
  • Figure 6 shows somatic cell nuclear transfer of cynomolgus monkey cumulus cells and activation treatment and reprogramming of reconstructed eggs
  • Figure 7 shows more mitochondrial SNP locus analysis of fibroblast nuclear transfer cloned monkey "ZhongZhong” "HuaHua” (for Figure 4D)
  • Figure 8 shows genetic analysis of cumulus cell somatic cell nuclear transfer cloned monkeys "A” and "B";
  • Figure 9 shows the preparation of BMAL1 gene editing monkey fibroblasts.
  • A Schematic diagram of gene editing preparation of cynomolgus monkey fibroblasts.
  • B BMAL1 edited monkey A6.
  • D A6 fibroblasts have normal karyotypes.
  • E A6 genotype analysis of monkey ear tip tissue, blood cells, and fibroblasts.
  • F Single cell genotyping analysis of A6 fibroblasts.
  • Figure 10 shows the BMAL1 gene edited cloned monkey construct.
  • A Schematic diagram of construction of a BMAL1 gene-edited cloned monkey.
  • C B1-B5, 5 cloned monkeys (S) from A6 fibroblasts, 5 cloned monkey ear tissue BMAL1 mutation analysis, 4 monkeys carrying heterozygous genes ("-8/-8, +4 , 2PM”), one monkey carries a homozygous mutant gene ("-8/-8") mutation.
  • Figure 11 shows the genotype analysis and expression analysis of cloned monkey BMAL1.
  • the inventors have unexpectedly developed a method for successfully constructing a somatic cell cloned animal of a non-human primate through extensive screening and experimentation through extensive and intensive research. Specifically, the inventors used a specific reprogramming activator (such as Kdm4d protein, mRNA encoding Kdm4d protein, DNA encoding Kdm4d protein, or a combination thereof) to reconstitute or reconstitute embryos (including genetically edited).
  • the constitutive or reconstituted embryo is subjected to a reprogramming process, and optionally in combination with a specific activating treatment agent (such as a calcium ion activator (such as ionomycin), a histone deacetylase inhibitor (TSA), 6- DMAP, or a combination thereof, activates the reconstructed egg to successfully obtain a somatic cloned animal (such as a monkey) of a non-human primate for the first time.
  • a specific activating treatment agent such as a calcium
  • reconstituted egg refers to a reconstituted egg obtained by injecting a somatic cell (from the nucleus of a somatic cell) into an enucleated oocyte.
  • reconstructed embryo As used herein, the terms "reconstructed embryo”, “reconstructed embryo” are used interchangeably and refer to an embryo formed by the development or differentiation of a reconstituted egg, particularly an embryo at the 2-16 cell stage. Typically, reconstituting an embryo refers to injecting a somatic cell (from the nucleus of a somatic cell) into an enucleated oocyte, and the reconstructed egg divides to form an embryo and all embryos at different cell stages from which the embryo develops. .
  • activation treatment refers to the activation treatment of a reconstituted or reconstituted embryo using suitable activation processing conditions to facilitate reconstituted or reconstituted embryos for cell division.
  • activation processing can be performed using activation processing conditions conventional in the art. Typically, it can be carried out with an activating treatment agent and activated at a certain temperature (e.g., 37 ⁇ 2 ° C) for a period of time (e.g., 0.1-24 hours).
  • a certain temperature e.g., 37 ⁇ 2 ° C
  • a period of time e.g., 0.1-24 hours
  • a typical activation treatment activation condition is the activation treatment at 37 ° C, 5% CO 2 .
  • the kind of the activating treatment agent is not particularly limited, and representative examples include, but are not limited to, a calcium ion activator, a histone deacetylase inhibitor, 6-DMAP, cycloheximide (Cycloheximide) CHX), or a combination thereof.
  • histone deacetylase inhibitors include, but are not limited to, TSA, scriptaid, or a combination thereof.
  • Representative calcium ion activators include, but are not limited to, ionomycin, calcium ionophore A23187, ethanol, barium chloride, or combinations thereof.
  • the concentration of the activating treatment agent is from 0.1 nM to 100 mM; preferably, from 1 nM to 50 mM;
  • the concentration is from 0.5 mM to 30 mM, preferably from 0.8 mM to 15 mM, more preferably from 1 to 10 mM.
  • the calcium ion activator has a concentration of the calcium ion activator of 0.2 to 80 ⁇ M, preferably 0.5 to 50 ⁇ M, more preferably 0.8 to 20 ⁇ M, still more preferably 1 to 15 ⁇ M. .
  • the concentration of the histone acetylase inhibitor (TSA) in the activation treatment agent is from 0.5 to 80 nM, preferably from 1 to 50 nM, more preferably from 3 to 30 nM.
  • the concentration of the ionomycin in the activation treatment agent is 0.2 to 80 ⁇ M, preferably 0.5 to 50 ⁇ M, more preferably 0.8 to 20 ⁇ M, still more preferably 1 to 15 ⁇ M.
  • the concentration of the cycloheximide (CHX) in the activating treatment agent is 0.2-80 ⁇ g/ml, preferably 0.5-50 ⁇ g/ml, more preferably 1-20 ⁇ g. /ml.
  • the activation process comprises: ionomycin treatment for 2-6 minutes, 6-DMAP treatment for 3-6 hours, and treatment with TSA for 5-15 hours.
  • the experimental results of the present invention unexpectedly show that the success rate of preparing somatic cloned animals of non-human primates can be significantly improved when TSA is used as an activator together with the specific reprogramming treatment agent of the present invention.
  • a preferred activation treatment method comprises: treating a somatically injected reconstituted embryo in 5 ⁇ M of ionomycin dissolved in a TH3 operating solution for a period of time (e.g., 2-20 minutes, preferably about 5) Minutes (preferably at 37 ° C and 5% CO 2 ), then transfer the embryos to a solution of 2 mM DMAP and 10 nM TSA dissolved in HECN-9 for a period of time (eg 3-8 hours) (preferably) Ground at 37 ° C and 5% CO 2 ). Then transfer the embryo to the hamster embryo
  • Culture medium 9 (HECM-9) was treated with a 10 nM TSA concentration for one end of the time (eg, 3-8 hours).
  • reprogramming process refers to the treatment of a reconstructed egg or a reconstructed embryo such that the state of transcription and/or translation of a particular gene in the treated reconstructed egg or reconstructed embryo changes, thereby Causes transcription and translation of genes involved in the development of fertilized eggs.
  • the inventors have unexpectedly discovered that directional reprogramming of reconstructed eggs can be performed very efficiently and accurately using only one specific substance (ie, Kdm4d, Kdm4a, or Kdm4b protein or its encoding mRNA or a combination thereof), and this The effect of reprogramming is extremely effective and no observable side effects (such as embryo malformations, miscarriage, etc.) are found.
  • a typical reprogramming process is such that the reprogramming process is performed using an inverted microscope at a temperature that maintains cell activity (e.g., room temperature, preferably, 25-35 ° C).
  • a temperature that maintains cell activity e.g., room temperature, preferably, 25-35 ° C.
  • the kind of the reprogramming treatment agent is not particularly limited, and representative examples include, but are not limited to, (a1) Kdm4d protein; (a2) mRNA encoding Kdm4d protein; (a3) DNA encoding Kdm4d protein. And (a4) any combination of the above (a1), (a2), (a3).
  • 10 2 -10 8 (preferably 10 4 -10 6 ) copies/cell of mRNA encoding the Kdm4d protein are injected into the reconstituted egg or the embryonic cell.
  • the mRNA encoding Kdm4d is in a solution having a concentration of 10-6000 ng/ ⁇ l (preferably, 50-4000 ng/ ⁇ l, more preferably 80-3000 ng/ ⁇ l). .
  • the experimental results of the present invention unexpectedly indicate that when Kdm4d protein or mRNA encoding Kdm4d protein is used as a reprogramming treatment agent, and optionally used together with the activation treatment agent of the present invention, The success rate of preparing somatic cloned animals of non-human primates is significantly improved.
  • the method of the invention comprises: after activation of the embryo from 6-DMAP and TSA, transfer to HECM-9 containing only TSA, and Kdm4d mRNA injection after 1-2 hours: embryo
  • the Kdm4d mRNA was injected into the activated embryos using a Piezo micro-operating system by transferring to a TH3 operating solution containing 5 ⁇ g/ml of cytochalasin CB.
  • the cells cultured in vitro can be first genetically manipulated, and then the transgenic positive cells are screened, and then the cells are used as somatic cell nuclear donor donor cells to obtain transgenic positive cloned animals.
  • Transgenic offspring are obtained by somatic cell nuclear transfer technology.
  • somatic cloned animals of various different primates can be efficiently prepared. These cloned non-human primates can be used as model animals for scientific research, drug screening, and the like.
  • the reconstituted egg or the reconstituted embryo may be unengineered or genetically engineered.
  • Representative genetic engineering operations include, but are not limited to, gene editing (eg, CRISPR/Cas9-based gene editing), genetic recombination, lentiviral transgenes, Bacterial Artificial Chromsomes (BAC) transgenes, TALEN-based transgenes.
  • the genetic manipulation can be carried out before, during and/or after preparation of the reconstructed egg. Furthermore, in the present invention, the genetic manipulation can also be carried out before, during and/or after preparation of the reconstructed embryo.
  • the reconstituted egg, reconstituted embryo or somatic cell cloned animal of the invention has a mutated gene (including a knock-in or knock-out gene).
  • AAVS1 site knocked in green fluorescent protein AAVS1-GFP Knock In.
  • CamkIIa site knocks into the light-sensitive ion channel protein CHR2: CamkIIa-CHR2-EYFP Knock In.
  • Vgat site knocks into the light-sensitive ion channel protein CHR2: Vgat-CHR2-EYFP Knock In.
  • Drd1 Dopamine receptor D1 knock-in cre sequence: Drd1-Cre Knock In
  • Drd2 Dopamine receptor D2 knock-in cre sequence: Drd2-Cre Knock In
  • Drd2 knock-in Chr2 sequence Drd2-Chr2-EYFP Knock In
  • somatic cell nuclear transfer also known as somatic cell cloning, refers to injecting a somatic cell nucleus into an enucleated oocyte, reprogramming the somatic cell nucleus in the oocyte and reconstituting it into a new embryo, thereby obtaining the animal. A method of the individual.
  • an animal model carrying a specific genetic modification can be obtained by genetically modifying a cultured somatic cell in vitro and then obtaining a cloned animal using the genetically modified somatic cell as a nuclear donor.
  • An animal model carrying a complex genetic modification can be obtained by this method, and the obtained F0 animal model has no chimerism and can be used without passage, and the animal model obtained by using the same strain cell has a uniform genetic background.
  • the present invention therefore utilizes somatic cell nuclear transfer techniques to construct genetically modified non-human primate models.
  • the present invention provides, for the first time, a method of constructing a somatic cell-derived non-human primate cloned animal by using a reprogramming activator (such as Kdm4d protein, mRNA encoding Kdm4d protein, DNA encoding Kdm4d protein, or Combination) and activation of treatment agents (such as calcium ion activators (such as ionomycin), histone deacetylase inhibitors (TSA), 6-DMAP, or a combination thereof) on reconstructed eggs (including genetic editing or not) Gene editing) performs activation and reprogramming processing to obtain somatic cloned animals (such as monkeys) of non-human primates.
  • a reprogramming activator such as Kdm4d protein, mRNA encoding Kdm4d protein, DNA encoding Kdm4d protein, or Combination
  • treatment agents such as calcium ion activators (such as ionomycin), histone deacetylase inhibitors (
  • a reprogramming activator such as H3K9me3 demethylase Kdm4d
  • H3K9me3 demethylase Kdm4d a reprogramming activator that can significantly enhance the developmental capacity of somatic cell nuclear transfer embryos in nuclear transfer embryos of non-human primates (such as monkeys).
  • the present inventors have found for the first time that 10-180 ng/ ⁇ l (preferably, 50-4000 ng/ ⁇ l, more preferably, 80-3000 ng/ ⁇ l) of activated embryo-activated reconstructed embryos at the 1-cell stage are injected.
  • the dose of H3K9me3 demethylase Kdm4d can significantly improve the developmental capacity of somatic cell nuclear transfer embryos. More than 50% of embryos develop into blastocyst stage, and 3% of blastocysts can develop into mature individuals.
  • the present inventors have found for the first time that nuclear transfer using cells edited by Crispr/Cas9 as a nuclear donor is highly efficient, and the obtained genetically edited non-human primate (e.g., monkey) has no off-target phenomenon.
  • Aborted fetal monkeys were removed from tissues such as viscera, head, limbs and tail. The remaining trunks were cut with scissors to the smallest possible size, and then in a cell culture medium containing 1 mg/ml DNase and 0.5 mg/ml Type IV collagenase. Digestion (DMEM + 10% FBS + antibiotic + non-essential amino acids + glutamine) for four hours (37 ° C, 5% CO 2 ). The digested and isolated cells were transferred to a 10 cm culture dish, and cultured until the cells were overgrown, and the isolated primary cells were digested, resuspended in a cell culture medium containing 10% DMSO, and stored overnight. Before performing somatic cell nuclear transfer, a cryopreserved primary cell is resuscitated and cultured in a 6-well plate. After the cell is overgrown for 3 days, it can be subjected to somatic cell nuclear transfer experiments.
  • the follicular fluid was taken from the crab monkey/rhesus monkey when it was taken, centrifuged and washed twice with TH3, and finally resuspended in a 4° refrigerator with a small amount of TH3.
  • oocyte donors Female cynomolgus monkeys with healthy and menstrual patterns of 6-12 years old were selected as oocyte donors. FSH was injected on the third day of menstruation, 25 IU each time, and two consecutive injections per day for 8 days. On the eleventh day of menstruation, 1000 IU of HCG was injected, and laparoscopic ovulation was performed 36 hours after the injection of HCG. Through laparoscopic surgery, a 2-8 mm diameter follicular fluid was aspirated by a vacuum aspirator, and the oocytes were picked out from the follicular fluid by a glass tube under a stereoscopic microscope and transferred to a CMRL-1066 mature medium for use.
  • Oocyte enucleation Transfer 15-20 MII oocytes to a glass-bottomed, oil-sealed TH3 manipulation drop containing 5 ⁇ g/ml CB and observe the oocyte nucleus under an inverted microscope assisted by a spindle imaging system. Position, using ovipositate to fix the oocyte to position the oocyte nucleus at 3 o'clock. The Pezon microscopy system was used to drive a 10 micron diameter flat needle to break the zona pellucida and aspirate the oocyte nucleus. After the end of the whole group of oocytes, the enucleated oocytes were transferred to HECM-9 medium.
  • Injecting fibroblasts into enucleated oocytes Transfer 15-20 enucleated oocytes to an oil-sealed TH3 drip containing 5 ⁇ g/ml CB, and fix the oocytes with egg-shaped needles. Point or 6 o'clock direction.
  • a laser-breaking zona pellucida system to break the zona pellucida into a narrow slit, use a 15-18 micron diameter oblique needle to aspirate fibroblasts and transfer the cells to HVJ-E for 10 seconds, then aspirate the cells through a transparent The narrow space is injected into the perivitelline space of the enucleated egg cells, and the somatic cells are fused into the oocyte in about 10 minutes.
  • Injecting cumulus cells into enucleated oocytes Transfer 15-20 enucleated oocytes to an oil-sealed TH3 operation drop containing 5 ⁇ g/ml CB, and fix the oocytes with egg-shaped needles, and place the polar body at 12 o'clock. Or 6 o'clock direction.
  • the transparent band was broken into a narrow slit by a laser-breaking zona pellucida system.
  • the cumulus cells were aspirated by a 10 ⁇ m diameter oblique needle and the cells were transferred to HVJ-E for 10 seconds, and then the cells were aspirated and narrowed through a transparent band. At the 9 o'clock position where the enucleated egg cells are injected, the somatic cells are fused into the oocyte in about 10 minutes.
  • Embryo activation can be performed 1- to 1.5 hours after fusion of somatic cells with enucleated oocytes. The embryos were transferred to TH3 containing 5 ⁇ M ionomycin for 5 minutes, then transferred to HECM-9 medium containing 2 mM 6-DMAP and 10 nM TSA for 5 hours, and then transferred to HECM-9 containing 10 nM TSA. After culturing for 5 hours, it was finally transferred to HECM-9 medium for culture.
  • Kdm4d mRNA injection After 5-6 hours of embryo activation, the activated embryos were transferred to an oil-sealed TH3 operation drop containing 5 ⁇ g/ml CB, and the embryos were fixed with an egg-shaped needle, and the Diazo microscopic operating system was used to drive the diameter 2-3.
  • a micron flat-mouth injection needle breaks the zona pellucida and penetrates into the embryo, breaking the embryonic cell membrane and injecting 1000 ng/ ⁇ l of Kdm4d mRNA into the embryo, each embryo is injected about 10 pl. After the end of the injection, the embryos were transferred to HECM-9 medium at 37 ° C, 5% CO 2 .
  • Embryo culture The cloned embryos were cultured in HECM-9 medium, transferred to HECM-9 medium containing 5% FBS at the stage of culture to 8 cells, and changed every other day until blastocysts appeared on days 7-8. .
  • Embryo transfer Healthy adult female monkeys in the large follicular phase or ovulation phase are selected for embryo transfer. 3-7 2 cell-blastocyst stage embryos were transplanted into the fallopian tubes of a female monkey by minimally invasive surgery.
  • the human Kdm4d gene CDS region was amplified from the plasmid carrying the Kdm4d gene sequence, and the amplified fragment was purified and recovered. In vitro transcription was carried out using the mMESSAGE mMACHINE T7ULTRA kit (Life Technologies, AM1345) kit, and the transcript was purified by MEGA clear kit (Life Technologies, AM1908) kit and dissolved in RNAse free water.
  • cynomolgus monkeys Macaca fascicularis
  • the use and care of cynomolgus monkeys is in accordance with the approval document of the “Genetic-Modified Monkey Model by Somatic Cell Nuclear Transfer (ION-2018002)” approved by the Animal Science Committee of the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences.
  • the monkeys used were placed in an air-conditioned environment (temperature: 22 ⁇ 1 ° C; humidity: 50% ⁇ 5% RH), 12 hours light / 12 hours dark cycle (07:00 to 19:00). All feeds were purchased. From Suzhou Anmufei Company, twice a day, the green material is mainly based on fruits and vegetables, and is replenished once a day.
  • Ear tissues, blood cells, and cultured fibroblasts from A6 monkeys were used for genotypic analysis.
  • the primer for the BMAL1 detection site was forward: ACCATCGGCTGCGTACACCTCTAT, reverse: ATTTCAGGTGTGAGCCACTCCACC, and we cloned the PCR product for TA analysis.
  • Karyotype analysis A6 fibroblasts grown in 10 cm culture dishes were treated with 100 ng/ml colchicine for 4-6 hours, then digested with 0.25% trypsin-EDTA, then treated with 0.075 M potassium chloride at 37 °C. 30 minutes. The hypotonic-treated cells were fixed in methanol and acetic acid (3:1) for 30 minutes and then dropped, and finally subjected to Giemsa staining and chromosome counting.
  • the method of collecting cynomolgus superoved and oocytes is as described above. Intramuscular injection of 25 IU of recombinant human follicle stimulating hormone in healthy female rhesus monkeys was performed twice daily from day 3 to day 11 of menstruation. On the evening of the 11th day, 1000 IU of human chorionic gonadotropin (hCG) was intramuscularly injected, and the next day, oocyte collection was performed by a laparoscope and a vacuum aspiration system. The collected oocytes were cultured in a pre-equilibrated HECM-9 medium. The second malignant oocyte in the second division is selected for SCNT.
  • hCG human chorionic gonadotropin
  • the spindle-chromosome complex was rapidly removed by a piezo-driven denuclearization needle under the Oosight system.
  • a laser was used to make a gap in the enucleated oocyte zona pellucida, and then the HVJ-E-infected fibroblasts were injected into the perivitelline space by a slanting needle and mediated by fusion.
  • Reconstituted nuclear transfer embryos were activated in TH3 (HEPES-buffered TALP medium containing 0.3% bovine serum albumin), first treated with TH3 containing 5 mM calcium ionophore for 5 minutes, and then containing 2 mM 6-dimethylamine. The TH3 was processed for 5h.
  • the nuclear transfer embryos were treated with 10 nM Trichostatin (TSA) for 10 h, and 10 pl of 1000 ng/ml Kdm4d mRNA was injected 6 h after activation.
  • TSA Trichostatin
  • the embryos were cultured in HECM-9 medium at 37 ° C with 5% carbon dioxide. When the embryo reached the 8-cell stage, it was transferred to HECM-9 medium supplemented with 5% fetal calf serum, and the medium was changed every two days until the blastocyst stage was reached. The embryos will be transplanted into the fallopian tubes of the mother monkeys (with ovulation or corpus luteum) synchronized in the menstrual cycle in a well-developed 2 to 8 cell stage.
  • Site-specific primers containing fluorescent dyes FAM/HEX/TMR
  • Fluorescent dye-labeled STR amplicons were diluted by a mixture of ROX500 and deionized formamide and then subjected to capillary electrophoresis on an ABI PRISM 3730 genetic analyzer to obtain raw data.
  • mtDNA Single nucleotide polymorphism (SNP) analysis, mtDNA was also extracted from monkey ear tissue samples. The mtDNA was amplified using a specific primer (F:CCACTTCACATCAAACCATCACTT R:CAAGCAGCGAATACCAGCAAAA), and the PCR product was used for sequencing and SNP analysis.
  • F:CCACTTCACATCAAACCATCACTT R:CAAGCAGCGAATACCAGCAAAA Single nucleotide polymorphism
  • BMAL1 primer forward primer; 5"-TAACCTCAGCTGCCTCGTTG-3", reverse primer; 5'-TATTCATAACACGACGTGCC-3
  • Example 1 Monkey nuclear transfer technology operation process establishment and optimization.
  • the nucleus of the oocyte of a large mouse can be clearly distinguished under a normal inverted microscope, and can be easily removed by a microscopic needle.
  • the nucleus of primate oocytes cannot be clearly distinguished under normal inverted microscope.
  • Previous methods of removing monkey oocyte nucleus rely on Hoechst staining and fluorescence localization and blind suction. Both methods are very large for embryo loss and can easily affect the subsequent developmental efficiency of the embryo. In this study, we used a polarized light-based spindle imaging system (Oosigt Imaging System) that clearly distinguishes the monkey oocyte nucleus without affecting oocyte quality.
  • Oosigt Imaging System polarized light-based spindle imaging system
  • a 10 micron microneedle driven by Piezo was used to break the zona pellucida and aspirate the monkey oocyte nucleus.
  • the laser-breaking zona pellucida system is used to assist the somatic cell injection, and the inactivated Sendai virus is used to induce the fusion of the fetal monkey somatic cells and the enucleated oocytes.
  • the fused somatic cell nucleus will re-agglomerate into the morphology of the oocyte nucleus.
  • the optimized embryo activation conditions can significantly enhance the developmental capacity of monkey somatic cell nuclear transfer embryos.
  • the embryo constructed by this method was transplanted into the female monkey receptor oviduct, and although many recipients were transplanted, the recipient of the monkey somatic cell nuclear transfer pregnancy was not obtained.
  • a normal cynomolgus monkey embryo and three somatic cell nuclear transfer embryos obtained by single sperm injection are first gathered together to make them co-develop, and 10 transplants are received.
  • three pregnant recipients were obtained and two surviving individuals were obtained, but by genetic analysis, it was found that only two surviving monkeys were derived from normal monkey embryos obtained by single sperm injection, while somatic cell nuclear transfer embryos did not participate in the individual. occur.
  • 3-4 embryos all transplanted with monkey somatic cells were brought together to make them co-develop. Although a number of pregnant recipients were obtained, all were aborted at an early stage and no surviving individuals were obtained.
  • Kdm4d mRNA enhances the developmental capacity of monkey nuclear transfer embryos
  • the 4-cell and 8-cell stage embryos obtained from normal monkey sperm injection (ICSI) and the monkey-nuclear transplantation 8-cell stage embryos injected with Kdm4d and Kdm4d.
  • Transcriptome sequencing was performed. By comparing the normal ICSI 4 cell stage and the 8-cell stage embryo transcriptome, the 8 cell stage expression level was higher than the 4 cell stage 5 times or more, and 3997 transcription activation regions were obtained. Next, 3997 regions of the 8-cell nuclear transfer embryo transcriptome without Kdm4d were analyzed, and 2465 regions were found to be unactivated. These 2465 regions were called reprogramming resistance regions. Comparative analysis of the reprogramming resistance zone in the transcriptome of 8-cell nuclear transfer embryos injected with Kdm4d revealed a significant increase in the expression level of 2178 regions.
  • female cynomolgus monkey fetal monkey primary fibroblasts were used as donor cells for the construction of somatic cell nuclear transfer cloned monkeys.
  • a total of 127 MII oocytes were manipulated, and 109 single pronuclear embryos were obtained after oocyte enucleation, somatic cell injection, and embryo activation (Fig. 6).
  • the 109 embryos were injected with Kdm4d mRNA, and 79 embryos of 2 cell phase, 2-4 cell phase, 8 cell phase, 8-16 cell phase, or blastocyst stage were transplanted to 21 recipient mothers.
  • 6 pregnant mothers were successfully obtained.
  • the four pregnant female monkeys there are gestational sacs and fetuses, while the other two pregnant females have only gestational sacs and no fetuses.
  • This example utilizes adult female cynomolgus monkey cumulus cells as a nuclear donor for the construction of clonal monkeys.
  • Example 7 Using the same procedure as in Example 7, a total of 290 MII stage oocytes were manipulated, and after ovary enucleation, somatic cell injection, and embryo activation, 192 single pronuclear embryos were obtained. The 192 embryos were injected with Kdm4d mRNA, and 181 2-cell-blastocyst embryos were transplanted into 42 recipient mothers. By B-ultrasound, 22 pregnant females were successfully obtained. Among them, 12 pregnant female monkeys have gestational sacs and fetuses, while the other 10 pregnant females have only gestational sacs and no fetuses.
  • the two most commonly used non-human primates in current scientific research include cynomolgus monkeys and rhesus monkeys. It has been suggested in the study of the above-described embodiments of the present invention that the above specific treatment method of the present invention can significantly improve the efficiency of mammalian nuclear transfer.
  • SNPs single nucleotide polymorphisms
  • STR microsatellite sequence analysis
  • Example 11 Construction of a non-human primate genetically modified animal model using somatic cell nuclear transfer technology (taking cynomolgus monkey/rhesus monkey as an example)
  • an animal model carrying a specific genetic modification can be obtained by genetically modifying a cultured somatic cell in vitro and then obtaining a cloned animal using the genetically modified somatic cell as a nuclear donor.
  • An animal model carrying a complex genetic modification can be obtained by this method, and the obtained F0 animal model has no chimerism and can be used without passage, and the animal model obtained by using the same strain cell has a uniform genetic background. Therefore, the use of somatic cell nuclear transfer technology to construct a genetically modified non-human primate model can solve all the problems mentioned above.
  • the genetically modified non-human primate models that have been developed by the present invention using this technology are:
  • AAVS1 site knocked in green fluorescent protein AAVS1-GFP Knock In.
  • CamkIIa site knocks into the light-sensitive ion channel protein CHR2: CamkIIa-CHR2-EYFP Knock In.
  • Vgat site knocks into the light-sensitive ion channel protein CHR2: Vgat-CHR2-EYFP Knock In.
  • Drd1 Dopamine receptor D1 knock-in cre sequence: Drd1-Cre Knock In
  • Drd2 Dopamine receptor D2 knock-in cre sequence: Drd2-Cre Knock In
  • Drd2 knock-in Chr2 sequence Drd2-Chr2-EYFP Knock In
  • Example 12 BMAL1 gene knockout cynomolgus monkey somatic cell preparation
  • ARNT-Like 1 is a transcription factor that regulates circadian rhythms.
  • BMAL1 gene-edited cynomolgus monkey individuals were obtained by the CRISPR/Cas9 method.
  • monkey A6 was selected as a donor cell-derived monkey for cloning because the monkey did not detect BMAL1 protein expression, and showed obvious physiological disease phenotype, including physiological cycle inhibition of blood hormones, frequent nocturnal activity, and rapid eye movement ( REM) Reduced and non-rapid eye movement sleep, as well as psychosis-related behavior. Therefore, skin fibroblasts of A6 monkeys were taken and cultured for SCNT (Fig. 9, A-C).
  • the karyotype of the cultured fibroblasts showed a normal diploid with 42 chromosomes (Fig. 9D).
  • Genotyping of ear tissue, blood cells and fibroblasts of A6 monkeys, two types of BMAL1 mutations were found by monoclonal sequencing of PCR products: "-8bp” and "-8bp, +4bp, 2bpPM” (Fig. 9E).
  • Further genotypic analysis of individual fibroblasts revealed a "-8bp/-8bp" homozygous gene mutation or a "-8bp/-8bp, +4bp, 2bpPM” heterozygous gene mutation in the BMAL1 gene (Fig. 9F). ).
  • SCNTs were performed using fibroblasts from macaque A6 edited by the BMAL1 gene. Mature oocytes are obtained by super-discharging female macaques. Then, Sendai virus is used to assist the fusion of individual fibroblasts with enucleated oocytes to obtain SCNT oocytes, and is incubated with calcium ionophore and 6-dimethylamine. To promote epigenetic reprogramming after nuclear transfer, SCNT embryos were incubated with the histone deacetylase inhibitor trichostatin (TSA) and simultaneously injected with H3K9me3 demethylation factor Kdm4d mRNA (Fig. 10A, B).
  • TSA histone deacetylase inhibitor
  • the BMAL1 genotype was analyzed by first taking the ear tip tissues of five cloned monkeys. Four cloned monkeys (B1, B3, B4, B5) were found to carry a homozygous gene mutation of BMAL1 gene ("-8bp/-8bp, +4bp, 2bpPM"), and one monkey (B2) carries a heterozygous gene of BMAL1 gene. Mutation ("-8 bp / -8 bp") (Fig. 10D). This is identical to the mutant genotype of the donor fibroblasts previously detected and the mutant genotype of monkey A6.
  • mtDNA mitochondrial DNA
  • Fig. 11A-E STR analysis of 29 loci showed that the donor fibroblasts of the five cloned monkeys were identical to the nuclear DNA of the monkey A6, but different from the nuclear DNA of the recipient monkey and the oocyte donor monkey (Fig. 11F). .
  • the present invention knocks out the rhythm-related important gene BMAL1 in the cynomolgus monkey genome by the CRISPR/Cas9 system, and obtains 5 gene-editing monkeys.
  • fibroblasts isolated from one of the well-characterized monkey skins were used as somatic cell nuclear donors, and five BMAL1 gene-edited cloned monkeys were successfully obtained.
  • the sequence alignment and microsatellite analysis of the first monkey and the cloned monkey and the comparison of mitochondrial DNA indicated that these cloned monkeys and the first monkey had the same genetic background.
  • Kdm4d as a demethylating enzyme of H3K9me3, can significantly improve the reprogramming efficiency of mice and monkeys by removing reprogrammed methylation regions in nuclear transfer embryos; Xist-mediated X chromosome silencing is likely in mice, It is also conserved in pigs and primates.
  • DNA remethylation and deletion of H3K27me3 may also be key factors affecting cloning efficiency.
  • the five cloned monkeys each had only one genotype, indicating that this chimeric phenomenon did not occur in the five cloned monkeys. Therefore, the present invention recognizes that the current cloning methods are mature, and the phenotype of these cloned monkeys will also Will be further analyzed.

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Abstract

本发明首次提供了一种非人灵长类的体细胞克隆动物的制备方法,具体地,包括步骤:(i)提供一重构卵,所述的重构卵来自所述非人灵长类动物;(ii)对所述重构卵进行激活处理,从而形成经激活的重构卵或经激活的由所述重构卵形成的重构胚胎;(iii)对(a)所述经激活的重构卵或(b)经激活的重构胚胎的胚胎细胞,进行重编程处理,从而获得经重编程的重构卵或经重编程的重构胚胎;和(iv)将所述经重编程的重构卵或经重编程的重构胚胎进行再生,从而获得非人灵长类的体细胞克隆动物。本发明的方法可以显著提高非人灵长类动物(如猴)的细胞核移植胚胎的发育能力。

Description

非人灵长类的体细胞克隆动物的制备方法 技术领域
本发明涉及哺乳动物繁殖技术领域,具体地,涉及非人灵长类的体细胞克隆动物的制备方法。
背景技术
由于进化上与人相近,非人灵长类的脑结构、功能活动等很多方面与人类高度相似。相对与其他实验动物,非人灵长类具有解决人类问题,特别是脑相关问题的独特优势:除了是一个很好的研究人脑正常脑高级功能的关键实验动物外,它们还是研究脑疾病机理和治疗方法的很好的模型动物。我国具有非常丰富的非人灵长类资源,为了充分利用非人灵长类来研究脑疾病以及人脑正常高级功能,需要利用转基因技术构建多种多样的非人灵长类脑疾病模型和光遗传等工具模型。
目前获得非人灵长类基因修饰动物模型最常用的方法是慢病毒感染卵母细胞法和可编辑核酸酶法(CRISPR-Cas9等)。尤其是CRISPR-Cas9为代表的基因编辑技术的飞速发展,使得科学家们对非人灵长类的基因编辑变得简便易行。即便如此,利用现有的转基因技术构建非人灵长类转基因动物模型仍然有很多关键的问题没有解决,比如,1,无论是利用慢病毒感染卵母细胞法获得转基因动物还是利用可编辑核酸酶技术获得基因敲除动物,其首代(F0)动物模型普遍存在嵌合体现象,这就导致用此类方法获得的首代动物模型不能用于科学研究。传统的大小鼠等啮齿类动物可以通过传代获得没有嵌合现象的F1代动物模型,而对非人灵长类动物模型而言,要获得F1代动物模型,一般需要5年左右的时间。2,对于携带复杂的基因修饰非人灵长类模型(条件敲除或基因敲入),目前通过胚胎注射可编辑核酸酶引入位点特异性的DNA双链断裂及同步注射同源片段使外源片段定点插入的方法在哺乳动物水平效率非常低下,在非人灵长类水平更是还没有成功的报道。3,无论是药物筛选还是机制机理研究,目前最常用的啮齿类动物模型以近交系为宜,这样可以尽量减少遗传背景差异性,保证实验结果的严谨精确。大小鼠可以通过不断的自交来实现遗传背景的一致,而对于具有漫长性成熟周期的非人灵长类来说,通过此自交来实现遗传背景的一致性肯定是不现实的。可以说,由于以上三个关键问题的存在, 虽然大家对非人灵长类基因修饰动物模型需要迫切,但是目前非人灵长类基因修饰动物模型尚未在科学研究中得到广泛应用。
目前体细胞来源的克隆猴尚未有成功报道。该课题之所以一直没有获得成功,主要原因有:1,技术要求高,灵长类核移植技术操作难度远高于目前已经成功的其他哺乳动物。2,克隆胚胎发育效率低,目前猴体细胞克隆胚胎的发育效率远远低于其他哺乳动物。
因此,本领域迫切需要成功构建一种体细胞来源的非人灵长类的克隆动物。
发明内容
本发明的目的在于提供一种体细胞来源的非人灵长类的克隆动物。
本发明的另一目的在于提供了一种优化非人灵长类体细胞核移植操作的流程和提升非人灵长类体细胞核移植效率的方法。并利用优化后的操作和方法得到了健康存活的体细胞核移植来源的克隆猴。
本发明第一方面,提供了一种非人灵长类的体细胞克隆动物的制备方法,包括步骤:
(i)提供一重构卵,所述的重构卵来自所述非人灵长类动物;
(ii)对所述重构卵进行激活处理,从而形成经激活的重构卵或经激活的由所述重构卵形成的重构胚胎,其中所述的重构胚胎处于2细胞期、4细胞期或8细胞期;
(iii)对(a)所述经激活的重构卵或(b)经激活的重构胚胎的胚胎细胞,进行重编程处理,从而获得经重编程的重构卵或经重编程的重构胚胎;和
(iv)将所述经重编程的重构卵或经重编程的重构胚胎进行再生,从而获得非人灵长类的体细胞克隆动物。
在另一优选例中,所述步骤(iii)中,用重编程激活剂进行重编程处理。
在另一优选例中,所述重编程激活剂选自下组:
(a1)Kdm4d蛋白;
(a2)编码Kdm4d蛋白的mRNA;
(a3)编码Kdm4d蛋白的DNA;和
(a4)上述(a1)、(a2)、(a3)的任意组合。
在另一优选例中,所述重编程激活剂包括选自下组的重编程激活剂:
(a1)Kdm4d蛋白;
(a2)编码Kdm4d蛋白的mRNA;
(a3)编码Kdm4d蛋白的DNA;和
(a4)上述(a1)、(a2)、(a3)的任意组合。
在另一优选例中,所述重编程激活剂还包括选自下组的重编程激活剂:
(b1)Kdm4a蛋白;
(b2)编码Kdm4a蛋白的mRNA;
(b3)编码Kdm4a蛋白的DNA;和
(b4)上述(b1)、(b2)、(b3)的任意组合。
在另一优选例中,所述重编程激活剂还包括选自下组的重编程激活剂:
(c1)Kdm4b蛋白;
(c2)编码Kdm4b蛋白的mRNA;
(c3)编码Kdm4b蛋白的DNA;和
(c4)上述(c1)、(c2)、(c3)的任意组合。
在另一优选例中,所述的重构卵或重构胚胎是未经基因工程操作或经过基因工程操作的重构卵或重构胚胎。
在另一优选例中,所述基因工程操作包括ZFN(zinc-finger nucleases)、TALEN或Crispr技术。
在另一优选例中,所述经过基因工程操作的重构卵或重构胚胎包括敲除BMAL1基因的重构卵或重构胚胎。
在另一优选例中,所述的重编程处理包括:将编码Kdm4d蛋白的mRNA注入所述的重构卵或所述重构胚胎的胚胎细胞中。
在另一优选例中,所述的重编程处理包括:将编码Kdm4a、Kdm4b或Kdm4d蛋白的一种或多种的mRNA注入所述的重构卵或所述重构胚胎的胚胎细胞中。
在另一优选例中,将10 2-10 8(较佳地10 4-10 6)拷贝/细胞的编码Kdm4d蛋白的mRNA注入所述重构卵或所述的胚胎细胞。
在另一优选例中,将10 2-10 8(较佳地10 4-10 6)拷贝/细胞的编码Kdm4a、Kdm4b或Kdm4d蛋白的一种或多种的mRNA注入所述重构卵或所述的胚胎细胞。
在另一优选例中,所述的重构胚胎处于4细胞期或8细胞期。
在另一优选例中,所述编码Kdm4d的mRNA处于一溶液中,所述mRNA的浓 度为10-6000ng/μl(较佳地,50-4000ng/μl,更佳地,80-3000ng/μl)。
在另一优选例中,所述编码Kdm4a或Kdm4b的mRNA处于一溶液中,所述mRNA的浓度为10-6000ng/μl(较佳地,50-4000ng/μl,更佳地,80-3000ng/μl)。
在另一优选例中,所述的再生在非人灵长类的代孕动物的子宫中进行。
在另一优选例中,所述的代孕动物的物种与所述重构卵的物种是相同的。
在另一优选例中,所述的再生在人工子宫中进行。
在另一优选例中,在所述方法中,所述步骤(ii)中,获得经激活的重构胚胎;以及步骤(iii)中,对所述重构胚胎进行重编程处理。
在另一优选例中,在所述方法中,所述步骤(iv)中,将经重编程的重构胚胎进行再生,从而获得非人灵长类的体细胞克隆动物。
在另一优选例中,所述步骤(iv)包括:
(iv1)将所述经重编程的重构卵在体外或体内培养,形成重构胚胎;和
(iv2)将所述重构胚胎移植到非人灵长类动物的输卵管中,从而获得所述非人灵长类的体细胞克隆动物。
在另一优选例中,所述的移植为未侵入性移植(即不造成任何创伤)。
在另一优选例中,所述的方法是非治疗性和非诊断性的。
在另一优选例中,所述重构卵用包括以下步骤的体外方法制备:
(a)提供一去核的非人灵长类动物的供质卵母细胞和一供核的非人灵长类动物的体细胞;
(b)用显微注射法将用于供核的非人灵长类动物的体细胞直接注射入所述的去核供质卵母细胞,形成重构卵。
在另一优选例中,所述体细胞选自下组:成纤维体细胞、卵丘细胞、胚胎干细胞、精原细胞、睾丸支持细胞、间充质干细胞、皮肤细胞、乳腺细胞、输卵管细胞、耳细胞、卵巢细胞、上皮细胞、内皮细胞、肌肉细胞、神经细胞、成骨细胞、或其组合。
在另一优选例中,所述步骤(ii)中,用激活处理剂进行激活处理。
在另一优选例中,所述的激活处理的激活条件如下所示:
在37℃、5%CO 2下,用离子霉素2-6分钟,6-DMAP 3-6小时,TSA 5-15小时。
在另一优选例中,所述激活处理剂选自下组:钙离子激活剂、组蛋白去乙 酰化酶抑制剂、6-DMAP、放线菌酮Cycloheximide(CHX)、或其组合。
在另一优选例中,所述组蛋白去乙酰化酶抑制剂选自下组:TSA、scriptaid、或其组合。
在另一优选例中,所述激活处理剂的浓度为0.1nM-100mM;较佳地,1nM-50mM。例如,对于离子霉素而言,其浓度为0.5mM-30mM,较佳地0.8mM-15mM,更佳地1-10mM。
在另一优选例中,所述激活处理剂中,所述组蛋白乙酰化酶抑制剂(TSA)的浓度为0.5-80nM,较佳地,1-50nM,更佳地,3-30nM。
在另一优选例中,所述步骤(ii)包括选自下组的一个或多个特征:
(i)在钙离子激活剂中激活0.5min-1h,较佳地,1min-30min,更佳地,2min-10min;
(ii)在TSA和6-DMAP中激活0.5h-20h,较佳地,0.8h-10h,更佳地,1h-8h;
(iii)在TSA中激活0.5h-24h,较佳地,0.8h-15h,更佳地,10-12h。
在另一优选例中,步骤(iii)中,对所述激活的重构卵再次激活2-10小时,较佳地,3-7小时。
在本发明第二方面,提供了一种试剂盒,包括:
(a)第一容器,所述第一容器中含有重编程激活剂,其中所述的重编程激活剂包括选自下组的重编程激活剂:
(a1)Kdm4d蛋白;
(a2)编码Kdm4d蛋白的mRNA;
(a3)编码Kdm4d蛋白的DNA;
(a4)上述(a1)、(a2)、(a3)的任意组合;
(b)第二容器,所述第二容器中含有激活处理剂;和
(c)标签或说明书。
在另一优选例中,所述重编程激活剂还包括选自下组的重编程激活剂:
(b1)Kdm4a蛋白;
(b2)编码Kdm4a蛋白的mRNA;
(b3)编码Kdm4a蛋白的DNA;和
(b4)上述(b1)、(b2)、(b3)的任意组合。
在另一优选例中,所述重编程激活剂还包括选自下组的重编程激活剂:
(c1)Kdm4b蛋白;
(c2)编码Kdm4b蛋白的mRNA;
(c3)编码Kdm4b蛋白的DNA;和
(c4)上述(c1)、(c2)、(c3)的任意组合。
在另一优选例中,所述标签或说明书中记载了本发明第一方面所述的方法。
在本发明第三方面,提供了一种本发明第二方面所述的试剂盒的用途,用于制备一试剂盒,所述试剂盒用于制备非人灵长类的体细胞克隆动物。
在本发明第四方面,提供了一种非人灵长类的体细胞克隆动物,用本发明第一方面所述的方法制备。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1显示了食蟹猴体细胞核移植操作流程优化;
其中,A.用作核供体的食蟹猴胎猴成纤维体细胞
B.纺锤体成像系统下的食蟹猴卵母细胞核(纺锤体-染色体复合体)
C.纺锤体成像系统辅助下利用Peizo显微操作系统去除食蟹猴卵母细胞核
D-F.激光辅助破透明带,HVJ-E病毒介导的供体细胞与去核卵母细胞融合
G.体细胞与去核卵母细胞融合后,细胞核重新形成的纺锤体-染色体复合体
H.核移植胚胎体外激活后形成单原核
I.优化条件后TSA处理并注射H3K9me3去甲基化酶Kdm4d mRNA的胚胎发育的囊胚。
图2显示了不同条件下利用胎猴成纤维细胞和成年猴卵丘细胞作核供体的效率统计;
其中,A.普通激活条件、组蛋白去乙酰化酶抑制剂TSA处理、TSA加H3K9me3去甲基化酶Kdm4d注射三种条件下成纤维细胞核移植胚胎囊胚发育统计
B.普通激活条件、组蛋白去乙酰化酶抑制剂TSA处理、TSA加H3K9me3去 甲基化酶Kdm4d注射三种条件下成纤维细胞核移植胚胎高质量囊胚发育统计
C.组蛋白去乙酰化酶抑制剂TSA处理、TSA加H3K9me3去甲基化酶Kdm4d注射两种条件下卵丘细胞核移植胚胎囊胚发育统计
D.组蛋白去乙酰化酶抑制剂TSA处理、TSA加H3K9me3去甲基化酶Kdm4d注射两种条件下卵丘细胞核移植胚胎高质量囊胚发育统计
E.转录组水平验证H3K9me3去甲基化酶Kdm4d提升猴体细胞核移植的效果;
图3显示了食蟹猴体细胞核移植胚胎移植怀孕率及胎儿发育统计;
其中,A,B.核移植怀孕受体B超图。A受体只有孕囊,无胎儿。B受体有孕囊和胎儿
C,D.两种不同细胞供体怀孕率统计和怀孕受体携带胎儿比例统计
E.携带胎儿的怀孕受体最终发育情况;
图4显示了体细胞核移植克隆猴遗传分析;
A,B.利用成纤维细胞得到的两只健康的克隆猴“ZhongZhong”和“HuaHua
C.两只克隆猴核基因组三个微卫星位点分析示意图
D,E两只克隆猴线粒体SNP分析示意图;
图5显示了食蟹猴胎猴成纤维细胞核移植;
其中,A.体细胞与去核卵母细胞融合后,细胞核重新形成的纺锤体-染色体复合体(对应图1G)
B.核移植胚胎体外激活后形成单原核(对应图1H)
C.常规条件激活后的胚胎发育的情况
D.常规条件激活并加TSA处理的胚胎发育的情况;
图6显示了食蟹猴卵丘细胞体细胞核移植以及对重构卵进行激活处理和重编程处理;
其中,A.食蟹猴卵丘细胞
B.卵丘细胞与去核卵母细胞融合后,细胞核重新形成的纺锤体-染色体复合体
C,D.核移植胚胎体外激活后形成单原核
E.常规条件激活并加TSA处理的胚胎发育的情况
F.优化条件后TSA处理并注射H3K9me3去甲基化酶Kdm4d mRNA的胚胎发育的囊胚;
图7显示了成纤维细胞核移植克隆猴“ZhongZhong”“HuaHua”更多线粒体SNP位点分析(对于图4D)
图8显示了卵丘细胞体细胞核移植克隆猴”A”和”B”遗传分析;
其中,A.两只克隆猴核基因组三个微卫星位点分析示意图
B和C.两只克隆猴多线粒体SNP位点分析。
图9显示了BMAL1基因编辑猴成纤维细胞制备。
(A)基因编辑食蟹猴成纤维细胞制备示意图。(B)BMAL1编辑猴A6。(C)来源于A6猴的皮肤纤维细胞Bar=200μm。(D)A6成纤维细胞核型正常。(E)A6猴耳尖组织、血液细胞、成纤维细胞基因型分析。(F)A6成纤维细胞单细胞基因型分析。
图10显示了BMAL1基因编辑克隆猴构建。
(A)构建BMAL1基因编辑克隆猴的示意图。(B)不同阶段的猴子SCNT胚胎,Bar=120μm。(C)B1-B5,来自由A6成纤维细胞进行SCNT的5只克隆猴(D)5只克隆猴耳组织BMAL1突变分析,4只猴子携带杂突变基因(“-8/-8,+4,2PM”),1只猴子携带纯合突变基因(“-8/-8”)突变。
图11显示了克隆猴BMAL1基因型分析及表达分析。
(A-E)B1至B5的单核苷酸多态性分析,显示线粒体DNA的SNPs与卵母细胞供体猴子相同,但与受体猴子和供体成纤维细胞不同。(F)5只克隆猴子(B1-B5)耳组织的短串联重复序列,表明其核DNA与供体成纤维细胞相同,但与卵母细胞供体和受体猴子不同。(G)克隆猴B1血液样本BMAL1表达的RT-PCR分析,显示其野生BMAL1转录本完全缺失。
具体实施方式
本发明人通过广泛而深入的研究,经过大量的筛选和尝试,首次意外地开发了一种可成功构建非人灵长类的体细胞克隆动物的方法。具体地,本发明人通过使用特定的重编程激活剂(如Kdm4d蛋白、编码Kdm4d蛋白的mRNA、编码Kdm4d蛋白的DNA、或其组合)对重构卵或重构胚胎(包括经过基因编辑的重构卵或重构胚胎)进行重编程处理,并且任选地配合使用特定的激活处理剂(如钙离子激活剂(如离子霉素)、组蛋白去乙酰化酶抑制剂(TSA)、6-DMAP、或其组合)对重构卵进行激活,从而首次成功地获得非人灵长类的体细胞克隆动物(如猴)。在此基础上,本发明人完成了本发明。
术语
典型地,“重构卵”指将体细胞(来自体细胞的细胞核)注射入去核的卵母细胞所获得的重构卵。
如本文所用,术语“重构胚胎”、“重构胚”可互换使用,指由重构卵发育或分化形成的胚胎,尤其是处于2-16细胞阶段的胚胎。典型地,重构胚胎指将体细胞(来自体细胞的细胞核)注射入去核的卵母细胞,并由所述重构卵分裂形成胚胎以及由此胚胎发育来的所有处于不同细胞阶段的胚胎。
激活处理
如本文所用,术语“激活处理”指采用合适的激活处理条件,对重构卵或重构胚胎进行激活处理,从而促使重构卵或重构胚胎进行细胞分裂。
在本发明中,可以采用本领域常规的激活处理条件进行激活处理。典型地,可以采用激活处理剂进行,在一定温度(如37±2℃)激活处理一段时间(如0.1-24小时)。
一种典型的激活处理的激活条件为在在37℃、5%CO 2下进行激活处理。
在本发明中,激活处理剂的种类没有特别限制,代表性的例子包括(但并不限于):钙离子激活剂、组蛋白去乙酰化酶抑制剂、6-DMAP、放线菌酮Cycloheximide(CHX)、或其组合。
代表性的组蛋白去乙酰化酶抑制剂包括(但并不限于):TSA、scriptaid、或其组合。
代表性的钙离子激活剂包括(但并不限于):离子霉素、钙离子载体A23187、乙醇、氯化锶、或其组合。
在另一优选例中,所述激活处理剂的浓度为0.1nM-100mM;较佳地,1nM-50mM;
例如对于离子霉素而言,其浓度为0.5mM-30mM,较佳地0.8mM-15mM,更佳地1-10mM。
在另一优选例中,所述激活处理剂中,所述钙离子激活剂的浓度为0.2-80μM,较佳地,0.5-50μM,更佳地,0.8-20μM,更佳地,1-15μM。
在另一优选例中,所述激活处理剂中,所述组蛋白乙酰化酶抑制剂(TSA)的浓度为0.5-80nM,较佳地,1-50nM,更佳地,3-30nM。
在另一优选例中,所述激活处理剂中,所述离子霉素的浓度为0.2-80μM,较佳地,0.5-50μM,更佳地,0.8-20μM,更佳地,1-15μM。
在另一优选例中,所述激活处理剂中,所述放线菌酮Cycloheximide(CHX)的浓度为0.2-80μg/ml,较佳地,0.5-50μg/ml,更佳地,1-20μg/ml。
在本发明的一个实施例中,所述的激活处理包括:离子霉素处理2-6分钟,6-DMAP处理3-6小时,以及用TSA处理5-15小时。
本发明的实验结果意外地表明,当采用TSA作为激活剂与本发明特定的重编程处理剂一起使用时,可以显著提高制备非人灵长动物的体细胞克隆动物的成功率。
在本发明中,一种优选的激活处理方法包括:将完成体细胞注射的重构胚胎在5μM溶于TH3操作液的离子霉素中处理一段时间(如2-20分钟,较佳地约5分钟)(较佳地在37℃和5%CO 2条件下),然后将胚胎转移到溶于HECN-9的2mM DMAP和10nM TSA浓度的液体处理一段时间(如3-8小时)(较佳地在37℃和5%CO 2条件下)。然后将胚胎转移到溶于hamster embryo
culture medium 9(HECM-9)的10nM TSA浓度的液体处理一端时间(如3-8小时)。
重编程处理
如本文所用,术语“重编程处理”指对重构卵或重构胚胎进行处理,从而使得经处理的重构卵或重构胚胎中的特定基因的转录和/或翻译的状态发生变化,从而导致与受精卵发育相关的基因发生转录和翻译。
对于灵长动物而言,由于基因组的复杂性,在本发明之前,虽然有人对重构卵(或重构胚胎)尝试了各种重编程处理和/或重激活处理,但是从未获得成功。另外,由于灵长动物基因组的复杂性(例如,目前对于灵长动物受精卵发育相关的基因知之甚少),在本发明之前,也无法预见能否成功对灵长动物的重构卵(或重构胚胎)进行有效而精确的重编程,从而产下体细胞克隆动物。
而本发明人意外地发现,仅用一种特定的物质(即Kdm4d、Kdm4a、或Kdm4b蛋白或其编码mRNA或其组合)可以非常有效而精准地对重构卵进行定向的重编程,而且这种重编程的效果极其有效且未发现存在可观察的副作用(比如胚胎畸形、流产等)。
一种典型的重编程处理的条件为在保持细胞活性的温度(如室温下,较佳地,25-35℃)利用倒置显微镜进行重编程处理。
在本发明中,重编程处理剂的种类没有特别限制,代表性的例子包括(但并不限于):(a1)Kdm4d蛋白;(a2)编码Kdm4d蛋白的mRNA;(a3)编码Kdm4d蛋白的DNA;和(a4)上述(a1)、(a2)、(a3)的任意组合。
在另一优选例中,将10 2-10 8(较佳地,10 4-10 6)拷贝/细胞的编码Kdm4d蛋白的mRNA注入所述重构卵或所述的胚胎细胞。
在另一优选例中,所述编码Kdm4d的mRNA处于一溶液中,所述mRNA的浓度为10-6000ng/μl(较佳地,50-4000ng/μl,更佳地,80-3000ng/μl)。
在一种优选的实施方式中,本发明的实验结果意外地表明,当采用Kdm4d蛋白或编码Kdm4d蛋白的mRNA作为重编程处理剂,并任选的与本发明的激活处理剂一起使用时,可以显著提高制备非人灵长动物的体细胞克隆动物的成功率。
在本发明的一个优选例中,本发明方法包括:将胚胎从6-DMAP和TSA中激活完成后,转移至只含有TSA的HECM-9中,1-2小时后进行Kdm4d mRNA注射:将胚胎转移至含有5μg/ml的细胞松弛素CB的TH3操作液中,利用Piezo显微操作系统将Kdm4d mRNA注射入激活后的胚胎中。
应用
利用本方法,可以首先对体外培养的细胞进行基因操作,然后筛选转基因阳性的细胞,再利用此细胞作为体细胞核移植供体细胞,即可获得转基因阳性克隆动物。通过体细胞核移植技术得到转基因后代,这些克隆动物不仅基因型一致,也能解决脱靶和嵌合等问题,同时由于首建个体即可用于后续研究,因此构建时间也基本上缩短为猴的怀孕期(约5.5个月)。
基于本发明方法,可以有效制备各种不同的灵长动物的体细胞克隆动物。这些克隆的非人灵长动物可以用作模型动物,用于科学研究、药物筛选等研究。
此外,在本发明中,所述的重构卵或重构胚胎可以是未经基因工程操作或经过基因工程操作的。代表性的基因工程操作包括(但并不限于):基因编辑(如基于CRISPR/Cas9的基因编辑)、基因重组、慢病毒转基因、Bacterial Artificial Chromsomes(BAC)转基因、基于TALEN的转基因。
在本发明中,所述的基因操作可以在制备重构卵之前、之中和/或之后进 行。此外,在本发明中,所述的基因操作也可以在制备重构胚胎之前、之中和/或之后进行。
在另一优选例中,本发明的重构卵、重构胚胎或体细胞克隆动物具有突变的基因(包括敲入或敲除的基因)。
代表性的突变情况包括(但并不限于):
基因敲入:
(1)AAVS1位点敲入绿色荧光蛋白:AAVS1-GFP Knock In。
(2)AAVS1位点敲入cre序列:AAVS1-Cre Knock In。
(3)AAVS1位点敲入LSL-CHR2序列:AAVS1-LSL-CHR2Knock In。
(4)CamkIIa位点敲入光敏感离子通道蛋白CHR2:CamkIIa-CHR2-EYFP Knock In。
(5)CamkIIa位点敲入钙成像蛋白Gcamp6s:CamkIIa-Gcamp6s Knock In。
(6)CamkIIa位点敲入cre序列:CamkIIa-Cre Knock In。
(7)Vgat位点敲入光敏感离子通道蛋白CHR2:Vgat-CHR2-EYFP Knock In。
(8)Vgat位点敲入钙成像蛋白Gcamp6s:Vgat-Gcamp6s Knock In。
(9)Vgat位点敲入cre序列:Vgat-Cre Knock In。
(10)Chat(Choline acetyltransferase)位点敲入cre序列:Chat-Cre Knock in
(11)Chat位点敲入Chr2序列:Chat-CHR2-EFYP Knock in
(12)Chat位点敲入Gcamp6s序列:Chat-gcamp6s Knock in
(13)Drd1(Dopamine receptor D1)敲入cre序列:Drd1-Cre Knock In
(14)Drd1敲入Chr2序列:Drd1-Chr2-EYFP Knock In
(15)Drd2(Dopamine receptor D2)敲入cre序列:Drd2-Cre Knock In
(16)Drd2敲入Chr2序列:Drd2-Chr2-EYFP Knock In
(17)GFAP位点敲入Chr2序列:GFAP-CHR2-EFYP Knock in
(18)GFAP位点敲入cre序列:GFAP-Cre Knock in
(19)GFAP位点敲入gcamp6s序列:GFAP-gcamp6s Knock in
(20)TH(hydroxytryptamine)位点敲入cre序列:TH-Cre Knock In
(21)TH位点敲入Chr2序列:TH-Chr2-EYFP Knock In
(22)Nestin位点敲入cre序列:Nestin-Cre Knock In
(23)其他组织或细胞特异的基因敲入;
点突变:
(1)SOD1 A4V点突变
(2)SOD1 H46R点突变
(3)SOD1 G93A点突变
(4)Foxp2 T327N+N349S点突变
基因敲除:
(1)克隆遗传背景一致的PRRT2基因敲除;
(2)克隆遗传背景一致的FMR1基因敲除;
(3)克隆遗传背景一致的ASPM基因敲除;
(4)克隆遗传背景一致的DISC1基因敲除;
(5)克隆遗传背景一致的MKRN3基因敲除;
(6)克隆遗传背景一致的SNCA基因敲除;
(7)克隆遗传背景一致的LRRK2基因敲除;
(8)克隆遗传背景一致的GBA基因敲除;
(9)克隆遗传背景一致的PRKN基因敲除;
(10)克隆遗传背景一致的PINK1基因敲除;
(11)克隆遗传背景一致的PARK7基因敲除;
(12)克隆遗传背景一致的VPS35基因敲除;
(13)克隆遗传背景一致的EIF4G1基因敲除;
(14)克隆遗传背景一致的Bmal 1基因敲除;
(15)克隆遗传背景一致的LRRK2+PINK1+PARK7基因敲除。
体细胞核移植
如本文所用,术语“体细胞核移植”又叫体细胞克隆,指将体细胞核注入去核的卵母细胞中,使体细胞核在卵母细胞中重编程并重构为新的胚胎,进而获得动物个体的一种方法。
在本发明中,通过在体外对培养的体细胞进行基因修饰,然后利用基因修饰的体细胞作为核供体获得克隆动物即可获得携带有特定基因修饰的动物模型。利用此方法可获得携带复杂基因修饰的动物模型,且获得的F0代动物模型不存在嵌合现象,不需要传代即可使用,另外利用同一株细胞获得的动物模型具有一致的遗传背景。因此本发明利用体细胞核移植技术来构建基因修饰非 人灵长类模型。
本发明的主要优点包括:
(1)本发明首次提供一种构建体细胞来源的非人灵长类的克隆动物的方法,通过使用重编程激活剂(如Kdm4d蛋白、编码Kdm4d蛋白的mRNA、编码Kdm4d蛋白的DNA、或其组合)和激活处理剂(如钙离子激活剂(如离子霉素)、组蛋白去乙酰化酶抑制剂(TSA)、6-DMAP、或其组合)对重构卵(包括基因编辑或未被基因编辑)进行激活和重编程处理,从而获得非人灵长类的体细胞克隆动物(如猴)。
(2)本发明首次发现,重编程激活剂(如H3K9me3去甲基化酶Kdm4d)在非人灵长类动物(如猴)的核移植胚胎中可以明显提升体细胞核移植胚胎的发育能力。
(3)本发明首次发现,对1细胞时期的经激活处理剂激活的重构胚进行注射10-6000ng/μl(较佳地,50-4000ng/μl,更佳地,80-3000ng/μl)剂量的H3K9me3去甲基化酶Kdm4d,可显著提升体细胞核移植胚胎的发育能力,有50%以上的胚胎发育到了囊胚阶段,有3%的囊胚可发育成成熟的个体。
(4)本发明首次发现,使用Crispr/Cas9编辑过的细胞作为核供体的核移植效率很高,并且所获得的基因编辑的非人灵长类动物(如猴)没有脱靶现象。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
除非另有说明,否则实施例中的材料或试剂均为市售产品。
通用方法
1.胎猴成纤维细胞分离培养
取流产胎猴去除内脏、头部、四肢和尾巴等组织,剩余躯干部用剪刀剪至尽可能小块,然后至于含有1mg/ml DNA酶和0.5mg/ml IV型胶原酶的细胞培养液中消化(DMEM+10%FBS+抗生素+非必须氨基酸+谷氨酰胺)四个小时(37℃,5%CO 2)。将消化分离得到的细胞转移至10厘米培养皿,并培养至细胞长满培 养皿,然后将分离得到的原代细胞消化后重悬于含有10%DMSO的细胞培养液冻存备用。进行体细胞核移植前,复苏一只冷冻保存的原代细胞并培养于6孔板中,等细胞长满后继续培养3天以后即可由于体细胞核移植实验。
2.雌性成年猴卵丘细胞分离准备
取食蟹猴/恒河猴取卵时的卵泡液,离心并用TH3洗两遍,最后用少量TH3重悬放置于4°冰箱备用。
3.猴超数排卵和卵母细胞收集
挑选6-12岁健康且月经规律的雌性食蟹猴作为卵母细胞供体。在月经第三天开始注射FSH,每次25IU,每天两次连续注射8天。月经第十一天注射1000IU的HCG,注射HCG36小时后开始腹腔镜取卵手术。通过腹腔镜手术,利用负压吸引器吸取2-8毫米直径的卵泡液,在体式镜下利用玻璃管将卵母细胞从卵泡液中挑选出来并转移至CMRL-1066成熟培养液中备用。
4.猴体细胞核移植操作流程
卵母细胞去核:将15-20枚MII期卵母细胞转移至玻璃底的且油封的含有5μg/ml CB的TH3操作滴中,在纺锤体成像系统辅助的倒置显微镜下,观察卵母细胞核位置,利用持卵针固定卵母细胞将卵母细胞核定位于3点钟方向。利用Piezo显微操作系统驱动直径10微米的平口针打破透明带,将卵母细胞核吸出。整组卵母细胞去核结束后,将去核的卵母细胞转移至HECM-9培养液。
成纤维体细胞注入去核卵母细胞:将15-20枚去核卵母细胞转移至油封的含有5μg/ml CB的TH3操作滴中,利用持卵针固定卵母细胞,极体置于12点或6点钟方向。利用激光破透明带系统将透明带打破形成一条窄缝,利用直径15-18微米的斜口针吸取成纤维细胞并将细胞转移至HVJ-E中浸泡10秒钟,然后将细胞吸出,通过透明带窄缝注入去核卵细胞的卵周隙中,该体细胞10分钟左右即会融合入卵母细胞。
卵丘细胞注入去核卵母细胞:将15-20枚去核卵母细胞转移至油封的含有5μg/ml CB的TH3操作滴中,利用持卵针固定卵母细胞,极体置于12点或6点钟方向。利用激光破透明带系统将透明带打破形成一条窄缝,利用直径10微米的斜口针吸取卵丘细胞并将细胞转移至HVJ-E中浸泡10秒钟,然后将细胞吸出,通过透明带窄缝注入去核卵细胞的9点钟位置,该体细胞10分钟左右即会融合入卵母细胞。
核移植胚胎体外激活:体细胞与去核卵母细胞融合后1-1.5小时即可进行 胚胎激活。将胚胎转移至含有5μM的离子霉素的TH3里放置5分钟,然后转移至含有2mM 6-DMAP和10nM TSA的HECM-9培养液里放置5小时,然后转移至含10nM TSA的HECM-9中培养5小时,最后转移至HECM-9培养液中培养。
Kdm4d mRNA注射:胚胎激活5-6小时后,将激活后的胚胎转移至油封的含有5μg/ml CB的TH3操作滴中,利用持卵针固定胚胎,利用Piezo显微操作系统驱动直径2-3微米的平口注射针打破透明带并深入胚胎中,打破胚胎细胞膜将1000ng/μl的Kdm4d mRNA注射入胚胎中,每枚胚胎注射约10pl。注射结束后,胚胎转移至HECM-9培养液37℃,5%CO 2培养。
5.克隆胚胎培养和胚胎移植
胚胎培养:克隆胚胎开始培养在HECM-9培养液中,在培养至8细胞阶段即转移至含有5%FBS的HECM-9培养液中,隔天换液直至到第7-8天囊胚出现。
胚胎移植:挑选处于大卵泡期或排卵期的健康成年雌性猴用于胚胎移植。通过微创手术将3-7枚2细胞-囊胚期胚胎移植到一只雌性猴的输卵管内。
6.Kdm4d mRNA体外转录
利用含T7启动子的引物
(F:TTAATACGACTCACTATAGGGATGGAAACTATGAAGTCTAAGGCCAACT(SEQ ID NO.:1),R:ATATAAAGACAGCCCGTGGACTTAGG(SEQ ID NO.:2))将人Kdm4d基因CDS区从携带Kdm4d基因序列的质粒上扩增下来,将扩增的片段纯化回收,并用mMESSAGE mMACHINE T7ULTRA kit(Life Technologies,AM1345)试剂盒进行体外转录,然后将转录产物用MEGA clear kit(Life Technologies,AM1908)试剂盒纯化后溶于RNAse free的水中。
7、动物伦理声明
食蟹猴(Macaca fascicularis)的使用和护理,符合中国科学院上海生命科学研究院动物委员会批准的名为“代基因改性的猴模型通过体细胞核移植(ION-2018002)的审批文件。在这个实验中,使用的猴子被安置在空调环境(温度:22±1摄氏度;湿度:50%±5%RH),12小时光明/12小时黑暗周期(07:00到19:00)。所有饲料均采购自苏州安慕飞公司,每天两次,青料以水果和蔬菜为主,每天补充一次。
8、成纤维细胞培养,基因分析和核型鉴定
从大腿外侧麻醉的BMAL1-edited首建猴A6皮肤上,剪下一小片皮肤。组 织用含有青霉素和链霉素的PBS清洗三次后切成小块(1-2平方毫米),6厘米培养皿中培养。10天后从组织外植体中长出的成纤维细胞传代到10厘米培养皿,此后每3天以1:3的比例传代。
取A6猴子的耳朵组织、血液细胞和培养的成纤维细胞被用来做基因型分析。BMAL1检测位点的引物为正向:ACCATCGGCTGCGTACACCTCTAT,反向:ATTTCAGGTGTGAGCCACTCCACC,我们将PCR产物进行TA克隆后进行测序分析。
核型分析:用100ng/ml秋水仙胺处理长在10厘米培养皿中的A6成纤维细胞4~6个小时,然后用0.25%trypsin-EDTA消化,然后用0.075M氯化钾在37℃处理30分钟。低渗处理过的细胞被固定在甲醇和醋酸(3:1)中30分钟然后滴片,最后进行Giemsa染色和染色体计数。
9、超排和胚胎收集
食蟹猴超排和卵母细胞的收集方法如前所述。在健康的雌性猕猴的肌肉注射25IU重组人类促卵泡素,从月经的第3天到第11天每天两次。在第11天的晚上肌肉注射1000IU的人类绒毛膜促性腺激素(hCG),第二天通过腹腔镜和负压抽吸系统,进行卵母细胞的收集。收集到的卵母细胞培养于在预平衡的HECM-9培养液。选取减数第二次分裂中期的卵母细胞来做SCNT。
10、体细胞核移植、胚胎培养、胚胎移植
纺锤体-染色体复合物在Oosight系统下被piezo-driven的去核针快速除去。用激光在去核的卵母细胞透明带上打一个豁口,然后再用斜口针将沾染了HVJ-E的成纤维细胞注入卵周隙并介导其融合。重建核移植胚胎在TH3(HEPES-buffered TALP培养基,含有0.3%牛血清白蛋白)中进行激活,先用含有5mM钙离子载体的TH3中处理5分钟,然后在含有2mM 6-二甲基胺的TH3中处理5h。核移植胚胎用10nM Trichostatin(TSA)处理10h,在激活后6h注射10pl 1000ng/ml Kdm4d mRNA。
胚胎培养在HECM-9培养液中,培养箱条件未37摄氏度,5%的二氧化碳。当胚胎达到8细胞时期时转移到加入5%胎牛血清的HECM-9培养基中,此后每两天换一次培养基,直到达到囊胚阶段。将发育优质的2至8细胞时期将胚胎移植到月经周期同步的母猴(有排卵点或者黄体)的输卵管中。
11、克隆猴的基因分析
从克隆猴的耳朵组织提取基因组DNA,用于短串联重复序列(STR)分析。含有荧光染料(FAM/HEX/TMR)的位点特异性引物被用于PCR扩增。荧光染料标记的STR扩增子被ROX500和去离子的甲酰胺混合物稀释,然后在ABI PRISM 3730 genetic analyzer上进行毛细管电泳,获得原始数据。结果原始数据用Gene Marker 2.2.0,产生出Excel文件,包括大小和基因型信息,DNA样本和波图。
单核苷酸多态性(SNP)分析,mtDNA也从猴子的耳朵组织样本中提取。用特定的引物扩增(F:CCACTTCACATCAAACCATCACTT R:CAAGCAGCGAATACCAGCAAAA)mtDNA,PCR产物用于测序和SNP分析。
12、RT-PCR
采集A6,B1和两只野生型猴子的各0.5毫升血液,用总RNA
Figure PCTCN2018123507-appb-000001
Reagent(Invitrogen)试剂盒提取mRNA。然后用总RNA用PrimeScript TM RT reagent Kit with gDNA Eraser(Perfect Real Time,Takara,Japan)试剂盒进行反转录为cDNA。用BMAL1引物(正向引物;5“-TAACCTCAGCTGCCTCGTTG-3”,反向引物;5‘-TATTCATAACACGACGTGCC-3”)来扩增野生BMAL1的目的片段201bp,后进行电泳分析。
实施例1猴核移植技术操作流程建立及优化。
大小鼠的卵母细胞的细胞核在正常倒置显微镜下即可清晰分辨,利用显微操作针可以轻松吸出去除。而灵长类卵母细胞的细胞核在正常倒置显微镜下无法清晰分辨,以前去除猴卵母细胞核的方法有依赖Hoechst染色后荧光定位法和盲吸法。这两种方法对于胚胎损失都是非常大的,很容易影响胚胎后续的发育效率。在本研究中,我们采用基于偏振光的纺锤体成像系统(Oosigt Imaging System),该系统可以清晰分辨猴卵母细胞核而不影响卵母细胞质量。经过大量的训练,在利用纺锤体成像系统清晰分辨猴卵母细胞核位置后,采用Piezo驱动的10微米的显微操作针来打破透明带并将猴卵母细胞核吸出。为了将体细胞注入去核的卵母细胞,利用激光破透明带系统辅助体细胞注射,利用灭活的仙台病毒诱导胎猴体细胞与去核卵母细胞的融合。融合后的体细胞核会重新凝集成卵母细胞核的形态。(图1A-H,图5 A,B)
实施例2组蛋白去乙酰化抑制剂对猴克隆胚胎发育效率影响的研究。
目前认为体细胞核移植效率低下的原因之一是体细胞DNA在在卵母细胞重编程过程中出现异常导致DNA的甲基化异常增高。而提高组蛋白乙酰化的水平可以降低DNA的甲基化水平。在本发明的实验中,将正常激活(离子霉素ionomycin和二甲基氨基吡啶6-DMAP)后的食蟹猴猴体细胞核移植胚胎体外培养,发现有4/30的胚胎发育到了囊胚阶段,但是四枚囊胚质量都很差,没有明显的内细胞团。而在胚胎激活中和激活后加入组蛋白去乙酰化酶抑制剂TSA(10nM,10h)后,发现加入TSA处理的胚胎有5/31发育到囊胚。虽然跟I/D激活的囊胚率相似,但是加入TSA组的囊胚质量有所提升,5枚囊胚中有2枚有内细胞团。(图2A,B,图5C,D)
实施例3优化胚胎激活条件对猴克隆胚胎发育效率影响的研究。
在人体细胞核移植克隆胚胎研究中发现,除了利用常规的离子霉素ionomycin和二甲基氨基吡啶6-DMAP来激活胚胎外,电刺激和嘌呤霉素也被报道可以明显提高人克隆胚胎的发育效率。在本发明的优化激活条件的实验中,综合利用电刺激、离子霉素、二甲基氨基吡啶、嘌呤霉素和组蛋白去乙酰化酶抑制剂TSA,发现54枚食蟹猴猴体细胞克隆胚胎有18(33.3/%)枚发育到了囊胚,其中有8枚囊胚有明显的内细胞团。
相对于简单的使用离子霉素和二甲基氨基吡啶激活胚胎以及实验离子霉素、二甲基氨基吡啶和TSA处理胚胎,优化后的胚胎激活条件可以明显的提升猴体细胞核移植胚胎的发育能力。将利用此方法构建的胚胎移植到雌性猴受体输卵管,尽管移植了多只受体,并没有得到猴体细胞核移植怀孕的受体。
实施例4胚胎聚合法对非人灵长类体细胞核移植效率的影响
体细胞核移植胚胎基因表达异常是其效率低下的重要原因,而Oct4基因在内细胞团(ICM)中的表达可以作为克隆胚胎内细胞团质量好坏的一个指征。本发明的研究发现,小鼠克隆胚胎囊胚期的细胞数目是正常IVF胚胎细胞数目的一半,同样小鼠克隆胚胎囊胚中ICM数目也约为IVF胚胎ICM数目的一半。克隆胚胎中ICM数目的多少跟Oct4基因在ICM中的表达密切相关。在本发明的猴体细胞核移植胚胎聚合实验中,首先将1枚通过单精子注射获得的正常食 蟹猴猴胚胎和三枚体细胞核移植胚胎聚集在一起使他们共同发育,在移植的10只受体中,得到了三只怀孕受体并获得两只出生存活个体,但是通过遗传分析,发现只两只存活猴全部来源于单精子注射获得的正常猴胚胎,而体细胞核移植胚胎没有参与个体的发生。接着将3-4枚全部是猴体细胞核移植的胚胎聚集在一起使他们共同发育。尽管得到了多只怀孕受体,但是全部都在早期流产,未获得存活个体。
实施例5 H3K9me3去甲基化酶对猴体细胞核移植效率的影响
体细胞核移植过程中卵母细胞对体细胞核重编程过程中的异常导致了其效率的低下。分析重编程异常来源并有针对性的解决这些异常则可能提升体细胞核移植的效率。
在本实施例中,利用食蟹猴胎猴成纤维细胞构建获得了38枚核移植胚胎,经过Kdm4d mRNA注射后,有17枚发育到了囊胚阶段,其中有11枚囊胚有明显的内细胞团(图1I,图2,A,B)。为测试Kdm4d在其他供体细胞核移植胚胎的效果,又利用成年猴卵丘细胞作为体细胞供体构建获得了33枚核移植胚胎,经过Kdm4d mRNA注射后,有24枚发育到了囊胚阶段,其中有15枚囊胚有明显的内细胞团(图6,图2,C,D)。
为进一步了解Kdm4d mRNA在提升猴核移植胚胎发育能力的机制,将正常猴单精子注射(ICSI)获得的4细胞期和8细胞期胚胎以及注射Kdm4d和未注射Kdm4d的猴核移植8细胞期胚胎进行转录组测序。通过对比正常ICSI 4细胞期和8细胞期胚胎转录组,将8细胞期表达水平高出4细胞期5倍以上的区域筛选出,得到3997个转录激活区域。接着分析未注射Kdm4d的8细胞核移植胚胎转录组这3997个区域,发现有2465个区域未被激活,称这2465个区域为重编程抵抗区。对比分析注射Kdm4d的8细胞核移植胚胎转录组中重编程抵抗区,发现有2178个区域表达水平有明显提升。
这些结果表明,H3K9me3去甲基化酶Kdm4d在猴核移植胚胎中可以明显提升体细胞核移植胚胎的发育能力。(图2E)
实施例6 H3K4me3去甲基化酶对猴体细胞核移植效率的影响
在本实施例中,对在食蟹猴猴体细胞核移植胚胎水平进行了验证。
一共注射了20枚猴体细胞核移植胚胎,得到了2枚囊胚,囊胚率只有10%, 说明H3K4me3去甲基化酶Kdm5b对猴体细胞核移植胚胎效率提升无明显效果。
实施例7利用食蟹猴胎猴成纤维细胞获得体细胞核移植克隆猴
在本实施例中,采用雌性食蟹猴胎猴原代成纤维细胞作为供体细胞进行体细胞核移植克隆猴的构建。
利用通用方法中的流程,一共操作了127枚MII期的卵母细胞,经过卵母细胞去核,体细胞注射以及胚胎激活等流程后,获得了109枚单原核的胚胎(图6)。将这109枚胚胎进行Kdm4d mRNA注射,并将其中79枚2细胞期、2-4细胞期、8细胞期、8-16细胞期、或囊胚期的胚胎移植到21只受体母猴,通过B超验证,成功得到了6只怀孕母猴。其中四只怀孕母猴子宫中有孕囊和胎儿,而另外两只怀孕母猴只有孕囊,未有胎儿。四只怀有胎儿的母猴中,两只早期流产。另外两只分别在155天(注:8-16细胞期进行植入代孕母体)和141天通过破腹产得到两只健康的仔猴。将这两只小猴命名为“中中”“华华”(注:2-4细胞期进行植入代孕母体)。两只仔猴目前已经出生28天和18天,健康状况良好。(图3,A-E,图4,A,B)
实施例8利用成年食蟹猴卵丘细胞获得体细胞核移植克隆猴
本实施例利用成年雌性食蟹猴猴卵丘细胞作为细胞核供体进行克隆猴的构建。
采用与实施例7相同的流程,一共操作了290枚MII期的卵母细胞,经过卵母细胞去核,体细胞注射以及胚胎激活等流程后,获得了192枚单原核的胚胎。将这192枚胚胎进行Kdm4d mRNA注射,并将其中181枚2细胞期-囊胚期的胚胎移植到42只受体母猴,通过B超验证,成功得到了22只怀孕母猴。其中12只怀孕母猴子宫中有孕囊和胎儿,而另外10只怀孕母猴只有孕囊,未有胎儿。12只怀有胎儿的母猴中,8只早期流产,两只分别在84天和94天流产。另外有两只成功发育至130天以上,于137和135天通过破腹产得到两只出生存活的仔猴。将这两只小猴命名为“A”“B”。仔猴A身体发育阻滞,出生后仅存活了3小时就因为呼吸衰竭死亡,仔猴B体型正常,出生后有正常的喝水喝奶行为。(图3,C,D,E)
实施例9利用成年恒河猴卵丘细胞获得体细胞核移植克隆猴
目前科学研究中最常用的两只非人灵长类包括食蟹猴和恒河猴。在本发明上述实施例的研究中已提示,本发明上述特定的处理方法可显著提升哺乳动物核移植效率。
在本实施例中,为了进一步验证H3K9me3去甲基化酶在非人灵长类体细胞核移植的效率,将所述通用方法应用到恒河猴体细胞核移植的胚胎、
结果
在注射了H3K9me3去甲基化酶的10枚恒河猴体细胞核移植胚胎中,有7枚发育都了囊胚阶段。另外,目前已通过将此胚胎移植成功获得了恒河猴体细胞核移植的怀孕受体并得到了发育至130天的恒河猴克隆猴“C”。该结果提示,本发明方法还适用于其他非人灵长类,尤其是猴子。
实施例10体细胞核移植来源的克隆猴的遗传分析
为验证五只出生仔猴的核基因组DNA和线粒体DNA的遗传来源,对这四只仔猴进行了单核苷酸多态性(SNP)和微卫星序列分析(STR)。通过对仔猴“中中”和“华华”、供体细胞、各自代孕母猴以及各自卵供体母猴的27个微卫星位点进行分析发现,“中中”和“华华”的基因组和供体细胞基因组是一致的,而和代孕母猴和卵供体母猴的基因组是不一致的。通过对仔猴“中中”和“华华”、供体细胞、各自代孕母猴以及各自卵供体母猴线粒体DNA上的ND3基因测序进行SNP分析,发现“中中”和“华华”的线粒体基因组和卵供体猴线粒体基因组是一致的,而和代孕母猴和卵供体母猴的线粒体基因组是不一致的。利用同样方法,对出生后死亡的仔猴“A”和“B”进行SNP和STR分析,发现仔猴“A”,“B”和“C”的核基因组和线粒体基因组来自于卵细胞和卵丘细胞供体猴,而和代孕受体不同。这些遗传分析证明了,这五只仔猴确实是通过克隆技术获得的克隆猴。(图4C,D,E,图7和图8)
通过对四只体细胞克隆猴,及他们的卵母细胞供体,体细胞供体和怀孕受体的27个STR位点进行分析发现,这四只体细胞克隆猴的核DNA确实是来自供体体细胞。
实施例11利用体细胞核移植技术构建非人灵长类基因修饰动物模型(以食蟹猴/恒河猴为例)
本发明之所以花费巨大精力攻克利用体细胞核移植技术来获得克隆猴,除 了其本身的科学意义之外,更看重的是该技术在构建基因修饰动物模型方面的优势。本发明通过在体外对培养的体细胞进行基因修饰,然后利用基因修饰的体细胞作为核供体获得克隆动物,即可获得携带有特定基因修饰的动物模型。利用此方法可获得携带复杂基因修饰的动物模型,且获得的F0代动物模型不存在嵌合现象,不需要传代即可使用,另外利用同一株细胞获得的动物模型具有一致的遗传背景。因此利用体细胞核移植技术来构建基因修饰非人灵长类模型可以解决上文提到的全部问题。目前本发明利用此技术已经在开展的基因修饰非人灵长类模型有:
1,利用体细胞核移植技术构建食蟹猴/恒河猴精确基因敲入模型:
(1)AAVS1位点敲入绿色荧光蛋白:AAVS1-GFP Knock In。
(2)AAVS1位点敲入cre序列:AAVS1-Cre Knock In。
(3)AAVS1位点敲入LSL-CHR2序列:AAVS1-LSL-CHR2Knock In。
(4)CamkIIa位点敲入光敏感离子通道蛋白CHR2:CamkIIa-CHR2-EYFP Knock In。
(5)CamkIIa位点敲入钙成像蛋白Gcamp6s:CamkIIa-Gcamp6s Knock In。
(6)CamkIIa位点敲入cre序列:CamkIIa-Cre Knock In。
(7)Vgat位点敲入光敏感离子通道蛋白CHR2:Vgat-CHR2-EYFP Knock In。
(8)Vgat位点敲入钙成像蛋白Gcamp6s:Vgat-Gcamp6s Knock In。
(9)Vgat位点敲入cre序列:Vgat-Cre Knock In。
(10)Chat(Choline acetyltransferase)位点敲入cre序列:Chat-Cre Knock in
(11)Chat位点敲入Chr2序列:Chat-CHR2-EFYP Knock in
(12)Chat位点敲入Gcamp6s序列:Chat-gcamp6s Knock in
(13)Drd1(Dopamine receptor D1)敲入cre序列:Drd1-Cre Knock In
(14)Drd1敲入Chr2序列:Drd1-Chr2-EYFP Knock In
(15)Drd2(Dopamine receptor D2)敲入cre序列:Drd2-Cre Knock In
(16)Drd2敲入Chr2序列:Drd2-Chr2-EYFP Knock In
(17)GFAP位点敲入Chr2序列:GFAP-CHR2-EFYP Knock in
(18)GFAP位点敲入cre序列:GFAP-Cre Knock in
(19)GFAP位点敲入gcamp6s序列:GFAP-gcamp6s Knock in
(20)TH(hydroxytryptamine)位点敲入cre序列:TH-Cre Knock In
(21)TH位点敲入Chr2序列:TH-Chr2-EYFP Knock In
(22)Nestin位点敲入cre序列:Nestin-Cre Knock In
(23)其他组织或细胞特异的基因敲入猴模型
2,利用体细胞核移植技术构建食蟹猴/恒河猴点突变模型:
(1)SOD1 A4V点突变
(2)SOD1 H46R点突变
(3)SOD1 G93A点突变
(4)Foxp2 T327N+N349S点突变
3,利用体细胞核移植技术克隆多只遗传背景一致的食蟹猴/恒河猴基因敲除疾病或发育障碍模型:
(1)克隆遗传背景一致的PRRT2基因敲除猴模型。
(2)克隆遗传背景一致的FMR1基因敲除猴模型。
(3)克隆遗传背景一致的ASPM基因敲除猴模型。
(4)克隆遗传背景一致的DISC1基因敲除猴模型。
(5)克隆遗传背景一致的MKRN3基因敲除猴模型。
(6)克隆遗传背景一致的SNCA基因敲除猴模型。
(7)克隆遗传背景一致的LRRK2基因敲除猴模型.
(8)克隆遗传背景一致的GBA基因敲除猴模型。
(9)克隆遗传背景一致的PRKN基因敲除猴模型。
(10)克隆遗传背景一致的PINK1基因敲除猴模型。
(11)克隆遗传背景一致的PARK7基因敲除猴模型。
(12)克隆遗传背景一致的VPS35基因敲除猴模型。
(13)克隆遗传背景一致的EIF4G1基因敲除猴模型。
(14)克隆遗传背景一致的Bmal1基因敲除猴模型。
(15)克隆遗传背景一致的LRRK2+PINK1+PARK7基因敲除猴模型。
4,利用体细胞核移植技术克隆多只遗传背景一致的用于研究阿尔茨海默症、帕金森症、肌萎缩侧索硬化症、自闭症、抑郁症、亨廷顿综合症等疾病的转基因和基因敲除食蟹猴/恒河猴模型。
实施例12 BMAL1基因敲除食蟹猴体细胞制备
ARNT-Like 1(BMAL1)是调节昼夜节律的转录因子。在本发明中,用CRISPR/Cas9方法获得了5只BMAL1基因编辑的食蟹猴个体。其中选择猴子A6作为供体细胞来源猴进行克隆,因为这只猴子没有检测到BMAL1蛋白表达,而且表现出明显的生理疾病表型,包括血液激素的生理循环抑制,夜间活动频繁,快速眼动(REM)减少和非快速眼动睡眠期,以及精神病相关行为。因此取A6猴子的皮肤成纤维细胞,培养后用于SCNT(图9,A-C)。
培养后的成纤维细胞的核型显示正常的二倍体,有42条染色体(图9D)。对A6猴子的耳尖组织,血液细胞和成纤维细胞进行基因型分析,通过PCR产物的单克隆测序,都发现了两种类型的BMAL1突变:“-8bp”和“-8bp,+4bp,2bpPM”(图9E)。然后进一步对单个成纤维细胞的基因型分析显示,BMAL1基因中存在“-8bp/-8bp”的纯合基因突变或“-8bp/-8bp,+4bp,2bpPM”的杂合基因突变(图9F)。
1、应用BMAL1编辑猴成纤维细胞进行SCNT
使用来自BMAL1基因编辑的猕猴A6的成纤维细胞进行SCNT。通过对雌性猕猴进行超排,获得成熟卵母细胞。然后利用仙台病毒辅助单个成纤维细胞与去核卵母细胞融合获得SCNT卵母细胞,并与钙离子载体和6-二甲基胺孵育。为了促进核移植后表观遗传重编程,将SCNT胚胎与组蛋白脱乙酰酶抑制剂trichostatin(TSA)孵育,并同时注射H3K9me3去甲基化因子Kdm4d的mRNA(图10A、B)。发现使用BMAL1基因编辑猴子A6的成纤维细胞进行SCNT,获得了较高比例的囊胚形成(10/17,58.8%)。在这些囊胚中,80%(8/10)形成明显的内细胞团(ICM),这是胚胎发育正常的标志。由于囊胚形成和ICM形成的效率较高,我们将325个SCNT胚胎在发育早期(2-8细胞)转移到65只代母猴身上,其中16只母猴怀孕。最终获得了5只出生存活克隆猴个体(B1-B5),现均在人工喂养下存活(目前51-141天)(图10C,表1)。还研究了SCNT中成纤维细胞培养的传代代数与克隆效率的关系,如表1所示,经过4次传代的成纤维细胞进行SCNT后的受体猴妊娠率和出生猴活产率最佳,说明克隆成功率可能与供体细胞培养的传代代数有关。
表1 BMAL1基因编辑成纤维细胞SCNT胚胎数据统计
Figure PCTCN2018123507-appb-000002
2、克隆猴基因型分析
首先取5只克隆猴的耳尖组织进行了BMAL1基因型的分析。发现4只克隆猴子(B1,B3,B4,B5)携带BMAL1基因的纯合基因突变(“-8bp/-8bp,+4bp,2bpPM”),1只猴子(B2)携带BMAL1基因的杂合基因突变(“-8bp/-8bp”)(图10D)。这与前面检测的供体成纤维细胞的突变基因型以及猴A6的突变基因型相同。
为了确定克隆猴的遗传来源,分析了线粒体DNA(mtDNA)的单核苷酸多态性和核DNA的短串联重复。发现克隆猴线粒体DNA的ND3基因都与各自卵母细胞供体猴相同,但不同于受体猴和供体成纤维细胞(图11A-E)。对29个位点进行STR分析,结果表明,5个克隆猴的供体成纤维细胞与猴A6的细胞核DNA相同,但与受体猴和卵母细胞供体猴的细胞核DNA不同(图11F)。
虽然全基因组测序和PCR分析证实了BMAL1基因编辑猴A6不存在脱靶现象,仍然对5只克隆猴的耳部组织的基因组DNA进行了脱靶分析。在预测的潜在靶外位点,没有发现突变。另外,在B1克隆猴的血液中也没有检测到野生型BMAL1转录本(其余4只克隆仍未到可以采血的年龄)。(图11G)。
讨论
本发明通过CRISPR/Cas9系统敲除了食蟹猴基因组中节律相关的重要基因BMAL1,并得到了5只基因编辑猴。在本研究中,从其中一只表现明显的猴子皮肤中分离得到的成纤维细胞作为体细胞核供体,成功获得5只BMAL1基因编辑克隆猴。通过对首建猴和克隆猴的序列比对和微卫星分析以及线粒体DNA的比较表明这些克隆猴和首建猴拥有相同的基因背景。这些结果表明,被CRISPR/Cas9编辑过的食蟹猴疾病模型可以被用来作为核移植的供体并得到遗 传背景一致的克隆猴,为相关疾病研究和模型构建提供了有利的前景。
申请人在之前的研究中,使用了来自一只流产的雌性胎猴的成纤维细胞作为核供体,并成功克隆出“中中”和“华华”。与此不同的是,在此项实验中从一只16个月的未成年猴皮肤中分离的成纤维,并首次证明了雄性细胞作为核供体的可行性。尽管目前的克隆效率仍很低,将通过进一步优化成纤维细胞培养条件,如发现成纤维细胞在经过几次传代之后的克隆效率会比原代细胞的克隆效率更高。另外,Kdm4d作为H3K9me3的去甲基化酶,可以通过去除核移植胚胎中重编程甲基化区域,显著提升小鼠和猴子的重编程效率;Xist介导的X染色体沉默很可能在小鼠、猪和灵长类动物中也是保守的。除此之外,DNA重新甲基化和H3K27me3的缺失也有可能是影响克隆效率的关键因素。
理论上来说,经过体外基因编辑和筛选后得到的阳性细胞,是作为核供体的最理想的选择。申请人在此项实验中使用了从成体编辑猴中分离的细胞,因为这只首建猴的BMAL1基因被完全敲除,并且表现出类似于人类戒律紊乱的表型,于是选择其作为核供体来源。经过检测发现首建猴主要有两种基因型,即8bp纯合敲除(“-8bp/-8bp”)和杂合的8bp敲除、4bp敲入及2bp点突变(“-8bp/-8bp,+4bp,2bpPM”)。这5只克隆猴每只都只有一种基因型,说明这一嵌合现象并没有在这5只克隆猴中出现,因此本发明认为目前的克隆方法很成熟,这些克隆猴的表型也将会被进一步分析。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种非人灵长类的体细胞克隆动物的制备方法,其特征在于,包括步骤:
    (i)提供一重构卵,所述的重构卵来自所述非人灵长类动物;
    (ii)对所述重构卵进行激活处理,从而形成经激活的重构卵或经激活的由所述重构卵形成的重构胚胎,其中所述的重构胚胎处于2细胞期、4细胞期或8细胞期;
    (iii)对(a)所述经激活的重构卵或(b)经激活的重构胚胎的胚胎细胞,进行重编程处理,从而获得经重编程的重构卵或经重编程的重构胚胎;和
    (iv)将所述经重编程的重构卵或经重编程的重构胚胎进行再生,从而获得非人灵长类的体细胞克隆动物。
  2. 如权利要求1所述的方法,其特征在于,所述步骤(iii)中,用重编程激活剂进行重编程处理。
  3. 如权利要求2所述的方法,其特征在于,所述重编程激活剂选自下组:
    (a1)Kdm4d蛋白;
    (a2)编码Kdm4d蛋白的mRNA;
    (a3)编码Kdm4d蛋白的DNA;和
    (a4)上述(a1)、(a2)、(a3)的任意组合。
  4. 如权利要求1所述的方法,其特征在于,所述的重编程处理包括:将编码Kdm4d蛋白的mRNA注入所述的重构卵或所述重构胚胎的胚胎细胞中。
  5. 如权利要求4所述的方法,其特征在于,将10 2-10 8(较佳地10 4-10 6)拷贝/细胞的编码Kdm4d蛋白的mRNA注入所述重构卵或所述的胚胎细胞。
  6. 如权利要求1所述的方法,其特征在于,在所述方法中,所述步骤(ii)中,获得经激活的重构胚胎;以及步骤(iii)中,对所述重构胚胎进行重编程处理。
  7. 如权利要求1所述的方法,其特征在于,在所述方法中,所述步骤(iv)中,将经重编程的重构胚胎进行再生,从而获得非人灵长类的体细胞克隆动物。
  8. 如权利要求1所述的方法,其特征在于,所述步骤(iv)包括:
    (iv1)将所述经重编程的重构卵在体外或体内培养,形成重构胚胎;和
    (iv2)将所述重构胚胎移植到非人灵长类动物的输卵管中,从而获得所述非人灵长类的体细胞克隆动物。
  9. 一种试剂盒,其特征在于,包括:
    (a)第一容器,所述第一容器中含有重编程激活剂,其中所述的重编程激活剂选自下组:
    (a1)Kdm4d蛋白;
    (a2)编码Kdm4d蛋白的mRNA;
    (a3)编码Kdm4d蛋白的DNA;
    (a4)上述(a1)、(a2)、(a3)的任意组合;
    (b)第二容器,所述第二容器中含有激活处理剂;和
    (c)标签或说明书。
  10. 一种权利要求9所述的试剂盒的用途,其特征在于,用于制备一试剂盒,所述试剂盒用于制备非人灵长类的体细胞克隆动物。
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