CN113355359A - 一种高效生产类克隆鸡的方法 - Google Patents

一种高效生产类克隆鸡的方法 Download PDF

Info

Publication number
CN113355359A
CN113355359A CN202110654978.XA CN202110654978A CN113355359A CN 113355359 A CN113355359 A CN 113355359A CN 202110654978 A CN202110654978 A CN 202110654978A CN 113355359 A CN113355359 A CN 113355359A
Authority
CN
China
Prior art keywords
cells
pgc
chicken
feather
induction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110654978.XA
Other languages
English (en)
Inventor
周静
左其生
李碧春
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yangzhou University
Original Assignee
Yangzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yangzhou University filed Critical Yangzhou University
Priority to CN202110654978.XA priority Critical patent/CN113355359A/zh
Publication of CN113355359A publication Critical patent/CN113355359A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/605—Nanog
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/608—Lin28
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Environmental Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Animal Husbandry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Toxicology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Veterinary Medicine (AREA)
  • Transplantation (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Rheumatology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明涉及一种高效生产类克隆鸡的方法，包括以下步骤：（1）、构建OCT4,SOX2,NANOG, LIN28四因子的慢病毒过表达载体并转染黑羽狼山鸡成纤维细胞CEF；（2）、利用诱导培养基将转染OSNL的狼山鸡成纤维细胞CEF诱导为iPSCs，对诱导形成的iPSCs进行传代并进行鉴定；（3）、利用诱导培养基将诱导形成的iPSCs进行诱导分化为PGC‑like，并进行鉴定；（4）、收集PGC‑like细胞并将其通过鸡胚血管注射的方法注射至孵化2.5天的隐性白羽鸡胚血管中，并通过冰冻切片方法确定PGC‑like能够在受体隐性白羽鸡中的迁移归巢；（5）、将注射黑羽狼山鸡PGC‑like细胞的隐性白羽受体鸡胚继续孵化至至出壳。通过微卫星技术证明黑羽后代来源于供体的PGC‑like细胞。通过本发明，可大量生产与供体遗传特征相同的类克隆鸡。

Description

一种高效生产类克隆鸡的方法
技术领域
本发明涉及一种高效生产类克隆鸡的方法，属于生物技术领域。
背景技术
体细胞克隆技术在哺乳动物上被广泛地用来研究细胞特性，在研究基因结构和功能、基因治疗、遗传病的发生和治疗、濒危动物物种复原等方面具有巨大的应用价值。然而，这一技术在鸟类和两栖类等卵生动物上无法实施，这极大的限制了禽类科研和生产研究相关的发展。而禽类PGCs移植技术提供了一种生产类克隆鸡的替代方案。2006年，Larval等进行了鸡异体间PGCs移植并产生了后代，证明了这是目前在家禽中唯一可高效实现体细胞克隆的途径。但是，原代分离的PGCs数量较少，远不能满足实际移植时的需求，这就限制了该技术的应用。通过ESCs诱导PGCs作为解决PGCs来源的技术仍然存在获取困难、数量少的问题。而体细胞重编程可将易于获得的皮肤或组织细胞诱导为iPSCs，研究亦表明全能干细胞能够在体外被诱导形成PGCs，这能够有效的解决目前鸡PGCs难以大量获取的关键问题。但是目前在家禽上尚未有明确的技术方案，这极大的限制了克隆技术在家禽上的推广应用，需要进一步的优化和改进。
发明内容
本发明的目的就是针对上述现有问题，提供一种高效生产类克隆鸡的方法。
本发明的技术方案是：一种高效生产类克隆鸡的方法，其特征是，包括以下步骤：
（1）、构建OCT4, SOX2, NANOG, LIN28四因子的慢病毒过表达载体并转染黑羽狼山鸡成纤维细胞CEF；
（2）、利用第一诱导培养基将转染四因子的狼山鸡成纤维细胞CEF诱导为iPSCs，在诱导的第5天细胞形态发生显著变化，逐渐聚集变圆，贴壁能力下降，此时将细胞转移至丝裂霉素处理后的CEF饲养层上培养，整个诱导过程持续21天；对诱导形成的iPSCs进行传代并进行鉴定；第一诱导培养基配方为Knockout DMEM、10%FBS、0.1mmol/Lβ-巯基乙醇、1%非必需氨基酸、1000IU/mLLIF、10ng/mLbFGF、5ng/mL SCF、1%的青链霉素、2%鸡血清；
（3）、利用第二诱导培养基将诱导形成的iPSCs进行诱导分化为PGC-like，并通过PAS糖原染色、间接免疫荧光进行鉴定；第二诱导培养基配方为基础因子培养基、40ng/mlBMP4、40ng/ml BMP8b 、50ng/ml EGF；
（4）、收集PGC-like细胞并将其通过鸡胚血管注射的方法注射至孵化2.5天的隐性白羽鸡胚血管中，并通过冰冻切片方法确定PGC-like能够在受体隐性白羽鸡中的迁移归巢；
（5）、将注射黑羽狼山鸡PGC-like细胞的隐性白羽受体鸡胚继续孵化至至出壳，即，F0代；饲养至性成熟并进行相互交配，收集种蛋并进行孵化，获得黑羽后代，并通过微卫星技术证明黑羽后代来源于供体的PGC-like细胞，至此完成类克隆鸡的生产。
步骤（2）中，对诱导形成的iPSCs进行传代并通过间接免疫荧光、碱性磷酸酶染色、染色体核型分析方法进行鉴定。
步骤（3）中，利用第二诱导培养基将诱导形成的iPSCs进行诱导分化为PGC-like，并通过PAS糖原染色、间接免疫荧光进行鉴定。
本发明方法先进科学，通过本发明，使用体细胞重编程的方法大量获得PGC-like细胞，高效生产嵌合体鸡的方法，弥补了禽类无法体细胞克隆的缺陷，为濒危物种的恢复以及物种资源保护等工作提供新思路。本方法适用于将鸡胚成纤维细胞重编程并定向诱导，类克隆鸡和嵌合体制备。采用本方法，可大量生产与供体遗传特征相同的类克隆鸡，解决克隆技术在禽类上应用困难的问题。通过本发明，在方法上，包括的步骤如下：
（1）构建OCT4, SOX2, NANOG, LIN28（OSNL）四因子的慢病毒过表达载体并转染黑羽狼山鸡成纤维细胞（CEF）；
（2）利用第一诱导培养基将转染OSNL的狼山鸡CEF诱导为iPSCs，在诱导的第5天细胞形态发生显著变化，逐渐聚集变圆，贴壁能力下降，此时将细胞转移至丝裂霉素处理后的CEF饲养层上培养，整个诱导过程持续21天；对诱导形成的iPSCs进行传代并通过间接免疫荧光、碱性磷酸酶染色、染色体核型分析等方法进行鉴定；
（3）利用第二诱导培养基将诱导形成的iPSCs进行诱导分化为PGC-like，并通过PAS糖原染色、间接免疫荧光进行鉴定；
（4）收集PGC-like细胞并将其通过鸡胚血管注射的方法注射至孵化2.5天的隐性白羽鸡胚血管中，并通过冰冻切片等方法确定PGC-like能够在受体隐性白羽鸡中的迁移归巢；
（5）将注射黑羽狼山鸡PGC-like细胞的隐性白羽受体鸡胚继续孵化至至出壳（F0代），饲养至性成熟并进行相互交配，收集种蛋并进行孵化，获得黑羽后代，并通过微卫星等技术证明黑羽后代来源于供体的PGC-like细胞，至此完成类克隆鸡的生产。
本发明的优越性在于：
1、本发明能够高效的将体细胞诱导为原始生殖细胞，解决其来源问题；
2、本发明生产类克隆鸡方法切实可行，可大量生产类克隆鸡，克服体细胞克隆技术在鸡上无法应用的缺陷；
3、为转基因鸡制备、濒危物种的恢复以及物种资源保护等工作提供全新的方法，在这些领域具有较大的推广生产潜能和经济效益。
附图说明
图1是本发明iPSCs的诱导过程示意图；
图2是本发明 PGC-like的诱导过程示意图；
图3是本发明PGC-like在受体中迁移的鉴定图；
图4 是本发明类克隆鸡产生的示意图。
具体实施方法
下面结合具体实施例对本发明进一步进行描述。但本发明的保护范围并不仅限于此：
一种高效生产类克隆鸡的方法，包括的步骤为：
（1）鸡OCT4, SOX2, NANOG, LIN28（OSNL）慢病毒过表达载体感染CEF；
以1.5×105/孔状态良好的黑羽狼山鸡CEF细胞量接种24孔板，用含10%FBS的DMEM进行培养；预计细胞密度为60%左右时，每孔添加300μL无血清DMEM培养基稀释后的病毒，以感染复数为10，按1:1:1:1比例混合四种病毒，聚凝胺的终浓度为5ng/mL，侵染成纤维细胞；感染6小时后添加含有5ng/mL聚凝胺的无血清DMEM培养基稀释病毒；感染24小时后更换为含有10%FBS的DMEM继续培养至72小时观察EGFP绿色荧光表达情况。
（2）鸡iPSCs细胞诱导培养方法；
将感染后的成纤维细胞在10%FBS培养基中培养至72小时。观察细胞状态，将培养基更换为诱导培养基1(Knockout DMEM+10%FBS+0.1mmol/Lβ-巯基乙醇+1%非必需氨基酸+1000IU/mLLIF+10ng/mLbFGF+5ng/mL SCF+1%的青链霉素+2%鸡血清)；诱导至第5天，细胞形态发生显著变化，逐渐聚集变圆，贴壁能力下降。将细胞转移至丝裂霉素处理后的CEF饲养层上培养；根据细胞状态，每日更换一半DMEM培养基；饲养层细胞脱落后进行传代，更换新的饲养层；诱导至形成明显单克隆集落后（图1）在体式显微镜下挑取单细胞克隆集落。用accutase消化10分钟后接种于铺有饲养层细胞的96孔板；在96孔板中培养，待饲养层细胞脱落后传代至48孔板中，用同样的方法传代至24孔板中培养。
（3）鸡iPSCs传代和鉴定；
以饲养层细胞即将脱落，iPSCs细胞产生明显的细胞克隆集落为标准进行传代培养；收集iPSCs细胞的培养基上清，用PBS轻柔清洗孔板底面清除多余的血清；200μLaccutase消化液加入孔板中，37℃消化5分钟，将消化后的细胞收集，与上清一同以1000g离心8分钟；弃去上清，在细胞沉淀中加入1mL的accutase消化液消化iPSCs克隆，于37℃消化10分钟，每3分钟震荡一次；消化结束后根据孔板数量加入3倍以上体积的因子培养基稀释accutase消化液，400目滤布过滤，接种于新的饲养层上；6小时后待iPSCs细胞粘附于新的饲养层细胞后，更换新的培养基，清除死亡的饲养层细胞；通过SSEA-1间接免疫荧光，碱性磷酸酶染色，染色体核型分析对iPSCs的生物学特征进行鉴定；
（4）鸡PGC-like细胞诱导培养和鉴定；
收集培养中的iPSCs上清，用accutase消化饲养层细胞3分钟后与上清一同以1000g离心8分钟；接种于100mm培养皿中，差速贴壁45分钟，收集上清，去除iPSCs中的饲养层细胞；以5×104/孔接种iPSCs细胞于孔板中，诱导培养基成分为基础因子培养基加40ng/mL BMP4，40ng/mL BMP8b，50ng/mL EGF诱导PGC-like形成（图2），每隔12小时更换一半的诱导培养基，整个诱导过程持续4天；通过PAS糖原染色、Cvh/C-kit间接免疫荧光对PGC-like进行生物学鉴定。
（5）鸡胚血管注射；
取发育至2.5天的隐性白羽鸡种蛋，用酒精棉球小心擦拭其钝端；在钝端开口放置在体式显微镜下，找到鸡胚血管；用显微注射针向血管中注射1μL细胞悬液(5000个/μL)；将20μL青链霉素溶液从开口中轻轻滴入；用医用纸胶带将开口封住，将种蛋移回孵化箱中钝端朝上孵化。
（6）冰冻切片检测鸡PGC-like细胞迁移归巢能力；
将样品放入冰冻切片托架上加入包埋剂涂抹均匀，使样品托保持平整；迅速将样品托放入液氮，冷冻固定至无气泡产生，约20秒；将冷冻固定好的样品放入冰冻切片机中使温度平衡；安装好刀片，以20μm的厚度进行修片；修到样品处后，以8μm的厚度进行切片，用载玻片附着，显微镜下观察（图3）。
（7）体细胞诱导重编程介导的转基因鸡生产；
将注射黑羽狼山鸡PGC-like细胞的隐性白羽受体鸡胚继续孵化至至出壳（F0代）；F0代饲养至性成熟，公母鸡进行交配，收集种蛋（F1代）孵化；观察并记录F1代鸡出壳时羽色形状及胫色等表型性状（图4）；F1代鸡1月龄时对其进行翅静脉采血，提取血液基因组进行基因组测序或微卫星检测。

Claims (3)

1.一种高效生产类克隆鸡的方法，其特征是，包括以下步骤：
（1）、构建OCT4, SOX2, NANOG, LIN28四因子的慢病毒过表达载体并转染黑羽狼山鸡成纤维细胞CEF；
（2）、利用第一诱导培养基将转染四因子的狼山鸡成纤维细胞CEF诱导为iPSCs，在诱导的第5天细胞形态发生显著变化，逐渐聚集变圆，贴壁能力下降，此时将细胞转移至丝裂霉素处理后的CEF饲养层上培养，整个诱导过程持续21天；对诱导形成的iPSCs进行传代并进行鉴定；第一诱导培养基配方为Knockout DMEM、10%FBS、0.1mmol/Lβ-巯基乙醇、1%非必需氨基酸、1000IU/mLLIF、10ng/mLbFGF、5ng/mL SCF、1%的青链霉素、2%鸡血清；
（3）、利用第二诱导培养基将诱导形成的iPSCs进行诱导分化为PGC-like，并通过PAS糖原染色、间接免疫荧光进行鉴定；第二诱导培养基配方为基础因子培养基、40ng/ml BMP4、40ng/ml BMP8b 、50ng/ml EGF；
（4）、收集PGC-like细胞并将其通过鸡胚血管注射的方法注射至孵化2.5天的隐性白羽鸡胚血管中，并通过冰冻切片方法确定PGC-like能够在受体隐性白羽鸡中的迁移归巢；
（5）、将注射黑羽狼山鸡PGC-like细胞的隐性白羽受体鸡胚继续孵化至至出壳，即，F0代；饲养至性成熟并进行相互交配，收集种蛋并进行孵化，获得黑羽后代，并通过微卫星技术证明黑羽后代来源于供体的PGC-like细胞，至此完成类克隆鸡的生产。
2.根据权利要求1所述的一种高效生产类克隆鸡的方法，其特征是，步骤（2）中，对诱导形成的iPSCs进行传代并通过间接免疫荧光、碱性磷酸酶染色以及染色体核型分析方法进行鉴定。
3.根据权利要求1所述的一种高效生产类克隆鸡的方法，其特征是，步骤（3）中，利用第二诱导培养基将诱导形成的iPSCs进行诱导分化为PGC-like，并通过PAS糖原染色和间接免疫荧光进行鉴定。
CN202110654978.XA 2021-06-11 2021-06-11 一种高效生产类克隆鸡的方法 Pending CN113355359A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110654978.XA CN113355359A (zh) 2021-06-11 2021-06-11 一种高效生产类克隆鸡的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110654978.XA CN113355359A (zh) 2021-06-11 2021-06-11 一种高效生产类克隆鸡的方法

Publications (1)

Publication Number Publication Date
CN113355359A true CN113355359A (zh) 2021-09-07

Family

ID=77533991

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110654978.XA Pending CN113355359A (zh) 2021-06-11 2021-06-11 一种高效生产类克隆鸡的方法

Country Status (1)

Country Link
CN (1) CN113355359A (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111534481A (zh) * 2020-05-18 2020-08-14 扬州大学 一种提高鸡胚成纤维细胞体外诱导重编程为iPS细胞效率的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006534A1 (en) * 1997-08-04 1999-02-11 University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts, Represented By Its Amherst Campus Production of avian embryonic germ (eg) cell lines by prolonged culturing of pgcs, use thereof for cloning and chimerization
US20140363467A1 (en) * 2011-06-10 2014-12-11 University Of Georgia Research Foundation, Inc. Avian induced pluripotent stem cells and their use
CN110205299A (zh) * 2019-06-14 2019-09-06 扬州大学 一种新型鸡cef重编程为pgc的诱导体系的建立方法
CN110283894A (zh) * 2019-06-26 2019-09-27 扬州大学 一种通过羽色结合分子遗传标记鉴别pgc移植后代鸡的鉴定方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006534A1 (en) * 1997-08-04 1999-02-11 University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts, Represented By Its Amherst Campus Production of avian embryonic germ (eg) cell lines by prolonged culturing of pgcs, use thereof for cloning and chimerization
US20140363467A1 (en) * 2011-06-10 2014-12-11 University Of Georgia Research Foundation, Inc. Avian induced pluripotent stem cells and their use
CN110205299A (zh) * 2019-06-14 2019-09-06 扬州大学 一种新型鸡cef重编程为pgc的诱导体系的建立方法
CN110283894A (zh) * 2019-06-26 2019-09-27 扬州大学 一种通过羽色结合分子遗传标记鉴别pgc移植后代鸡的鉴定方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RUIFENG ZHAO等: "Production of viable chicken by allogeneic transplantation of primordial germ cells induced from somatic cells", 《NAT COMMUN》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111534481A (zh) * 2020-05-18 2020-08-14 扬州大学 一种提高鸡胚成纤维细胞体外诱导重编程为iPS细胞效率的方法

Similar Documents

Publication Publication Date Title
ES2360595T3 (es) Medio de cultivo de células embrionarias totipotentes aviares.
RU2426784C2 (ru) Способ селекции кардиомиоцитов (варианты)
US6156569A (en) Prolonged culturing of avian primordial germ cells (PGCs) using specific growth factors, use thereof to produce chimeric avians
JP2003506076A5 (zh)
AU738357B2 (en) Production of avian embryonic germ (EG) cell lines by prolonged culturing of PGCS, use thereof for cloning and chimerization
KR20020029096A (ko) 조류의 시원 생식 세포를 이용한 미분화된 조류 세포 배양생산 방법
CN108384749B (zh) 鸡性腺原始生殖细胞快速分离与建系的方法
JP2002536012A (ja) 鳥類の多能性胚性生殖細胞株
US20050244958A1 (en) Production of avian embryonic germ (EG) cell lines by prolonged culturing of PGCs, use thereof for cloning and chimerization
CN107760654A (zh) 一种无血清、无饲养层的小鼠诱导多能干细胞的培养基及其方法
US20040226058A1 (en) Prolonged culturing of avian (chicken) primordial germ cells (PGCs) using specific growth factors, use thereof to produce chimeric avians (chickens)
CN115747140A (zh) 一种诱导鸡胚胎干细胞分化生成原始生殖细胞的方法
KR100569168B1 (ko) 조류 정원줄기세포의 배양방법 및 이에 의해 수득한 조류정원줄기세포
CN113355359A (zh) 一种高效生产类克隆鸡的方法
JP4343946B2 (ja) 鳥類の始原生殖細胞の生殖系列転移効率の改善方法
CN111304156A (zh) 一种诱导型滋养层干细胞及其制备方法和应用
CN112553258A (zh) 一种鸡成纤维细胞转分化为原始生殖细胞介导生产转基因鸡的方法
CN109342153A (zh) 一种水牛精原干细胞样细胞的免疫荧光染色方法
CN107236703B (zh) 一种饲养层细胞、培养方法及应用
CN113249303B (zh) 血管内皮细胞生长因子在促进鸡原始生殖细胞增殖和迁移中的应用
CN114410572A (zh) 一种从鸡胚血中快速分离高纯度原始生殖细胞的方法
WO1996006160A1 (fr) Procede de culture de cellules aviaires et lignee cellulaire ainsi obtenue
CN113337456A (zh) 诱导分化培养基以及多能干细胞诱导分化为原始生殖细胞的方法
KR20090013543A (ko) 정조 줄기 세포의 분리방법
Zhao et al. Production of clone-like chicken by primordial germ cells induced from somatic cells

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20210907

RJ01 Rejection of invention patent application after publication