WO2018196143A1 - Procédé d'accroissement du rendement en porc maigre par retard du moment d'expression d'un gène - Google Patents

Procédé d'accroissement du rendement en porc maigre par retard du moment d'expression d'un gène Download PDF

Info

Publication number
WO2018196143A1
WO2018196143A1 PCT/CN2017/089556 CN2017089556W WO2018196143A1 WO 2018196143 A1 WO2018196143 A1 WO 2018196143A1 CN 2017089556 W CN2017089556 W CN 2017089556W WO 2018196143 A1 WO2018196143 A1 WO 2018196143A1
Authority
WO
WIPO (PCT)
Prior art keywords
myod
gene
pigs
pig
expression
Prior art date
Application number
PCT/CN2017/089556
Other languages
English (en)
Chinese (zh)
Inventor
莫德林
张续勐
陈瑶生
刘小红
何祖勇
丛佩清
邱柏钦
郭慈琳
Original Assignee
中山大学
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 中山大学 filed Critical 中山大学
Publication of WO2018196143A1 publication Critical patent/WO2018196143A1/fr

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
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • 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
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, 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
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • 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
    • 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

Definitions

  • the present invention relates to the field of molecular genetics and, in particular, to a method for increasing lean meat production by delaying gene expression time.
  • primary muscle fibers Compared with pigs of different breeds, the number of primary muscle fibers and the ratio of secondary muscle fibers to primary muscle fibers of larger white pigs were significantly higher than those of small pigs. Therefore, between different body types of pigs, primary muscle fibers may have a greater impact on the total number of muscle fibers. In summary, primary muscle fibers play a decisive role in the amount of meat produced after birth in pigs with different meat production.
  • the family of myogenic regulatory factors is a family of genes that play a decisive role in myogenic processes.
  • the family has four members: MyoD, Myf5, Myogenin, and MRF4, which are characterized by a highly conserved basic helical loop helix domain with potential for myogenic regulation.
  • Myf5 is first expressed in the embryo and complements MyoD as the initial determinant of myoblasts.
  • Studies have found that transgenic mice lacking the above two genes do not form any muscle, but the loss of either of the above two genes alone will not have much effect on myogenic muscle, indicating that Myf5 and MyoD have certain functions. Overlap and Complementary.
  • Myoblasts isolated from knockout MyoD mice found that myoblasts knocked out of MyoD proliferated for a longer period of time, leading to a delay in differentiation. Recent studies have shown that the site of MyoD binding to Myf5 binding to the downstream gene is almost identical, but Myf5 lacks a stronger activation domain than MyoD, so there is no such strong ability to cause downstream gene expression as MyoD.
  • Zhao Xiao et al. used morphological observation and transcriptome sequencing of the longissimus dorsi muscle tissue from 35 days old embryos of Guangdong local pig breeds and foreign lean-type white pigs to 180 days after birth. It was found that the blue-tang pigs had primary muscle fibers in the embryos for 35 days, while the Changbai pigs had primary muscle fibers in the embryos for 49 days, but in the subsequent muscle development, the blue-tang pigs developed slower than the white pigs and were born. The amount of meat produced afterwards is also lower than the latter.
  • transcriptome sequencing they found that the myogenic determinant was expressed earlier in the early morning of the Lantang pig embryo than the Landrace pig, including the MyoD gene.
  • Yuqiang Zhao and others compared the other long-term muscle tissue of the local pig breed Tongcheng pig in western China and western lean pig breed Yorkshire pig (Large white pig) embryos for 30 days until 5 weeks after birth. Similar to the case of blue pond pigs, histomorphology found that Tongcheng pigs with less meat production also had higher numbers and higher density of myoblasts than Yorkshire pigs during the embryonic period of 30 days, but after the second In the development of round muscle, the secondary muscle fibers of England pigs develop faster than Tongcheng pigs.
  • the number of primary muscle fibers in the embryonic stage determines the total number of muscle fibers, and the total number of muscle fibers affects the amount of meat after birth. At the same time, the total number of muscle fibers has been determined during the embryonic stage. Therefore, the development of primary muscle fibers in the embryonic stage of pigs is decisive for the amount of meat after birth. influences.
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a method for increasing the lean meat yield of pigs by delaying the expression time of the gene.
  • a method for increasing the lean meat yield of pigs by delaying the expression time of the gene, delaying the expression time of the myogenic differentiation factor MyoD in the pig embryo, thereby increasing the meat production of the pig.
  • the MyoD gene is postponed for one week in pigs to achieve an effect of increasing meat production.
  • the present invention simulates the early expression of MyoD gene in Chinese local pig breeds in a mouse-derived C2C12 myoblast cell line.
  • the expression of MyoD gene is 48 h and 24 h in advance, respectively, and the number of myotubes produced by differentiation is reduced, and the earlier expression is The fewer the number of myotubes produced.
  • we postpone the MyoD gene in pigs based on the actual results of a one-week expression of lean-type Landrace pigs compared to the small pig Wuzhishan pig MyoD. Expressed for 1 week to achieve an effect of increasing meat production.
  • the invention adopts the longest muscle-like tissue of the long-term muscles of the Changbai, Lantang and Wuzhishan pig embryos at the early 18th, 21st, 28th, 32nd, 35th and 42th day of the embryo production, and the myotube marker protein detection is performed.
  • the time of occurrence of primary muscle fibers of three breeds of pigs was found to be different: Wuzhishan pig first appeared primary muscle fibers in embryos 32 days, and blue pond and Landrace pigs showed primary muscle fibers in embryos 35 days, while the number of primary muscle fibers of Lantang pigs was significantly higher than that. Landrace pig.
  • MyoD was the earliest and highest in the Wuzhishan pig with the lowest meat production (the embryo began to express at 21 days), and in the Changbai pig with the most meat production. The expression was the latest and lowest (the embryo began to express in 28 days).
  • MyoD expression time we verified the expression of MyoD and the expression time in the C2C12 and NIH3T3 cell lines by overexpressing the MyoD plasmid, and the results were completely in line with expectations, ie in two Overexpression of MyoD in a cell line inhibits cell proliferation and reduces myotubes produced after differentiation.
  • a method for delaying the expression of MyoD is a technique for utilizing dox-induced Cre-LoxP knock-in of the MyoD gene in local pig breeds.
  • MyoD-LacZ-Stop-Cas9-KI pigs obtained by crossing MyoD knock-in pigs constructed by Cre-LoxP pigs with CRISPR-Cas9 technology, feeding dox to sows at different embryonic stages, induced the timing of MyoD gene expression, The local pig breed MyoD gene expression time was delayed.
  • a lentiviral-mediated RNA interference technique a lentiviral interference vector for constructing the MyoD gene, was used to inject a lentiviral interference vector into a sow of a local pig breed at different embryonic stages to delay the expression of MyoD.
  • the result of these methods is that the myoblasts have more time to proliferate, which in turn differentiates more muscle fibers and increases lean meat production in local pig breeds.
  • the present invention has the following beneficial effects:
  • the invention finds for the first time that the MyoD of pigs with higher meat production is later expressed, and the time of primary muscle fibers is also late, so that myoblasts have more time to proliferate, and the number of muscle fibers produced after differentiation is smaller than that of pigs with less meat production. There are many species, and eventually the amount of meat produced after birth is more. Based on this important finding, the present invention can affect or even change the expression time of MyoD in an animal by biotechnological means, thereby obtaining a new breed of livestock and poultry with a large amount of meat.
  • Figure 2 shows the transcriptome sequencing results of major genes/miRNAs affecting myoblasts in five stages of three breeds of pig embryos.
  • A-B MRFs and MEFs family genes;
  • C marker genes during myogenic differentiation;
  • D genes that inhibit myoblasts;
  • E myogenic-specific miRNAs that promote myoblasts.
  • Figure 4 is a graph showing the efficiency of overexpression of the MyoD gene.
  • D The proportion of proliferating cells in total cells.
  • E Overexpression of the MyoD gene affects the cell cycle.
  • Figure 5 is the effect of overexpression of the MyoD gene on myogenic differentiation at different time points.
  • A The MyoD gene was overexpressed 2 days before differentiation (-2d) and 1 day before differentiation (-1d), and the pCMV empty plasmid was used as a negative control. At the same time, differentiation was induced, and the expression level of MYHC protein was detected by Western-blot.
  • B Overexpression of MyoD gene at different time points, myotube immunofluorescence results.
  • C Quantitative results of gray scale scanning of MYHC protein for Western-blot of Figure A.
  • Figure 6 is a diagram of overexpression of MyoD to fuse NIH3T3 cells to form myotubes.
  • A After overexpressing MyoD, the mRNA expression level of MyoD was examined.
  • B After 48 h of transfection, the level of MyoD protein was measured.
  • C Detection of mRNA levels of key factors of myogenic differentiation after 7 days of induced differentiation.
  • D After induction of differentiation for 7 days, myotube formation was observed under white light.
  • E After overexpression of MyoD, differentiation was induced for 7 days, and the expression of MyHC was detected by immunofluorescence.
  • F After overexpression of MyoD, differentiation was induced for 7 days, and the expression of MyHC was detected at the protein level. .
  • *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, n 3.
  • Ruler size 100 ⁇ m.
  • Figure 7 is a graph overexpressing MyoD and inhibiting proliferation of NIH3T3 cells.
  • A Real-time label-free cell detection system detects the proliferation of NIH3T3 cells compared with the control group after overexpression of MyoD.
  • B Count the number of cells in the control group and the overexpression group at different times of GM.
  • C After over-expression of MyoD, PI staining Color, flow cytometry analysis of cell cycle, overexpression of MyoD compared with the control group, NIH3T3 cells G1 arrest, S phase cells decreased.
  • D Immunofluorescence detection of EdU positive cells and statistics.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.
  • the three breeds of pigs are abbreviated as: Changbai pig (LR), Lantang pig (LT) and Wuzhishan pig (WZS); the six embryonic periods are respectively abbreviated as: 18 days (LR1/LT1/WZS1), 21 days (LR2/LT2/WZS2), 28 days (LR3/LT3/WZS3), 32 days (LR4/LT4/WZS4), 35 days (LR5/LT5/WZS) and 42 days (LR6/LT6/WZS6).
  • paraffin section and HE staining (1) fixed: 4% paraformaldehyde placed for 24 to 48 hours; (2) dehydration: the sample is placed in the embedding box, marked, and then placed in a fully automatic dehydrator , the dehydration step is: 70% ethanol: 2h; 80% ethanol: 2h; 95% ethanol: 30min; anhydrous ethanol: 2.5h; 50% absolute ethanol + 50% transparent agent: 30min; transparent agent: 2.5h; % clearing agent + 50% paraffin wax: 30min; paraffin wax: 5h; (3) embedding: the dehydrated tissue is placed in a steel embedding box, the melted paraffin is added by the embedding machine, and solidified into wax on the refrigeration platform.
  • the dried slices are stored in a refrigerator at 4 ° C or dyed; (6) Dewaxing: slides with paraffin sections are placed in a clearing agent for 10 min, which can be repeated once; (7) Rehydration: Slides Soaked in 100%, 95%, 85%, 70%, 50% ethanol, PBS and pure water for 2min, occasionally oscillate; (8) HE staining: put the slide into hematoxylin 1-5min, pure Wash water for 1 min, add activator for 30s, rinse with pure water for 1min, eosin stain for 30s-2min, rinse with tap water for 1min; (9) Cover: After the slide is naturally air-dried, drop 20 ⁇ L of neutral resin in the slide specimen center, use The tweezers carefully cover the slide and label the sample name for observation.
  • Steps (1) to (7) are the same as steps (1) to (7) above (the reagents used in the subsequent steps are all from the immunohistochemistry kit); (8) the sliced specimen is air-dried in the tissue. Two drops of Peroxidase Blocking Reagent were added dropwise for 5 min; the slides were rinsed to the absence of residual liquid and rinsed with PBS for 5 min; (9) Three drops of Serum Blocking Reagent G (blocked serum) were added to the tissue block for 15 min, then the glass was removed.
  • image acquisition and data processing (1) image acquisition: upright fluorescence microscope, using 10 ⁇ 40 times magnification to observe the shooting, and label the ruler; (2) data processing: each experiment at least 3 times, the results used Mean ⁇ standard deviation (Mean ⁇ SD) indicates that the difference between the two groups was calculated using Student's test, and multiple sets of differences were examined by ANOVA.
  • the mapping software was GraphPad Prism 6, the data analysis was SPSS (version 20), the statistical analysis was performed by two-sided test, and the differential expression was: *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001.
  • Example 2 Transcriptome sequencing of major genes/miRNAs affecting myoblasts in five stages of three breeds of pig embryos
  • Sample collection same as the sample collection part of Example 1.
  • embryonic RNA extraction (1) embryonic meat-like tissue added 1mL Trizol, placed in the tissue disruptor to fully oscillate; (2) add 0.2 times the volume of chloroform in the centrifuge tube and vortex for 15s, rest at room temperature for 3min; 3) Then centrifuge the tube at 4 ° C, 12000g, centrifuge for 15min; (4) take out from top to bottom into the water phase (colorless transparent), the middle layer (white), the organic phase (pink), with a pipette Transfer the liquid in the aqueous layer to a new 1.5 mL centrifuge tube; (5) Inject an equal volume of pre-cooled isopropanol into the centrifuge tube, mix by inversion, and place at -20 ° C for 30-60 min; (6) Then centrifuge the tube at 4 ° C, 12000g, centrifuge for 15min; (7) take out the centrifuge tube, there will be a white precipitate at the bottom, discard the supernatant, wash the pellet with 1mL of
  • transcriptome sequencing experimental steps extract the sample total RNA and digest the DNA with DNase I, then enrich the eukaryotic mRNA with magnetic beads with Oligo (dT); add the interrupting reagent to interrupt the mRNA at the appropriate temperature in the Thermomixer A short fragment was obtained, and the stranded cDNA was synthesized by using the broken mRNA as a template, and then a two-stranded synthesis reaction system was used to synthesize the double-stranded cDNA, and the kit was used for purification and recovery, sticky end repair, and the 3' end of the cDNA plus the base. A "connection joint and then fragment size selection, the final PCR amplification; library were used and after Agilent 2100Bioanalyzer ABI StepOnePlus Real-Time PCR System quality inspection, using Illumina HiSeq TM 2000 or other sequencer for sequencing.
  • the information analysis process data 2000 obtained by the sequencing Illumina HiSeq TM or called raw reads raw data, for raw reads subsequently proceeds quality (the QC), to determine whether the data is suitable for subsequent sequencing analysis.
  • quality control clean reads are obtained by filtering, and clean reads are compared to the reference sequence using SOAPaligner/SOAP2 [16] .
  • the distribution and coverage of the reads on the reference sequence are used as indicators for judging whether the comparison result passes the second quality control. If the second quality control is passed, subsequent analysis such as gene expression is performed.
  • transcriptome data analysis the calculation of gene expression using RPKM [17] method (Reads per kilobase transcriptome per million mapped reads), the calculation formula is: In the formula, RPKM(X) is assumed to be the expression level of gene X, then C is the only number of reads aligned to gene X, N is the only total number of reads aligned to the reference gene, and L is the number of bases of the gene X coding region. As a standardized treatment, RPKM method can eliminate the influence of sequencing difference and gene length on the calculation of gene expression. The calculated gene expression can be used to directly compare gene differential expression between different samples.
  • Sample collection same as the sample one sample collection part.
  • Embryo RNA extraction same as Example 2 embryo RNA extraction step.
  • RNA Reverse transcription of RNA to obtain cDNA (for specific methods, refer to the procedures of the prior art or various commercial kits), the cDNA is placed at -20 ° C, and diluted 5 times when used.
  • the reaction procedure was: pre-denaturation at 95 ° C for 10 min; denaturation at 95 ° C for 5 s; annealing at 60 ° C for 1 min; Extend 72 ° C, 30 s; 40 cycles.
  • the dissolution curve was analyzed: 95 ° C, 5 s; 65 ° C, 15 s; 95 ° C, 0 s.
  • Table 1 shows the reaction system of qPCR
  • Table 2 shows the qPCR primer sequence
  • Upstream primer (5'to 3') Downstream primer (5'to 3') GAPDH GCCTCCAAGGAGTAAGAAAC GAAATTGTGAGGGAGATGCT MyoD ACCGCTCCGCGACGTAGATT GCGAGTGTTCCTCGGGCTTT
  • tissue protein extraction a small number of tissue blocks placed in the tissue disruption tube added to the magnetic beads, cut with scissors before the addition, add 400 ⁇ L of PMSF containing protein lysate to the tissue In the crushing tube, homogenate on the tissue disrupter, then placed on ice, repeat the tissue as much as possible, and lyse for 30 min; transfer the lysate to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 4 min at 5 °C. Then, the supernatant was dispensed with a 0.5 mL centrifuge tube and stored at -20 ° C; (2) Protein quantification: The protein sample concentration was uniform using the Coomassie Brilliant Blue method.
  • Transfection (1) C2C12 cells were inoculated into 6-well plates one day prior to transfection, with a density of approximately 30% to 50%, cultured in complete medium; (2) Transfection reagent preparation: diluted with 250 ul OPti-MEM 5ul of miRmimic or 10ul inhibitor and the corresponding control miRNC; another 250ul OPti-MEM diluted 5ul lipofectamine2000 TM and allowed to stand at room temperature for 5 minutes; (3) gently mix the two in (2) for 15 minutes at room temperature To form a transfection complex; (4) Add the transfection complex in (3) to the cells, add the medium, mix gently, and replace the fresh medium after 6 hours.
  • Real-time label-free cell detection Cell proliferation was analyzed using a Real-Time Cell Analyzer. Instrument calibration: Preheat the instrument, then add 50 ⁇ L of DMEM containing 10% FBS to each well of the proliferation assay plate (E-Plate16), replace the instrument, and adjust the baseline. Cell proliferation assay: The treated cell digestion count was added to the proliferation assay plate, and 8000 cells were added to each well. Finally, the culture medium of each well was filled with 180 ul in 10% FBS DMEM, and each group was repeated 3 times. The test was set for 5 min, and the cell proliferation curve was drawn after 72 h.
  • Instrument calibration Preheat the instrument, then add 50 ⁇ L of DMEM containing 10% FBS to each well of the proliferation assay plate (E-Plate16), replace the instrument, and adjust the baseline.
  • Cell proliferation assay The treated cell digestion count was added to the proliferation assay plate, and 8000 cells were added to each well. Finally, the culture medium of each well was filled with 180 ul in 10% FBS DMEM, and each
  • EDU proliferation assay (1) EDU is diluted with 1:1000 in 10% DMEM medium, 300 ul per well in a 12-well plate, and incubated for 2 h in a cell culture incubator; (2) Washed with 500 ul of PBS per well 2 times, 5min/time; (3) Each well was fixed with 500ul 4% paraformaldehyde for 10min at room temperature; (4) Washed twice with PBS for 5min/time; (5) 500ml with 0.5% tritonX-100 per well at room temperature Incubate for 10 min; (6) repeat step (4); (7) 300 ul / well Apollo staining reaction solution (according to instructions), incubate at room temperature for 30 min in the dark; (8) wash once with PBS for 5 min; (9) at room temperature, 300 ⁇ L DAPI staining per well for 5 min; (10) PBS washing 3 times, 5 min/time; (11) photographed under an inverted fluorescence microscope.
  • PI cell cycle detection (1) the day before, the cells were digested and placed in 1.5ml EP tube, washed with PBS, remove the medium and trypsin; (2) add 1ml 70% pre-cooled to each tube Ethanol was fixed overnight at 4 ° C; (3) Centrifuge for 5 minutes at 1500 r/s to collect ethanol, and wash with ethanol for 3 times for 5 minutes each time; (4) Add 1 ml of PI working solution per well, incubate at room temperature for 30 min in the dark. (5) Filter on machine detection.
  • RNA extraction from cells refer to the TRIzol extraction method commonly used in the art or use a commercially available extraction kit; (2) RNA inversion to cDNA: Reference Example 3; (3) SYBR Green ⁇ Real-time PCR: Refer to Example 3.
  • Plasmid DNA extraction Refer to the Genstar plasmid extraction kit instructions.
  • the annealing procedure was 95 ° C, 10 min; 85 ° C, 1 min; 75 ° C, 1 min; 65 ° C, 1 min; 55 ° C, 1 min; 45 ° C, 1 min; 30 ° C, 1 min.
  • C Double digestion and ligation of the vector: The vector was double-digested and linearized by restriction enzyme endonuclease. The digestion reaction system was digested with agarose gel at 37 ° C for 4 hours, and then recovered. The linearized vector and gene fragment were prepared according to the system of Table 6 and connected overnight at 16 ° C in a water bath.
  • Transformation and identification (1) DH5 ⁇ competent cells were taken out from the -80 °C refrigerator, melted on ice, and the ligation product was added to the clean bench, gently shaken to mix, ice bath for 30 min; (2) 42 ° C Immediately after heat shock for 45 s, ice bath for 2 min; (3) 600 ⁇ L of anti-LB liquid medium was added to each tube, and cultured at 37 ° C and 220 rpm for 30 min; (4) 3000 ⁇ g, 1 min, centrifuged to discard the supernatant, and the cells were removed.
  • Example 3 Refer to the relevant content of Example 3 for the rest of the Western blot operation steps.
  • F Data processing and statistical analysis: Unless otherwise specified, each biological experiment was repeated at least 3 times, and the experimental results were expressed as mean ⁇ standard error (Mean ⁇ SEM); experimental mapping using Prism 6 software, statistical analysis using SPSS ( Version 21), Statistical analysis between the two groups of data was performed using a two-sided test. Statistical analysis between the three groups of data was performed using ANOVA and expressed as: significant difference *: p ⁇ 0.05; extremely significant difference **: p ⁇ 0.01; :p ⁇ 0.001.
  • Table 3 shows the PCR reaction system
  • Table 4 is a single-strand annealing reaction system
  • Table 5 is a double enzyme digestion system
  • Induction of differentiation After the cells are completely overgrown, the medium is discarded and replaced with DMEM medium containing 2% horse serum, and the medium is changed every two days.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Environmental Sciences (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'accroissement du rendement en porc maigre par retard du moment d'expression d'un gène, en particulier, par retard du moment d'expression du gène myoblastique déterminant MyoD dans des embryons de porc précoces, afin que le moment de formation des myofibres primaires des embryons précoces soit reporté, et que le nombre total des myofibres soit accru, augmentant de là le rendement en viande maigre après la naissance.
PCT/CN2017/089556 2017-04-28 2017-06-22 Procédé d'accroissement du rendement en porc maigre par retard du moment d'expression d'un gène WO2018196143A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710295934.6A CN107974462A (zh) 2017-04-28 2017-04-28 一种通过延迟基因的表达时间来提高猪瘦肉产量的方法
CN201710295934.6 2017-04-28

Publications (1)

Publication Number Publication Date
WO2018196143A1 true WO2018196143A1 (fr) 2018-11-01

Family

ID=62012192

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/089556 WO2018196143A1 (fr) 2017-04-28 2017-06-22 Procédé d'accroissement du rendement en porc maigre par retard du moment d'expression d'un gène

Country Status (2)

Country Link
CN (1) CN107974462A (fr)
WO (1) WO2018196143A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023644A1 (fr) * 1995-12-22 1997-07-03 Cofok B.V. Gene de la myogenine du porc et procede d'identification de polymorphismes lies au developpement musculaire
CN104388465A (zh) * 2014-09-24 2015-03-04 华南农业大学 Mdfi在调控猪骨骼肌生长发育中的应用
CN106535630A (zh) * 2014-04-28 2017-03-22 重组股份有限公司 猪中的多重基因编辑

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023644A1 (fr) * 1995-12-22 1997-07-03 Cofok B.V. Gene de la myogenine du porc et procede d'identification de polymorphismes lies au developpement musculaire
CN106535630A (zh) * 2014-04-28 2017-03-22 重组股份有限公司 猪中的多重基因编辑
CN104388465A (zh) * 2014-09-24 2015-03-04 华南农业大学 Mdfi在调控猪骨骼肌生长发育中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COUTINHO, L.L. ET AL.: "Delayed Somite Formation in a Quail Line Exhibiting Myofiber Hyperplasia is Accompanied by Delayed Expression of Myogenic Regulatory Factors and Myosin Heavy Chain", DEVELOPMENT, vol. 117, no. 2, 1 February 1993 (1993-02-01), pages 563 - 569, XP055527804, ISSN: 1477-9129 *
TE PAS, M.F.W. ET AL.: "Influences of Myogenin Genotypes on Birth Weight, Growth Rate, Carcass Weight, Backfat Thickness, and Lean Weight of Pigs", JOURNAL OF ANIMAL SCIENCE, vol. 77, no. 9, 30 September 1999 (1999-09-30), pages 2352 - 2356, XP055527805, ISSN: 0021-8812 *
TE PAS, M.F.W. ET AL.: "Transcriptome Expression Profiles in Prenatal Pigs in Relation to Myogenesis", JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY, vol. 26, no. 2-3, 1 July 2005 (2005-07-01), pages 157 - 165, XP019256279, ISSN: 1573-2657 *

Also Published As

Publication number Publication date
CN107974462A (zh) 2018-05-01

Similar Documents

Publication Publication Date Title
Zhang et al. Longitudinal epitranscriptome profiling reveals the crucial role of N6-methyladenosine methylation in porcine prenatal skeletal muscle development
CN110551720B (zh) 基于美洲大蠊Dsx基因设计的dsRNA、其制备方法、编码基因及应用
CN111154763B (zh) 长链非编码RNA lncMGPF在调控猪肌肉发育功能中的应用
CN108676894B (zh) 与猪胴体肉品质相关的circRNA标志物及其应用
CN113186306A (zh) 一种与羊的皮肤毛囊成熟相关的miRNA及应用
CN110885823B (zh) 一种长链非编码RNA猪Lnc-000649及其应用
CN105063023B (zh) 一种锌指核酸酶介导的猪mstn基因突变序列及其应用
CN110511903A (zh) 一种miR-21-5p靶向Smad7调控猪卵巢颗粒细胞的分子机制研究方法及其应用
CN105648050B (zh) 一种快速鉴定山羊绒周期发育的方法及其分子靶标和应用
CN109988846B (zh) 一种适于红螯螯虾性腺组织mRNA石蜡切片原位杂交的方法
WO2018196143A1 (fr) Procédé d'accroissement du rendement en porc maigre par retard du moment d'expression d'un gène
CN116355898B (zh) miRNA-133在调节绵羊胚胎毛囊发育中的应用
CN115851972B (zh) 一种绵羊毛囊发育标志物miR-23b及其应用
CN104232648A (zh) 一种靶向脂肪酸合成酶的调控因子及其应用
CN114292850B (zh) 一种与山羊成肌细胞增殖和分化相关的circRNA及其应用
CN113832235A (zh) 一种与仔猪细菌性腹泻抗性相关的lncRNA标志物及其应用
CN113005095A (zh) 基于circ-Atp5BP调控卵巢颗粒细胞的方法
CN113583964A (zh) 利用miR-212及其靶基因调控卵巢颗粒细胞的方法
CN107881237B (zh) 肺癌诊断标记物microRNA-4317及在药物和诊断试剂盒中的应用
CN105435242A (zh) 人miR-23a在制备细胞生长和/或衰老调控剂中的应用
CN110592232A (zh) 一种与猪肉pH值性状相关的分子标记及应用
CN112574996B (zh) 一种长链非编码rna aagncr及其应用
CN114774417B (zh) 促日本血吸虫宿主肝脏纤维化的miRNA分子及miRNA拮抗剂和应用
CN114561466B (zh) Fto在诊治骨肉瘤中的应用
CN116426525B (zh) 一种抑制鸡Wnt3a基因表达的ASO及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17906789

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 05.03.2020)

122 Ep: pct application non-entry in european phase

Ref document number: 17906789

Country of ref document: EP

Kind code of ref document: A1