WO2005028655A1 - Procede d'augmentation de l'efficacite d'un animal transgenique - Google Patents

Procede d'augmentation de l'efficacite d'un animal transgenique Download PDF

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Publication number
WO2005028655A1
WO2005028655A1 PCT/CN2003/001099 CN0301099W WO2005028655A1 WO 2005028655 A1 WO2005028655 A1 WO 2005028655A1 CN 0301099 W CN0301099 W CN 0301099W WO 2005028655 A1 WO2005028655 A1 WO 2005028655A1
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Prior art keywords
transgenic
gene
cells
animal
dual
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PCT/CN2003/001099
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English (en)
Chinese (zh)
Inventor
Ning Li
Guochun Gong
Yunping Dai
Baoliang Fan
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Ning Li
Guochun Gong
Yunping Dai
Baoliang Fan
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Application filed by Ning Li, Guochun Gong, Yunping Dai, Baoliang Fan filed Critical Ning Li
Priority to AU2003292863A priority Critical patent/AU2003292863A1/en
Publication of WO2005028655A1 publication Critical patent/WO2005028655A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0273—Cloned animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8771—Bovine embryos
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/101—Bovine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins

Definitions

  • the invention relates to a method for improving the production efficiency of transgenic animals in the technical field of animal transgene.
  • Animal genetic research is a research hotspot in the field of biotechnology, and its application scope has penetrated into basic research, agriculture, medicine and many other fields.
  • Animal transgenic research refers to the process of integrating foreign gene structures constructed according to specific purposes into the host animal's genome with various experimental means, expressing them during the development of the animal, and transmitting them to the offspring through reproductive cells. Such an animal stably integrated with an artificially introduced foreign gene in its genome is called a transgenic animal.
  • transgenic domestic animals because the production cycle of all domestic animals is relatively long, the time from obtaining the original transgenic animal (founder) to forming a transgenic animal group with productive capacity will increase production costs again. It is precisely because the efficiency of producing transgenic domestic animals is much lower than that of transgenic mice, and the cost is extremely high. Therefore, many researches on transgenic animals are limited to mice, and they dare not get involved in domestic animals.
  • site-specific modifications to specific sites in the animal genome including site-specific insertions and site-specific knockouts (knock-in or knock-out), have the following advantages: 1) For specific target genes Site-specific modification can more accurately study the expression regulation of specific genes; 2) can avoid the random insertion of foreign genes to cause the inactivation of endogenous functional genes in the host; 3) can overcome the position of integration sites when foreign genes are randomly integrated Effects of effects on foreign gene expression.
  • Site-directed gene modification using embryonic stem cell technology has been widely used in mice. Although the current embryonic stem cell technology is more mature in mice, embryonic stem cells have not been successfully isolated in domestic animals, so that it has not been possible to use the technology to perform site-specific modification of human genes.
  • somatic cell nuclear transfer technology opens an effective way to produce transgenic animals.
  • the biggest feature of this technical route is mainly that the gene transfer step is advanced to the somatic cell culture stage.
  • Cell gene transfer refers to the integration of exogenous target genes into the genome of cells through methods such as electroporation, liposome transfection, calcium phosphate precipitation, and microinjection, and the use of specific screening markers to amplify and enrich transgenes in large quantities Cells to obtain transgenic cell lines. All cloned animals that are directly bred from such identified somatic cells integrated with the gene of interest as nuclear donors must be all transgenic animals.
  • the advantages of producing transgenic livestock through somatic cell nuclear transfer are obvious: First, the method is simple and easy to obtain.
  • Oocytes can be obtained from slaughterhouses, and animal somatic cells are more abundant. Secondly, because most of the animals produced are transgenic animals, the number of surrogate mothers is greatly reduced, and the production cost is reduced. Third, if the various phenotypic characteristics of the transgenic animals are confirmed, the same donor can be used Cells are used as nuclear donors for large-scale replication, thereby shortening the time from the acquisition of primary transgenic animals to the formation of productive transgenic animal populations; more valuablely, the use of this technology can also be used to obtain genetically modified domestic animals. Under current conditions, this is not possible with other transgenic methods.
  • somatic cell nuclear transfer technology to the production of genetically modified livestock are obvious, and have been successful in the production of genetically modified animals such as sheep, cattle, goats, and pigs, there are also limitations in this technology system.
  • the advantages of this technical route are mainly reflected in that the gene transfer step is advanced to the somatic cell culture stage, and the identified somatic cells integrated with the target gene are directly used as nuclear donors for somatic cell nuclear transfer. All cloned animals must be transgenic animals.
  • related studies have shown that because many non-transgenic cells are protected by drug-resistant substances produced by surrounding transgenic cells and survive, the drug resistance obtained by drug screening has been reduced. Not all of the cells are integrated with foreign genes, and not all cloned animals cultivated with these drug-resistant cells are transgenic animals.
  • the purpose of the present invention is to provide a method which can effectively improve the production efficiency of transgenic animals.
  • the application of this method will greatly improve the reliability of transgenic animals.
  • the method for improving the production efficiency of a transgenic animal provided by the present invention is to produce a transgenic animal by using a somatic cell nuclear transfer technology including a gene transfer step.
  • the gene transfer utilizes the ability to simultaneously express enhanced green fluorescent protein genes and neomycin resistance.
  • the dual-marker selection vector for sex genes was implemented.
  • the neomycin resistance gene is used for the screening and enrichment of transgenic cells; the enhanced green fluorescent protein gene is used to effectively screen out the transgenic blastocysts for embryo transfer, to ensure that the cloned animals cultivated are all transgenic animals.
  • the transgenic animals in the method may be mammals such as transgenic domestic animals and transgenic mice.
  • the transgenic livestock can be transgenic rabbits, transgenic cattle, transgenic sheep, transgenic pigs, etc., especially transgenic cattle, which is of great significance.
  • the above dual-marker selection vector may include a cytomegalovirus IE enhancer, a human elongation factor 1 ⁇ promoter pEF321, EGFP-IRES-NEO, and tailing signals and multiple cloning sites of SV40; its nucleotide sequence may be in the sequence listing Sequence 1. The 5th, 1st and 29th bases in sequence 1 are multiple cloning sites.
  • the dual-label selection vector can be introduced into recipient cells by electroporation.
  • the conditions for introducing the recipient cells by electroporation may be an electric field strength of 1-1.4 kV / cm, a pulse time of 0.8-1.2 ms, preferably an electric field strength of 1.2 kV / cm, and a pulse time of 1 ms.
  • Figure 1 is a physical map of the dual-labeled selection vector plasmid pCE-EGFP-IRES-NE0.
  • Figure 2 is a schematic diagram of the efficient production process of somatic clone-mediated transgenic cattle.
  • Figure 3 is the conversion efficiency curve of different electric field strength when the pulse time is 0.8ms
  • Figure 4 is the transformation efficiency curve at different pulse times when the electric field intensity is 1.2 kV cm- 1 .
  • Figure 5A is the PCR identification electrophoresis map of three transgenic cloned cattle.
  • Figure 5B is Southern Blot identification electrophoresis map of three transgenic cloned cattle
  • Figure 6A1 is a bright field photograph of the expression of the green fluorescent protein gene in bovine transgenic fallopian tube epithelial cells
  • Figure 6A2 is a dark field photograph of the expression of the green fluorescent protein gene in bovine transgenic fallopian tube epithelial cells
  • Figure 6B1 is a bright field photograph of the expression of the green fluorescent protein gene in the transgenic cloned blastocyst
  • Figure 6B2 is a dark field photograph of the expression of the green fluorescent protein gene in the transgenic cloned blastocyst.
  • Figure 6C1 is a bright field photograph of the expression of the green fluorescent protein gene in skin tissue samples of transgenic cloned cattle
  • Figure 6C2 is a dark field photograph of the expression of the green fluorescent protein gene in skin tissue samples of transgenic cloned cattle
  • Figure 6D1 is a bright field photograph of the expression of the green fluorescent protein gene in fibroblasts derived from Leva's skin tissue mass
  • Figure 6D2 is a dark field photograph of the expression of the green fluorescent protein gene in fibroblasts derived from Leva's skin tissue mass
  • Cell and embryo culture vessels were purchased from Costar or Nunc; DMEM / F12, Trypsin were purchased from Gibco, FBS was purchased from Hyclone, and other drugs were purchased from Sigma unless otherwise specified.
  • the fetuses of Holstein cows were taken from the Beijing Dairy Center, and the cattle ovaries came from slaughterhouses around Beijing.
  • plasmid pCE321-FL purchased from Clontech
  • plasmid pEGFP-IRESneo purchased from Clontech
  • CMV-IE Enhancer cytomegalovirus enhancer
  • EF321 human Elongation factor 1 ⁇ promoter
  • plasmid site pCE-EGFP-IRES-NEO-dNdB 5 of the gene component CE-EGFP-IRES-NEO was inserted into the regulatory site to form the plasmid pCE-EGFP-IRES-NEO-dB.
  • the prokaryotic part of the plasmid pBCl was ligated with the multiple cloning site (MCS) to form the plasmid pBM.
  • the plasmid pCE-EGFP-IRES-NEO-dB and plasmid pBM were double-digested with Wil and ⁇ EI, and the gene component CE-EGFP-IRES-NE0 was inserted into the plasmid pBM to form a dual-marker selection vector pCE-EGFP-IRES-NEO. After enzymatic digestion and identification, the constructed dual-marker selection vector pCE-EGFP-IRES-NE0 was completely consistent with the experimental design.
  • the physical map of the dual-marker selection vector pCE- EGFP- IRES- NE0 constructed in this example and capable of simultaneously expressing two marker genes of enhanced green fluorescent protein (EGFP) and neomycin resistance gene (Neo f ) is shown in Figure 1
  • MCS is a multicloning site
  • CMV-IE Enhancer is a cytomegalovirus enhancer
  • EF321 is a human elongation factor 1 ⁇ promoter
  • EGFP is an enhanced green fluorescent protein gene
  • IVS is an artificial intron
  • RES is an internal The ribosome entry site
  • Nee is the neomycin resistance gene
  • polyA is the tailing signal of SV40
  • Ara is the ampicillin resistance gene
  • Ori is the origin of pBR322 replication.
  • sequence 1 The full sequence is shown as sequence 1 in the sequence listing, from the 5th and 1st to 29th bases are polyclonal sites; from the 5 ⁇ to 46th and 420th bases are the cytomegalovirus enhancer sequences;
  • the 5 'terminal 423-1616 bases are human elongation factor 1 ⁇ promoter sequence;
  • the 5 terminal 1645-2364 bases are the enhanced green fluorescent protein gene sequence;
  • the 5 ⁇ terminal 2381-2676 bases are artificial Intron sequence; from 5 and terminal 2702 to 3287 bases are internal ribosome entry site sequence;
  • from 5 'terminal 3313 to 4116 bases are neomycin resistance gene sequences; from 5 and 4129
  • Position ⁇ 4346 base is SV40 tailed signal sequence; bases 4346 to 10217 from base 5 and terminal 4 are pHC79 cosmid vector sequences; bases 4578 to 5438 from base 5 and 5 are ampicillin resistance gene sequence ; 5BR-6255 bases from the 5 ⁇ end are PBR322 sequence of
  • the neomycin resistance gene is used for the screening and enrichment of transgenic cells; the enhanced green fluorescent protein gene is used to effectively screen the transgenic blastocysts for embryo transfer, to ensure that the cloned animals are all transgenic animals IRES ( small internal ribosome entry site of RNA viruses) ensure EGFP and Neo f can be expressed simultaneously.
  • the plasmid P CE- EGFP- IRES- NEO was double digested with Sail and cl to obtain a linear dual-label selection vector, purified and recovered by QIAEX II kit from QIAGEN, and dissolved in sterilized ultrapure water.
  • a May Holstein cow fetus was taken from the Beijing Dairy Center. The entire uterus was transported back to the laboratory. The fetus was removed, washed with PBS and 70% alcohol several times, and the epithelial tissue sample was taken from the fetal fallopian tube and cut into about 1 mm 3 Small pieces, washed with DMEM / F12 2 times, and then planted in batches in 25cm 2 culture flasks containing 1mL DMEM / F12 + 10% FBS.
  • DMEM / F12 + 10% FBS to 6 mL
  • culture in a 37 ° C, 5% CO 2 incubator for 6 to 7 days change the solution once every 2 days, and after the cells grow confluent, digest with 0.25% trypsin for 2 to 3 times.
  • the batches were frozen at DMEM / F12 + 20% FBS + 10 ° / oDMSO.
  • a bovine fetal fallopian tube epithelial cell line was established through in vitro culture operations such as primary culture, subculture, and freezing.
  • CE- EGFP- IRES- NE0 by double selection marker plasmid pCE- EGFP- IRES- NE0 via S a R ⁇ scl and digested target gene by comparing the different electric field strength (E), different pulse time (t )
  • E electric field strength
  • t pulse time
  • the screening concentration of G418 was determined by the sensitivity test of bovine fetal fallopian tube epithelial cells to different concentrations of G418 toxicity.
  • OlU / mL bLH + Wg / mL estradiol was washed twice, and then the cumulus-oocyte-complex was put into a four-well plate containing mature solution at 50-60 pieces / well, and cultured at 38.5 ° C, 5% C0 2 after maturation culture tank 18- 20h, mature oocytes were placed in 0.1% hyaluronidase after shaking 2-3mi n, then the inner glass tube by gently pipetting the cumulus cells and oocytes Completely detached, choose oocytes with complete morphology, uniform cytoplasm, and excrete the first polar body as cytoplasmic receptors.
  • the oocytes with the first polar body were transferred into an operating solution containing M199 + 10% FBS + 7.5 g / mL cytochalasin B, and a glass needle was used above the polar body under a 200x microscope to make the transparent Cut a small opening with a glass tube with an inner diameter of 20 ⁇ m to remove the chromosomes in the first polar body and the oocytes below it, and put them in a solution of M199 + 20% FBS three times. Store in the incubator.
  • the serum-starved 2--4d transgenic cells were digested with 0.25% trypsin for 2-4 minutes, and fetal fallopian tube epithelial cells with a diameter of 10-12 ⁇ m were selected for use.
  • 20 ⁇ m-diameter glass tubes were transferred to the nucleated oocytes Inside the cell zona pellucida, then put it into 0.3M Mannitol, 0.15mmol / L Ca 2+ , 0.15ramol / L Mg 2+ solution for 3-5min, then put it into the fusion tank, and rotate the egg cell
  • the donor cell and oocyte contact surface is perpendicular to the electric field, and the fusion is performed under the condition that the field strength of the DC pulse is 2.5 kV / cm, the pulse time is 10 ⁇ 5 , the number of pulses is 2 and the pulse interval is Is.
  • the instrument was ECM-2001 from BTX Co., Ltd.), the reconstructed embryos were quickly transferred into M199 + 10% FBS solution, and the fusion rate was observed after 0.5 hours, and the fusion embryos were selected for the next activation. deal with.
  • the well-formed GFP cloned blastocyst on day 7 was transferred into the uterine horn of the recipient cow.
  • B-ultrasounds were performed on recipient cows at 30 days after transplantation to determine conception, and rectal tests were performed at 60 and 90 days after transplantation to determine pregnancy rates.
  • PCR amplification of transgenic and non-transgenic cloned bovine genomic DNA samples using a pair of PCR primers upstream: 5 '-TGC AGT GCT TCA GCC GCT AC -3, downstream: 5, one CTC AGG TAG TGG TTG TCG GG -3'
  • the genomic DNA of ordinary Holstein dairy cows was used as a negative control, and the double-labeled vector plasmid pCE-EGFP-IRES-NE0 was used as a positive control.
  • PCR conditions 94 ° C 5 rain; 94 ° C 30 sec, 62 ° C 30 sec, 72 ° C 30 sec, 30 cycles; 72 ° C 7 min.
  • the full length of the amplified product is a DNA fragment of about 384 bp.
  • M is an lkb ladder
  • 1 is a transgenic cloned cow "Lewa”
  • 2 is a transgenic cloned cow "Jiumei”
  • 3 is a transgenic cloned cow "8C2”
  • 4 is a non-transgenic cloned cow " ⁇ ⁇ ”
  • 6 is a positive plasmid
  • 7 is sterilized distilled water.
  • the probe used for hybridization is the ⁇ -P 32 dCTP isotope-labeled dual-labeled vector plasmid Bglll to digest the recovered product.
  • the normal Holstein dairy cow genomic DNA was used as a negative control, and the double-labeled vector pCE- EGFP- IRES-NE0 was used as a positive control.
  • the positive hybridization signal was 1. 6kb fragment.
  • Bovine transgenic fallopian tube epithelial cells as a nuclear donor were added with G418 one day after transformation. Positive clones appeared after 14 days of screening with G418 and observed under blue light under a fluorescence microscope to detect the expression of GFP. The results are shown in Figures 6A1 and 6A2, indicating that the expression of green fluorescent protein (GFP) was found after the fallopian tube epithelial cells were transformed.
  • GFP green fluorescent protein
  • Tissue samples were obtained from the lower edge of the ear of the transgenic cloned cow "Lewa", cut into small pieces of about 1 mm 3 , washed with EM / F12 2 times and planted in batches in 25 cm 2 containing 1 mL of DMEM / F12 + 10% FBS.
  • DMEM / F12 + 10% FBS was added to 6 M1, and culture in a 37 ° C, 5% C02 incubator for 6-7 days, and change the solution every 2 days.
  • the cells are separated from the skin tissue, observe the expression of GFP in the cells.
  • the results are shown in Figures 6D1 and 6D2, indicating that there is expression of green fluorescent protein in the cells.
  • the invention constructs a dual-marker selection vector that can be used for both enrichment of transgenic cells and screening of transgenic blastocysts; optimized conditions for electroporation-mediated transformation methods of animal somatic genes; and using the marker gene of green fluorescent protein, Effectively screen transgenic blastocysts for embryo transfer, and ensure that 100% of the cloned animals are transgenic animals, thereby establishing a set of methods that can effectively improve the production efficiency of somatic clone-mediated transgenic livestock and overcome somatic cell cloning. Skill Not all the cloned clones that exist in the production of transgenic animals by surgery are shortcomings of transgenic animals.
  • the present invention establishes a set of methods that can effectively improve the production efficiency of somatic clone-mediated transgenic animals, especially transgenic livestock, for the production of medicinal proteins, nutritional proteins and industrial proteins for transgenic animals, especially transgenic livestock, and xenotransplantation,
  • somatic clone-mediated transgenic animals especially transgenic livestock
  • for the production of medicinal proteins, nutritional proteins and industrial proteins for transgenic animals especially transgenic livestock, and xenotransplantation

Abstract

L'invention concerne un procédé d'augmentation de l'efficacité de production d'un animal transgénique. Ce procédé comprend une technique de transfert de cellules somatiques qui consiste en une étape de transfert nucléaire se caractérisant par une sélection réussie de cellules transgéniques et d'embryons par un type de vecteur sélectif doublement marqué, qui co-expriment une protéine fluorescente améliorée (EGFP) et des gènes résistants à la néomycine (NeoR). Le procédé de l'invention est destiné à être utilisé pour améliorer l'efficacité de production d'animaux transgéniques, notamment d'animaux domestiques transgéniques. Ce procédé peut être utilisé dans la transplantation d'organe hétérospermique, la production de protéine médicinale, de protéine nutritive et de lait humanisé, ainsi que dans la construction de modèles animaux de divers types de maladies.
PCT/CN2003/001099 2003-09-23 2003-12-22 Procede d'augmentation de l'efficacite d'un animal transgenique WO2005028655A1 (fr)

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AU2003292863A AU2003292863A1 (en) 2003-09-23 2003-12-22 A method for increasing the efficiency of transgenic animal production

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CN03160031.X 2003-09-23
CN 03160031 CN1274814C (zh) 2003-09-23 2003-09-23 一种提高转基因动物生产效率的方法

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CN1891821B (zh) * 2005-04-21 2010-07-07 李宁 转人溶菌酶基因的动物细胞的生产方法
CN100445379C (zh) * 2005-04-21 2008-12-24 李宁 转人α-乳清蛋白基因的动物细胞的生产方法
CN106719435B (zh) * 2016-11-24 2019-07-12 南京师范大学 一种携带增强型绿色荧光蛋白标记的转基因家兔及其构建方法

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CN1274814C (zh) 2006-09-13
CN1600851A (zh) 2005-03-30

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