WO2012151718A1 - Promoteur de gène de myostatine de porc et ses applications - Google Patents

Promoteur de gène de myostatine de porc et ses applications Download PDF

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Publication number
WO2012151718A1
WO2012151718A1 PCT/CN2011/000831 CN2011000831W WO2012151718A1 WO 2012151718 A1 WO2012151718 A1 WO 2012151718A1 CN 2011000831 W CN2011000831 W CN 2011000831W WO 2012151718 A1 WO2012151718 A1 WO 2012151718A1
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WO
WIPO (PCT)
Prior art keywords
sequence
dna molecule
promoter
dna
cell
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PCT/CN2011/000831
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English (en)
Chinese (zh)
Inventor
毕延震
郑新民
乔宪凤
刘西梅
张立苹
华文君
李莉
肖红卫
周荆荣
魏庆信
Original Assignee
湖北省农业科学院畜牧兽医研究所
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Application filed by 湖北省农业科学院畜牧兽医研究所 filed Critical 湖北省农业科学院畜牧兽医研究所
Priority to PCT/CN2011/000831 priority Critical patent/WO2012151718A1/fr
Priority to CN201180004689.XA priority patent/CN102725408B/zh
Publication of WO2012151718A1 publication Critical patent/WO2012151718A1/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
    • C12N15/63—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
    • 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

Definitions

  • the invention relates to the field of biotechnology, in particular to a porcine myostatin gene promoter and application thereof. Background technique
  • transgenic technology In the past decade, the rapid development and wide application of transgenic technology, especially the combination of gene targeting and somatic cell cloning technology, has made the genetic modification of transgenic animals a reality.
  • the key to this technology is to allow site-specific integration and stable expression of foreign genes on the genome of transgenic animals, and the expression of foreign genes is closely related to the transcriptional activity of their promoters. Therefore, obtaining a promoter with stable expression activity will greatly promote transgenic animal research.
  • the types and quantities of promoters currently used in transgenic animals are very limited, mainly from viral promoters, such as cytomegalovirus (CMV), simian virus 40 (SV40), simple blisters.
  • a constitutive promoter such as herpes simplex virus HSV.
  • Another advantage of using the endogenous promoter of transgenic animals is that the "in situ" site-specific integration of the foreign gene can be achieved by gene targeting, driven by the endogenous promoter "in situ” to express the foreign gene. Control at a certain physiological level. Therefore, research in this area as soon as possible has important application value for the research and production of GM animals.
  • the myostatin gene was first cloned in mice by McPherron et al. in 1997. This gene belongs to the TGF- ⁇ family and is a transforming growth factor. Gene knockout proves that the inactivation of this gene causes muscle tissue hyperplasia and weight gain in mice; and the mice can survive normally and have fertility. Subsequently, it was found in cattle, sheep and other animals that the main function of the myostatin gene is to negatively regulate muscle growth and development, and the inactivation of myostatin does not cause abnormalities in the physiological functions of the above animals. Therefore, myostatin is a candidate modified gene for transgenic animals that has proven to be a phenotypic effect and relatively safe.
  • the DNA molecule provided by the present invention which is derived from a pig, is a DNA molecule according to any one of the following 1) -4):
  • the DNA molecule shown in 4) is the DNA molecule shown in SEQ ID NO: 1 in the Sequence Listing.
  • the stringent conditions were hybridization in a 6XSSC, 0.5% SDS solution at 65 °C followed by one wash with 2 XSSC, 0.1% SDS and 1X SSC, 0.1% SDS.
  • a recombinant vector, expression cassette, transgenic cell line or recombinant strain containing the DNA molecule is also within the scope of protection of the present invention.
  • the recombinant vector is as follows 1) or 2):
  • Primer pairs that amplify the DNA molecule are also within the scope of the invention.
  • the primer pair is as follows 1) or 2) :
  • the animal cell is a mouse cell or a human cell.
  • the mouse cell is a mouse myoblast
  • the human cell is a human cervical cancer cell.
  • the mouse myoblast is a C2C12 cell
  • the human cervical cancer cells are Hela cells.
  • Figure 1 is a schematic diagram of the tissue structure and promoter candidate regions of porcine myostatin gene.
  • Figure 2 shows the amplification of the porcine myostatin gene promoter.
  • Figure 3 is a porcine myostatin gene promoter reporter vector digestion verification
  • Figure 4 shows the cloning, mutation and activity assay of porcine myostatin gene promoter.
  • Figure 5 is a functional verification of the expression of green fluorescent protein driven by the porcine myostatin gene promoter.
  • Figure 6 shows the activity assay of porcine myostatin gene promoter reporter vector in human cultured cells. The best way to implement the invention
  • pGL3-bas ic both purchased from pGL3-bas ic, pGL3-p:romote:r, Promega (USA) pGL3- promoter expression firefly incinerated pRL-TK reporter vector photozyme, and pRL-TK reporter vector expresses Renilla fluorescence Prime enzyme reporter gene high fidelity polymerase K0D Toyobo (Japan) amplification of porcine myostatin gene promoter plus sub
  • ThermoCycler PCR Thermo- Fisher nucleic acid PCR amplification Thermostat, shaker Shanghai Zhicheng Bacterial culture
  • Each sample was added with 300 ⁇ l DNA extraction buffer (Tris-Hcl 10 mM, EDTA 10 mM, SDS 2%, NaCl 300 mM, pH 8.0), 8 ⁇ l proteinase K (10 mg/ml), and digested at 55 ° C for 6 hours.
  • DNA extraction buffer Tris-Hcl 10 mM, EDTA 10 mM, SDS 2%, NaCl 300 mM, pH 8.0
  • 8 ⁇ l proteinase K 10 mg/ml
  • the mixture was extracted twice with phenol, centrifuged at 10, OOO rpm for 10 minutes, and the supernatant was removed to remove the protein and the lower phenol.
  • the extract was again extracted with chloroform/isoamyl alcohol phenol, and the supernatant was taken by centrifugation at 10, OOO rpm for 10 minutes.
  • the precipitate was washed once with 75% cold ethanol, and centrifuged for 5 minutes to remove the ethanol to take a precipitate.
  • the precipitate is fully dried at 4 ° C, added with three distilled water ⁇ ⁇ ⁇ ⁇ , electrophoresis check quantitative, -20 ° C frozen storage available
  • the pig genomic DNA was obtained by PCR amplification.
  • M is the molecular weight standard of lkb DNA, and the length is 0.5 kb, lkb, 1. 5 kb, 2 kb, 3 kb, 4 kb, 5kb, 6kb, 8kb, lOkb, PCR amplification to obtain 1. lkb fragment.
  • the PCR product was sent to the Huada gene for sequencing, and the sequencing primer was Glprimer2.
  • the PCR product had the nucleotide shown in SEQ ID NO: 1 in the sequence listing, and the gene of the PCR product was named MSTN.
  • 1031-1038 is the 5' end transcriptional element TATA box (5' end TATA box), from the 5' end 1056-1063 to the 3' end transcriptional element TATA box (3 ' end TATA box) ), from the 5' end to the 991-996 position for the 3'-end transcriptional element CAAT box.
  • Sequence 1 can also be synthesized manually.
  • FIG. 1 is a schematic diagram showing the tissue structure and promoter candidate region of porcine myostatin gene. It can be seen that the porcine myostatin gene contains three exons and two introns, and the full length is 3789 bp. The exon lengths were 373 bp, 374 bp and 381 bp, respectively; the intron lengths were 1809 bp and 1980 bp, respectively.
  • the sequence immediately upstream of exon 1 contains a TATA cassette (2) and a CAAT box (1), and a MEF (myocyte enhancer factor) binding sequence. 3) Cloning and identification of porcine myostatin gene promoter fragment
  • reaction conditions were: 16 ° C, and allowed to stand overnight.
  • the ligation product was transformed into Escherichia coli DH5a according to standard methods, and cultured overnight at 37 ° C to obtain a transformant.
  • the transformants obtained above were identified by colony PCR using the MSTN-F and MSTN-R primers, and the recombinant which can obtain the lkb amplification product was a positive plasmid;
  • the positive plasmid was digested with Mlu I and Bgl II, and pGL3-basi C was used as a control.
  • the results are shown in Fig. 3.
  • lanes 1, 3 are pGL3-basic
  • 2, 4 is a p-port GL3_basic plasmid digested with Mlul and BglU; it can be seen that the pGL3-basi C plasmid has no insert, so the plasmid is electrophoretically displayed after double digestion. It is a single strip with a length of 4. 8 kb, which is consistent with the theoretical analysis.
  • Lanes 5, 7, 9 are recombinants, 6, 8, and 10, respectively, 5, 7, and 9 recombinants were digested with Mlul and BglU; it can be seen that lanes 6, 8, 10 were digested and electrophoresed. The results showed that 1. lkb (shown by the black arrow on the right, the brightness was weak) and the 4.8 kb two fragments were positive plasmids, which was consistent with the theoretical analysis, indicating that lanes 5, 7, and 9 were positive plasmids.
  • the plasmid which was positive by PCR and restriction enzyme digestion was sent for sequencing. The result was that the plasmid was inserted into the plasmid between the Mlu I and Bgl II cleavage sites of pGL3-baisc, and the plasmid was named. For pGL3-MSTN.
  • mutant vector adopts a Dpnl-mediated point mutation method
  • the reaction system of the mutant PCR is as follows:
  • reaction conditions are:
  • Sequence 2 or Sequence 3 can also be synthesized manually.
  • the reaction was carried out in a water bath at 37 ° C for 2 hours.
  • the ⁇ digested product was separately transformed into E. coli competent cells DH5 a according to standard procedures.
  • the linear digested product has a homology arm at both ends, which can be cyclized in E. coli. This is the principle of the classical mutation method. Developed by Stratagene, USA, three transformants were obtained: positive plasmid 1, positive plasmid 2, transformant 3.
  • the plasmid of transformant 1 was extracted and sent for sequencing. As a result, the plasmid contained the sequence 2 in the sequence listing, and the plasmid was inserted between the Mlu I and Bgl II cleavage sites of pGL3-basic.
  • the plasmid was named PGL3-TATA1;
  • the plasmid of transformant 2 was extracted and sent for sequencing.
  • the plasmid contained the sequence 3 in the sequence listing, which was obtained by inserting sequence 3 in the sequence listing between the Mlu I and Bgl II cleavage sites of pGL3-basic.
  • the plasmid of transformant 3 was extracted and sent for sequencing.
  • the plasmid contained the sequence 4 in the sequence listing, which was obtained by inserting the sequence 4 in the sequence listing between the Mlu I and Bgl II cleavage sites of pGL3-basic.
  • the plasmid was named pGL3-3-boxes.
  • transfection complexes were prepared according to the system listed in the table, and dropped into 24-well plates, supplemented with 400 ⁇ l complete medium, placed. Incubate at 37 ° C under 5% CO 2 . Cells were harvested 48 h after transfection. Luciferase activity assays were performed according to Promega's DLR Assay kit instructions.
  • the transfection system is as follows:
  • FIG. 4 A schematic diagram of the partial structure of each reporter vector and the relative ratio of enzyme activity are shown in Figure 4.
  • the TATA box and the CAAT box are key components of the transcription of the porcine myostatin gene, and the deletion of the three components can lead to the report. Loss of gene activity (not significantly different from the enzyme activity of the negative control pGL3-basi C ).
  • Two TATA boxes were deleted, respectively, and they were all able to efficiently drive transcription. It was proved that these two TATA boxes are effective elements for the transcription initiation of porcine myostatin gene (mutant and wild type enzyme activities are almost identical), but the two are not superimposed. effect.
  • *, p ⁇ 0.05 the difference is significant, indicating that the DNA molecule shown in SEQ ID NO: 2 and the DNA molecule shown in SEQ ID NO: 3 are also promoters.
  • ThermoCycler PCR Thermo-Fi she nucleic acid PCR amplification instrument r
  • the pGL3-MSTN plasmid DNA was used as a template, and a specific primer with a predetermined restriction site (promoter upstream primer and promoter downstream primer) was used to amplify the porcine myostatin gene promoter sequence.
  • the reaction system was:
  • reaction conditions are:
  • the lj PCR product was subjected to 1% agarose gel electrophoresis to obtain a 1122 bp PCR product.
  • reaction conditions were: 16 ° C, and allowed to stand overnight.
  • the ligation product was transformed into E. coli DH5a according to standard methods and cultured overnight at 37 ° C to obtain a transformant.
  • the plasmid of the transformant was extracted, and the primer upstream of the promoter and the primer downstream of the promoter were subjected to PCR amplification to obtain a 1122 bp fragment as a positive plasmid.
  • the positive plasmid was digested with Nhel to obtain 1122 bp.
  • the fragment was further proved to be a positive plasmid, and the plasmid which was positive by PCR and restriction enzyme digestion was sent for sequencing, and the result was that the plasmid 1 was inserted between the sel and Nhel cleavage sites of pEGFP-Cl.
  • the plasmid was named pMSTN-EGFP.
  • the constructed pMSTN-EGFP reporter vector was transfected (transfection method is described in detail in the kit). Mouse myoblast C2C12 was observed 24 hours later, and pEGFP-Cl was used as a control.
  • the transfection system is as follows:
  • transfected complexes were prepared separately in the system listed, and were added to a 24-well plate, supplemented with 400 ⁇ l of complete medium, and cultured at 37 ° C and 5% CO 2 . Cells were harvested 48 h after transfection. Luciferase activity assays were performed according to Promega's DLR M Assay kit instructions.
  • the transfection system is as follows:
  • porcine myostatin gene promoter reporter vector PGL3-MSTN was transfected into human cervical cancer Hela cells, and the transcriptional activity of the porcine myostatin gene promoter in human cells was examined by a reporter gene. The results showed that the cloned promoter fragment was highly active in human cells, demonstrating that the identified porcine myostatin gene promoter is efficiently expressed in mammalian cells.
  • the experiments of the present invention prove that the promoter provided by the present invention can drive the expression of the reporter gene firefly luciferase, and can also be used to construct the promoter into the reporter vector and then transfect the pig source and the human cultured cell.
  • reporter gene testing the activity and efficiency of the promoter are identified by accurate quantitative methods, and the problems of introducing foreign genes after the promoter, solving the unsteadiness of the expression of foreign genes in transgenic pigs, and the unpredictability of positional effects are provided. A reliable and valuable genetic resource.

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Abstract

La présente invention concerne un promoteur de gène de myostatine de porc et ses applications. Le fragment d'ADN décrit dans la présente invention qui est dérivé de porcs est l'une quelconque des molécules d'ADN suivantes 1) à 4) : 1) la molécule d'ADN de SEQ ID NO: 2 décrite dans la liste de séquences ; 2) la molécule d'ADN de SEQ ID NO: 3 décrite dans la liste de séquences ; 3) la molécule d'ADN qui s'hybride avec la séquence d'ADN dans 1) ou 2) dans des conditions strictes et a une fonction de promoteur ; 4) la molécule d'ADN qui a plus de 90 % d'homologie avec la séquence d'ADN dans 1) ou 2) et a une fonction de promoteur. Les essais dans la présente invention montrent que le promoteur décrit dans la présente invention peut induire l'expression de gène rapporteur de luciférase de luciole. Le promoteur peut également être intégré dans le vecteur rapporteur et ensuite le vecteur est transfecté dans des cellules de porc et humaines cultivées, et l'activité et l'efficacité du promoteur sont identifiées par des procédés quantitatifs précis par test de gène rapporteur.
PCT/CN2011/000831 2011-05-11 2011-05-11 Promoteur de gène de myostatine de porc et ses applications WO2012151718A1 (fr)

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CN201180004689.XA CN102725408B (zh) 2011-05-11 2011-05-11 猪肌抑素基因启动子及其应用

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113151502A (zh) * 2021-05-18 2021-07-23 华中农业大学 一种与猪活体产肉性状关联的遗传标记及应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103382505B (zh) * 2013-08-07 2015-09-30 贵州大学 利用双荧光素酶报告基因检测启动子活性的方法
CN105779592B (zh) * 2016-03-22 2019-05-28 国家粮食局科学研究院 用于谷蠹鉴定及其遗传学分析的微卫星标记

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000001810A1 (fr) * 1998-07-07 2000-01-13 New Zealand Pastoral Agriculture Research Institute Limited Nouvelles sequences promotrices de gene myostatine
WO2007067616A2 (fr) * 2005-12-06 2007-06-14 Amgen Inc Utilisations d'antagonistes de la myostatine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000001810A1 (fr) * 1998-07-07 2000-01-13 New Zealand Pastoral Agriculture Research Institute Limited Nouvelles sequences promotrices de gene myostatine
WO2007067616A2 (fr) * 2005-12-06 2007-06-14 Amgen Inc Utilisations d'antagonistes de la myostatine

Non-Patent Citations (4)

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Title
DATABASE GENBANK 2 April 2007 (2007-04-02), STINCKENS,A. ET AL., accession no. F490989 *
STINCKENS,A. ET AL.: "Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity.", ANIMAL GENETICS., vol. 39, no. 6, December 2008 (2008-12-01), pages 586 - 596 *
XU, CHENG ET AL.: "Analysis of myostatin gene structure, expression and function in zebrafish.", THE JOURNAL OF EXPERIMENTAL BIOLOGY., vol. 206, 2003, pages 4067 - 4079 *
YU, ZHENGQUAN ET AL.: "Comparative analysis of the pig BAC sequence involved in the regulation of myostatin gene.", SCIENCE IN CHINA SER. C LIFE SCIENCES., vol. 48, no. 2, 2005, pages 168 - 180 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113151502A (zh) * 2021-05-18 2021-07-23 华中农业大学 一种与猪活体产肉性状关联的遗传标记及应用
CN113151502B (zh) * 2021-05-18 2022-06-03 华中农业大学 一种与猪活体产肉性状关联的遗传标记及应用

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