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WO2019113743A1 - Procédé de modification génétique - Google Patents

Procédé de modification génétique Download PDF

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Publication number
WO2019113743A1
WO2019113743A1 PCT/CN2017/115481 CN2017115481W WO2019113743A1 WO 2019113743 A1 WO2019113743 A1 WO 2019113743A1 CN 2017115481 W CN2017115481 W CN 2017115481W WO 2019113743 A1 WO2019113743 A1 WO 2019113743A1
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WIPO (PCT)
Prior art keywords
zebrafish
microinjection
oocyte
situ
stage
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PCT/CN2017/115481
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English (en)
Chinese (zh)
Inventor
孟安明
吴小童
沈炜敏
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Tsinghua University
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Tsinghua University
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Priority to PCT/CN2017/115481 priority Critical patent/WO2019113743A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection

Definitions

  • the present invention relates to the field of biotechnology, and in particular, to genetic modification methods.
  • zebrafish is one of the typical developmental biological model organisms, and its eggs are fertilized in vitro and early embryo development.
  • the female fish expels the unfertilized mature egg, while the male fish expels the sperm, and the two complete the fertilization process in vitro.
  • the early embryos of the zebrafish are transparent for easy observation and experimental manipulation.
  • the existing embryonic microinjection technique is a microinjection of fertilized eggs that have been successfully fertilized in vitro. It is an injection of a glass needle that has been subjected to mitosis after fertilization in a perforated injection tray which is solidified by agar and heated by a glass capillary pipette under a microscope.
  • the injection needle is fixed on the needle holder, and the operation is simple and convenient.
  • the injected substances may be mRNA, protein, DNA, antisense oligonucleotide (Morpholino), and other small molecules, which can inject a large number of embryos in a short time (on average, 200 embryos are injected every 30 minutes). After the injection is completed, the embryos are washed out from the agar plate, placed in embryo-specific culture water, and cultured in an incubator at 28-30 °C.
  • the early embryos of the zebrafish enter a rapid cleavage phase, which is approximately 45 minutes after fertilization to the first cell division; followed by approximately ten rapid cell divisions, 15 minutes apart; then the embryonic zygote
  • the genes begin to activate in large quantities, and the embryos begin to move in the primitive gut (Kimmel et al., 1995).
  • the existing microinjection technique is limited by the rapid division of early embryos, the injection operation must be completed within 40 minutes, and the reagents injected into the embryos are not fully utilized and are diluted by rapidly dividing cells; so that individual cells The amount of the injected reagent is rapidly decreased, and the efficiency of action is lowered.
  • Tol2 transposase mRNA and plasmid DNA are also diluted after injection because of rapid division of embryonic cells, resulting in transgene insertion occurring only in some cells, and also requiring a lot of time and effort. Screening is required to obtain stable transgenic lines (Hamlet et al., 2006; Kawakami, 2007). More importantly, when several or even dozens of functionally redundant genes need to be knocked out at the same time; or when multiple transgenic fish are obtained, embryonic microinjection technology takes a lot of time and effort, sometimes even impossible.
  • Microinjection techniques for embryos are also widely used in other species. For example, mice (Mice) and Xenopus (Xenopus), because mouse embryos are developed in vivo, microinjection techniques involve the sacrifice of female donors to take eggs or embryos, and then in vitro fertilization of the injected eggs. (Embryns do not require in vitro fertilization), and finally the surviving embryos are transplanted into the uterus of the recipient female mice for output; this operation is very complicated, with low success rate and extremely high technical requirements.
  • the egg microinjection technique is also to take the egg out of the mother, inject it and then put it into the mother to continue to develop and produce; and the microinjection of the embryo is also facing the same problem as the microinjection of the zebrafish embryo.
  • the injected oocytes or fertilized eggs are taken out from the mother for in vitro culture and microinjection, and then fertilized in vitro or transplanted into another mother to continue development.
  • the inventors of the present application advanced the injection time to a period in which the egg was not mature, and by direct injection into the mother, the inventors found that the in situ microinjection of the oocyte precursor stimulates a series of stress responses. The survival rate of oocytes will decrease significantly.
  • the inventors of the present application succeeded in overcoming some of the responses that immature oocytes were stimulated after being injected in vivo by screening and attempting a series of injection buffers, acclimation before the injection of model animals, and exploration conditions after injection. In the stimulating reaction, the oocytes after injection can finally survive naturally and mature naturally in the mother to be fertilized.
  • the inventor of the present application succeeded in the in vivo injection of oocytes by taking zebrafish as an example.
  • the oocyte is sufficiently fully involved in gene editing (eg, Cas9 mutation and transgene) in the egg within about 40 hours from injection to maturation. .
  • This application is the first to realize the gene editing of immature oocytes, which provides a new technical means for the study of maternal gene function.
  • the inventors of the present application can obtain mutants or transgenic fish in which almost all cell genomes are edited in the F0 generation; therefore, the inventors can see a phenotype consistent with homozygous mutants in the injected contemporary embryos, and are stable. Expressed transgenic map.
  • the genetically engineered method of the present application shortens the acquisition time of mutants and transgenic fish to 2-3 months, and does not require large-scale screening work.
  • the present invention proposes a genetic modification method.
  • the method comprises: in situ microinjection of a genetic engineering agent into immature oocytes in a model animal.
  • the time of in situ microinjection is advanced to a period in which the egg is not yet mature, and the oocyte after injection is naturally matured in the mother body by direct injection in the mother body. , it is discharged and fertilized, and it is almost in F0 generation.
  • Mutants with cell genomes are edited, and the phenotype consistent with homozygous mutants and the stably expressed transgene map can be seen in the injected contemporary embryos.
  • the mutant acquisition time is shortened to 2 - 3 months, and no large-scale screening work is required.
  • the genetic modification method may further include at least one of the following additional technical features:
  • the genetic engineering reagent comprises at least one selected from the group consisting of gRNA, plasmid, mRNA, protein, dye, and Morpholino.
  • the above small molecule substance can be more conveniently injected into the oocyte in situ, and the genome editing efficiency is further improved.
  • the model animal is a zebrafish.
  • Zebrafish early embryos are transparent, easier to observe and experimentally manipulate, and zebrafish oocytes take approximately 40 hours from injection to maturation, which is sufficient for gene editing in oocytes (eg Cas9 mutations and transgenes). The genetic modification success rate is further improved.
  • the age of the zebrafish is 6 months to 1 year.
  • the zebrafish which is 6 months to 1 year old, is a rigid mature zebrafish.
  • the zebrafish is in a good state. After microinjection of the immature oocytes, the survival rate of the eggs is further increased.
  • the method before the in-situ microinjection treatment, the method further comprises: acclimating the zebrafish of the age of 6 months to 1 year; wherein the acclimation treatment is performed as follows (1) let the female zebrafish mate once a week so that the oocyte matures to form a regular cycle; (2) after the oocyte matures to form a regular cycle, let the female zebrafish rest for two weeks, so that Oocytes are accumulated.
  • the female and its oocytes are in a good state, facilitating microinjection procedures and subsequent recovery and development.
  • the zebrafish is fasted within 2-3 hours prior to the in situ microinjection.
  • zebrafish death is prevented during anesthesia before zebrafish injection.
  • said zebrafish prior to said in situ microinjection, is anesthetized and placed on a moist sponge which contains 5.4 mM KCl, 136.8 mM NaCl. Obtained after infiltration of a solution of 4.2 mM NaHCO 3 , 0.44 mM KH 2 PO 4 , 0.25 mM Na 2 HPO 4 and 0.5% by mass of BSA.
  • the inventors have found that placing the anesthetized zebrafish on a sponge infiltrated with the above buffer prevents the egg from being activated during micromanipulation or that the female is excessively dry.
  • the method further comprises: restoring the zebrafish to a rearing treatment and a mating treatment, wherein the restoring feeding treatment is performed by: (1) the zebra Fish were housed separately in water containing 32 ⁇ g/ml anesthetic and 10 units/ml Penicillin and 10 ⁇ g/ml Streptomycin to facilitate recovery of the zebrafish; (2) conditional zebrafish at 28 °C Feeding for 2 to 3 hours; (3) placing the zebrafish obtained in step (2) in water containing 16 ⁇ g/ml of anesthetic and 10 units/ml of Penicillin and 10 ⁇ g/ml of Streptomycin for overnight incubation; (4) Steps ( 3) The obtained zebrafish is placed in a containing 8 The ⁇ g/ml anesthetic and the 10 units/ml Penicillin and 10 ⁇ g/ml Streptomycin in water were separately reared for 10 to 12 hours to obtain the zebra
  • the anesthetic is Triaine.
  • the inventors have found that under the gradient dosage of the above anesthetic agent, the zebrafish is in a relatively calm state and the state of the zebrafish can be gradually restored, which facilitates subsequent mating, and the survival rate of the fertilized egg is further improved after mating.
  • the mRNA, DNA, dye and/or Morpholino are pre-diluted in a buffer solution containing 5.4 mM KCl, 136.8 mM NaCl, 4.2 mM NaHCO 3 , 0.44 mM KH 2 PO 4 , 0.25 mM Na 2 HPO 4 and 100 nM melatonin.
  • the inventors have found that the above buffer solution can alleviate the physical stimulation, pH change and oxidation of the egg by the injection operation; and can effectively improve the survival rate and quality of the injected oocyte.
  • the immature oocyte is a stage I, stage II or stage III oocyte.
  • the immature oocyte is a stage I or stage II oocyte, and further comprising: after the zebrafish completes mating, the zebrafish body is not treated by in situ microinjection.
  • the oocytes are matured and excreted by signal stimulation. In this way, the eggs of stage I and II are stimulated to further mature.
  • the oocytes in stages I and II can be imaged normally by in situ microinjection of different mRNAs, but the efficiency is slightly lower than that of oocytes in phase III.
  • FIG. 2 is a schematic flow chart of a oocyte microinjection reagent and a flow chart of a specific technique and a photograph of the implementation thereof according to an embodiment of the present invention
  • FIG. 3 is a schematic view showing a recovery method of female fish after injection according to an embodiment of the present invention.
  • Figure 4 is a comparison of the formulation and effect of the buffer used for injection according to an embodiment of the present invention, and the recovery of the ability of the female to lay eggs one month after the injection;
  • Figure 5 is a time flow diagram of a Phase I or II egg injection in accordance with an embodiment of the present invention.
  • zebrafish is taken as an example to describe the genetic modification method and its application in detail.
  • the BSA is infiltrated to prevent the oocyte from being activated during the operation, or the female is excessively dry. Subsequently, the abdomen was opened with a surgical scissors, the oocyte was exposed, and a microinjected glass needle (which was obtained by the applicant to stretch the glass capillary pipette) was used.
  • the diameter of the injected droplet is about 1.875 ⁇ m and the volume is about 0.216 nl.
  • the droplet size is significantly lower than the droplet size in the microinjection of the fertilized egg (refer to Fig. 2a).
  • the inventor can inject 30-50 oocytes.
  • the wound is then sutured with a surgical suture needle (see Figure 2b).
  • the females were housed separately in water containing 32 ⁇ g/ml of anesthetic and 10 units/ml of Penicillin, and 10 ⁇ g/ml of Streptomycin.
  • the fish slowly recovered consciousness, they began to swim and then placed in the fish room (guaranteed temperature is about 28 °C).
  • the concentration of the anesthetic was lowered to 16 ⁇ g/ml overnight. The next day, the fish can be fed normally.
  • the fish After the feeding, the fish is exchanged for water, and the concentration of the anesthetic is further reduced to 8 ⁇ g/ml until the evening of the fish (see Fig. 3). At the same time, the concentrations of Penicillin and Streptomycin did not change.
  • the inventors mate females with common wild-type males, and the method of matching fish is no different from the general method (Kimmel et al., 1995). On the third morning, the separator was removed and the embryos were collected for subsequent cultivation and research.
  • the inventors used this technique to inject mRNA, DNA, dyes, and Morpholino. Since the immature oocyte volume is small relative to the embryo and the regulation ability is weak, the injected substance must be diluted in a specific buffer solution containing: 5.4 mM KCl, 136.8 mM NaCl, 4.2 mM NaHCO 3 , 0.44 mM. KH 2 PO 4 , 0.25 mM Na 2 HPO 4 , and 100 nM melatonin.
  • the above solution can alleviate the physical stimulation of the egg by the injection operation, the pH change and the oxidation; and can effectively improve the survival rate and quality of the injected oocyte (see Fig. 4).
  • Females that have finished laying eggs can be returned to the system for rearing without additional care; after 4 weeks, the females are able to mate again and lay eggs, and the number and quality of eggs laid are not significantly different from those of wild-type females. (Figure 4).
  • the above operation is directed to oocytes in the stage III of developmental stage.
  • Microinjection of the oocytes in stage I or II can also be performed by the above microinjection technique; the approximate operation procedure is the same, except that Since the time required for oocyte maturation is different, a slight adjustment to the above steps is required.
  • (1) The preparation process of female fish is the same, However, when injecting, choose to inject I or II oocytes; (2) the injected females will still mate the next night, and the third day will be the uninjected mature stage III oocytes. And excreted to stimulate the further maturation of the I and II oocytes; (3) in the following 2-3 weeks, the female and the male are mated at the same time each week, and the embryo produced is injected.
  • the I and II oocytes mature and develop (see Figure 5).
  • the genome of the embryo obtained by the inventor's CRISPR/Cas9 system injecting the ntla gene was successfully edited; more than 60% of the genomes were edited using the results obtained by the prior art.
  • the embryos obtained by the genetic modification method described in Example 1 all have a typical tailless phenotype, and the embryos obtained by the original microinjection technique only have a part of the embryo to obtain a similar phenotype, and the remaining embryos only have a shortened tail or almost Normal phenotype.
  • the inventors used the Tol2 transgenic system to construct transgenic fish in the technology platform of egg injection, and found that the three genetically modified plasmids were injected simultaneously with the genetic modification method described in Example 1, and the expression profiles of the three embryos obtained were complete, and the fluorescence intensity was complete. High; shows that gene integration occurs in most cells. And after raising the embryo to sexual maturity, it was found that 50% of its offspring had a complete transgene expression profile for each gene (since the oocyte injection can only edit the maternal genome, so the F0 generation has half of the genome.
  • the wild-type male parent was edited; then only half of the copies of the F1 embryo were edited), and about 8% of the offspring were able to express three transgenes simultaneously, enough to stabilize the triple-transgenic line.
  • only a few cells in the prior art have fluorescence, and the proportion of embryos successfully integrated into the germ cells is even lower or even impossible.
  • Example 1 It can be seen that the genetic modification method described in Example 1 has a significantly better editing ability for the genome than the existing fertilized egg microinjection technique.
  • the prior art injection of Morpholino of dnmt1 found that the DNA methylation level did not change significantly, and the embryo development was normal.
  • the level of embryonic DNA methylation was significantly down-regulated at an early stage, and even during the dot period (4.3 hours after fertilization), DNA methylation signals were almost undetectable by immunofluorescence.
  • the inventors used high-throughput sequencing to detect the level of methylation of whole-genome DNA, and found that during the 256-cell period (2.5 hours after fertilization) and the dot period, the methylation level was reduced to about 20% of the original level.
  • the result of this experiment is the first phenotype in the zebrafish that has a significantly down-regulated level of whole-genome DNA methylation in early embryos.
  • embryos were reported 84 hours after fertilization. It can be seen that a gene such as dnmt1 has a high accumulation amount of maternal RNA, a high level of translation at an early stage of development, and a homozygous mutation in a zygote, according to an embodiment of the present invention.
  • the genetic modification method has an irreplaceable advantage.

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Abstract

La présente invention concerne un procédé de modification génétique comprenant : la micro-injection in situ d'un réactif de modification génique dans des ovocytes immatures dans un animal modèle. Par le procédé de modification génique décrit ci-dessus, la micro-injection est avancée jusqu'à une période dans laquelle l'œuf n'est pas encore mature, et par injection directe dans le corps parent, après que les ovocytes injectés arrivent à maturité naturellement dans le corps parent et sont déchargés et fertilisés, un mutant qui peut être édité dans presque tous les génomes cellulaires peut être obtenu dans la génération F0, et un phénotype cohérent avec des mutants homozygotes et une carte transgénique exprimée de manière stable peuvent être constatés dans les embryons injectés simultanément. Par comparaison avec l'art antérieur, le temps d'acquisition du mutant est raccourci à 2-3 mois, et un travail de criblage à grande échelle n'est pas nécessaire.
PCT/CN2017/115481 2017-12-11 2017-12-11 Procédé de modification génétique Ceased WO2019113743A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001050847A2 (fr) * 1999-12-23 2001-07-19 Pangene Corporation Production d'organismes recombines
CN1334870A (zh) * 1998-08-11 2002-02-06 夏威夷大学 通过胞质内精子注射进行哺乳动物基因转移
EP1234021A2 (fr) * 1999-10-27 2002-08-28 Mount Sinai Hospital Procedes et compositions ameliorant le potentiel de developpement d'oocytes et de zygotes
CN101175858A (zh) * 2004-12-17 2008-05-07 国立农业研究所-Inra 生产具有靶向基因组修饰的卵母细胞或卵的体外方法
CN105274144A (zh) * 2015-09-14 2016-01-27 徐又佳 通过CRISPR/Cas9技术得到敲除铁调素基因斑马鱼的制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1334870A (zh) * 1998-08-11 2002-02-06 夏威夷大学 通过胞质内精子注射进行哺乳动物基因转移
EP1234021A2 (fr) * 1999-10-27 2002-08-28 Mount Sinai Hospital Procedes et compositions ameliorant le potentiel de developpement d'oocytes et de zygotes
WO2001050847A2 (fr) * 1999-12-23 2001-07-19 Pangene Corporation Production d'organismes recombines
CN101175858A (zh) * 2004-12-17 2008-05-07 国立农业研究所-Inra 生产具有靶向基因组修饰的卵母细胞或卵的体外方法
CN105274144A (zh) * 2015-09-14 2016-01-27 徐又佳 通过CRISPR/Cas9技术得到敲除铁调素基因斑马鱼的制备方法

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