CN102382827A - shRNA segment for efficiently inhibiting expression of myostatin gene and construction method of transgenic sheep - Google Patents
shRNA segment for efficiently inhibiting expression of myostatin gene and construction method of transgenic sheep Download PDFInfo
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- CN102382827A CN102382827A CN2011103138999A CN201110313899A CN102382827A CN 102382827 A CN102382827 A CN 102382827A CN 2011103138999 A CN2011103138999 A CN 2011103138999A CN 201110313899 A CN201110313899 A CN 201110313899A CN 102382827 A CN102382827 A CN 102382827A
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Abstract
The invention discloses a shRNA segment for efficiently inhibiting the expression of a myostatin gene and a construction method of a transgenic sheep. The construction method of a transgenic sheep comprises the following steps: using the shRNA of a targeted sheep myostatin gene to construct a shRNA expression vector, transfecting a sheep fibroblast, then carrying out resistance screening to obtain a sheep fibroblast integrated with the shRNA expression vector, and obtaining the transgenic sheep by utilizing a somatocyte cloning technology and an embryonic implantation technology. Tests prove that the expression of the myostatin gene of the transgenic sheep is silenced, and the expression quantity is obviously decreased.
Description
Technical field
The present invention relates to the shRNA fragment of a kind of efficient inhibition myostatin genetic expression and utilize the RNA perturbation technique to make up the method for the reticent Transgenic Sheep of myostatin gene, belong to biological technical field.
Background technology
Improve animal meat productivity and lean ratio is the target that Animal Genetics scholar, animal reproduction scholar and Animal nutrition scholar pursue always.Can improve animal meat productivity and lean ratio through improving the feed nutrition ratio with technique means such as using additive, but effect is not remarkable limitedly, but also has some food-safety problems.Therefore, it is significant to Developing of Animal Industry to seek an approach that improves the animal meat production safely and effectively.
(myostatin MSTN), belongs to the growth and differentiation factor beta superfamily member to myostatin, is the main negative regulatory factor of muscle growth differentiation.Hyperplasia and hypertrophy have widely taken place in the muscle of myostatin gene knock-out mice, and the Skelettmuskel weight of mouse increases 2-3 doubly.But other phenotypes do not have significant difference with wild-type.World-renowned Belgian blue ox and Piemonte ox with " two flesh " phenotype all is because due to this gene generation spontaneous mutation, their meat yield will exceed about 30% than wild ox.Simultaneously, " two flesh " ox has the muscle group hypertrophy, and subcutaneous lipids is few, and more fatty deposits is arranged in the medium phenotypic characteristic of myofiber.These researchs show and utilize gene engineering method blocking-up myostatin gene function, are a kind of animal meat yield and new ways of improving meat matter of improving.
The RNA perturbation technique is a kind of new technology in the transcriptional level inhibition of gene expression, through express can with target gene paired siRNA, can realize the silence of target gene.At present, RNA disturbs silencer to be expressed on the different genes of various species and has obtained fully proving, and realizes using.
Summary of the invention
The objective of the invention is to design, filter out the shRNA fragment of the one section efficient myostatin of inhibition gene.This fragment called after shMSTN3, shMSTN3 can significantly suppress the expression of myostatin in cell.
Another object of the present invention is to provide a kind of RNA of utilization perturbation technique to make up the method for the reticent Transgenic Sheep of myostatin gene.Expression vector transfection sheep inoblast with the shMSTN3 that makes up; Go out the low sheep inoblast of myostatin clpp gene with the cell screening technology screening, and utilize body-cell neucleus transplanting and embryo transfer technology to prepare the Transgenic Sheep of myostatin gene silencing.
The above-mentioned RNA perturbation technique that utilizes makes up the reticent Transgenic Sheep method of myostatin gene, realizes through following technical scheme:
(1) the segmental screening of shRNA of inhibition myostatin genetic expression
Designed the shRNA fragment of 3 target myostatin genes; Behind these 3 shRNA fragment transfectional cells; Expression with myostatin in the quantitative fluorescent PCR pair cell detects, and the result shows that shMSTN3 can significantly suppress the expression of myostatin in cell.
(2) structure of sheep shRNA specific expression carrier
Use the method that merges PCR, obtain the amalgamation and expression box (U6-shMSTN3) of promotor U6 and shMSTN3, should express box and be cloned among the pMD18-T, produce the pU6-shMSTN3 carrier.
(3) the fibroblastic structure of myostatin gene silencing sheep
With pU6-shMSTN3 transfection sheep inoblast, the cell with after the Xin Meisu screening transfection screened after 7-10 days, with the resistant cell clone enlarged culturing that forms.Utilize neomycin gene primer pair cell clone to carry out PCR and identify, thereby obtain myostatin gene silencing sheep inoblast.
(4) structure of myostatin gene silencing Transgenic Sheep
Utilizing somatic cell nuclear transfer technique is the reconstruct embryo with obtaining cell construction in (3), again through embryo transfer technology with embryo transfer in the replace-conceive ewe, the lamb of birth is carried out PCR identifies, confirm successfully to make up the Transgenic Sheep of myostatin gene silencing.
The present invention relates to utilize the expression of the reticent sheep myostatin gene of RNA perturbation technique in somatocyte.Utilizing somatic cell nuclear transfer technique, making up a kind of myostatin gene silencing Transgenic Sheep, this Transgenic Sheep has the lean ratio height, the characteristics fast of growing.
The present invention utilizes RNA perturbation technique and somatic cell clone technique to make up the low transgene clone sheep of myostatin clpp gene; Explore the sheep that preparation has " two flesh " proterties, for the final improved seeds domestic animal of cultivating meat productivity, lean ratio and meat quality better lays the foundation.
Description of drawings
Fig. 1 pU6-shMSTN3 carrier collection of illustrative plates;
The fibroblastic PCR of sheep that Fig. 2 is integrated with shMSTN3 identifies;
The PCR of Fig. 3 Transgenic Sheep identifies.
Embodiment
Below in conjunction with specific embodiment, the present invention is elaborated.
The embodiment 1 efficient segmental screening of shRNA that suppresses myostatin genetic expression
1.1 the segmental design of target myostatin gene shRNA and synthetic
The siRNA of 3 target myostatin genes of applying biological information software design; The siRNA sequence is added loop and terminator sequence; Convert shRNA into, be respectively shMSTN1, shMSTN2, shMSTN3, with shMSTN1, shMSTN2; The shMSTN3 subclone obtains the pGenesil-shRNA expression vector to pGenesil-1 (brilliant match company) carrier.
1.2shRNA transfection sheep inoblast
Previous day is cultivated the sheep inoblast with DMEM (10%FBS) in transfection in 12 orifice plates, when treating that cell grows into the 80-90% abundance, with liposome 2000, with the shRNA expression vector transfection sheep inoblast in 1.8ug/ hole.After the transfection 48 hours, extract total RNA of transfectional cell and control cells ,-80 ℃ of preservations.
1.3 the expression of fluorescence quantitative PCR detection myostatin
Myostatin expression conditions in the cell of fluorescence quantitative PCR detection transfection shRNA and the control cells (non-transfected cells).With random primer and the reverse transcription test kit total RNA to the reverse transcription cell, the eDNA of acquisition is used for quantitative fluorescent PCR with the nucleic acid quantification appearance after quantitatively.Amplification myostatin gene use MSTN-F (5 '-ATCCGATCTCTGAAACTTGACAT-3 ') and MSTN-R (5 '-AGTCCTTCTTCTCCTGGTTCTG-3 ') primer.Internal control gene GAPDH use GAPDH-F (5 '-GGCGCCAAGAGGGTCAT-3 ') and GAPDH-R (5 '-GTGGTTCACGCCCATCACA-3 ') primer.Quantitative PCR has used SYBR Green dyestuff, after reaction system is 95 ℃/5min; 95 ℃/15s, 56 ℃/15s, 72 ℃/10s is totally 30 circulations; The Ct value of myostatin is by the GAPDH homogenization.Myostatin relative expression level is quantitative through Δ Δ CT numerical method.The result shows, shMSTN1, shMSTN2 and shMSTN3 suppress the expression of 81%, 56% and 90% myostatin transcriptional level respectively.Wherein shMSTN3 suppresses most effective, compares with control group myostatin genetic expression to have significant difference.
The structure of embodiment 2 sheep shRNA specific expression carriers
2.1shRNA the structure of expression vector
Referring to Fig. 1, use and merge round pcr, U6 promotor and shMSTN3 fragment are realized merging.The PCR reaction conditions is: behind the 95 ℃/5min; 95 ℃/30s, 58 ℃/30s, 72 ℃/1min is totally 30 circulations, and 72 ℃ of 7min. identify PCR product row agarose gel electrophoresis.Reclaim test kit with gel and reclaim the U6-shMSTN3 gene fragment.To reclaim the product subclone to the pMD18-T carrier, produce the pU6-shMSTN3 carrier.
The fibroblastic structure of embodiment 3myostatin gene silencing sheep
3.1 the fibroblastic preparation of sheep
Sheep embryo when gathering fertilization 35-45d,, aseptic taking-ups fetus, after containing two anti-PBS liquid washings 3-4 time, removal head, tail, four limbs and internal organ part, remainder is put into the 10mm plate, is cut into 1mm
3Organize fragment, after placing 4 hours in the incubator, add the DMEM nutrient solution 39.5 ℃, 5%CO2 saturated humidity in the CO2 incubator that contain 20% foetal calf serum and cultivate, cultivate behind the 24h according to the cell growing state and carry out that going down to posterity of cell cultivated or frozen.
3.2 fibroblastic transfection of sheep and screening
1d before the transfection, with the sheep fetal fibroblast with 5 * 10
5The density of/mL adds and contains in DMEM (antibiotic-free) substratum of 10% calf serum, is inoculated in 6 orifice plates.When treating that cell 80%～90% merges, substratum is changed to the DMEM substratum of unparalleled anti-, serum-free, with pU6-shMSTN3 carrier transfection sheep fetal fibroblast.Behind the transfection 36h, the dilution proportion that 1 hole passes the 4-6 hole goes down to posterity, and screens with 500 μ g/mL G418 in 24 hours.Screen after 3-5 days, the part cell can begin the death that comes off.Begin to occur cell clone after 10 days, after the cell enlarged culturing, frozen subsequent use.
3.3 the PCR of positive cell identifies
With Xin Meisu neo gene order design primer, be used to detect the sheep fibroblast cloning.Primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized, and primer sequence is neo-P1 (ATTCGGCTATGACTGGGCACAAC) and neo-P2 (ACACCCAGCCGGCCACAGT).As primer, be template with the positive cell lysate, carry out pcr amplification, reaction conditions is: 95 ℃ of 5min, 95 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 2min40sec circulate 36 times, 72 ℃ of 10min, PCR product length is 585bp.The cell clone that can pcr amplification goes out the 585bp exogenous genetic fragment is identified as the transgenic positive cell, is contrast with water as pcr template simultaneously.Referring to Fig. 2, the result obtains the transgenic cell clone that 4 strains are integrated with foreign gene altogether.
The structure of embodiment 4myostatin gene silencing Transgenic Sheep
4.1 oocyte maturation is cultivated:
The sheep ovary is collected in the slaughterhouse in a large number; After transporting the laboratory back, form dirt settling such as tissue through saline water washing, expel stagnation after, adopt the sectility method from the ovarian follicle of 2-5mm; Separation configuration is complete; Tenuigenin is fine and close, the color and luster homogeneous, and cumulus cell is at the ovarian cumulus more than 3 layers or 3 layers-ovocyte complex body (COCs).At 38.5 ℃, 5%CO2 cultivates under the saturated humidity, adopts droplet culture systems maturation in vitro in oocyte maturation liquid OM to cultivate 22-24h.
4.2 nuclear transplantation:
Remove granulosa cell with 0.1% Unidasa piping and druming digestion, the ovocyte of selecting to discharge first polar body carries out stoning in cytochalasin, donorcells (sheep inoblast) is injected under the zona pellucida of enucleation oocyte.
4.3 electricity merges
Put vertical with the sense of current contact surface of donorcells and enucleation oocyte.It is 120v/mm that the electricity that electricity merges swashs parameter, and 3 times electric pulse inductor cell merges with enucleation oocyte mutually.
4.4 the embryo activates and cultivates
6-DMAP activates the reconstruct embryo with the Ionomycin associating, transfers in the SOFaa nutrient solution and cultivates, and every 48h half amount is changed liquid 1 time, inspection spilting of an egg rate behind the 48h, 7d record of search blastaea developmental state.
4.5 embryo transfer
Select to grow the good 2-8 cell stage transgene clone embryo of form, through first bend of surgical graft to the 3rd day nearly fimbriae tubae in acceptor sheep both sides of spontaneous estrus.With quadrat method vitelline sphere and blastaea are transplanted to spontaneous estrus 6d, 7d, 8d acceptor sheep horn of uterus.Acceptor sheep operation back is observed it and is returned the feelings situation, and the 30-45d of sheep behind operation transplantation to not returning feelings carries out the pregnant inspection of rectum with B ultrasonic, to identify acceptor sheep gestation situation.
The identification and analysis of embodiment 5myostatin gene silencing Transgenic Sheep
5.1 Transgenic Sheep PCR identifies
Extract the sheep genome with a day root DNA genome test kit, carry out pcr amplification with Xin Meisu neo gene order primer neo-P1 (ATTCGGCTATGACTGGGCACAAC) and neo-P2 (ACACCCAGCCGGCCACAGT), reaction conditions is: 95 ℃ of 5min; 95 ℃ of 30sec; 60 ℃ of 30sec, 72 ℃ of 2min40sec circulate 36 times; 72 ℃ of 10min, PCR product length is 585bp.The sheep that can pcr amplification goes out the 585bp exogenous genetic fragment is identified as transgene clone sheep, is that pcr template is contrast with non-transgenic sheep genomic dna and water simultaneously.Referring to Fig. 3, the result obtains 2 transgene clone sheep.
5.2 the expression analysis of Transgenic Sheep myostatin
Extract the total RNA of sheep muscle tissue, carry out reverse transcription with random primer and reverse transcription test kit, the cDNA of acquisition is used for quantitative fluorescent PCR with the nucleic acid quantification appearance after quantitative.Amplification myostatin gene use MSTN-F (5 '-ATCCGATCTCTGAAACTTGACAT-3 ') and MSTN-R (5 '-AGTCCTTCTTCTCCTGGTTCTG-3 ') primer.Internal control gene GAPDH use GAPDH-F (5 '-GGCGCCAAGAGGGTCAT-3 ') and GAPDH-R (5 '-GTGGTTCACGCCCATCACA-3 ') primer.Quantitative PCR has used SYBR Green dyestuff, after reaction system is 95 ℃/5min; 95 ℃/15s, 56 ℃/15s, 72 ℃/10s is totally 30 circulations; The Ct value of myostatin is by the GAPDH homogenization.Myostatin relative expression level is quantitative through Δ Δ CT numerical method.The result shows that the myostatin gene expression amount has reduced by 71% and 49% respectively in 2 transgene clone sheep.
Should be understood that, concerning those of ordinary skills, can improve or conversion, and all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention according to above-mentioned explanation.
Claims (4)
1. the shRNA fragment that efficiently suppresses myostatin genetic expression: shMSTN3, its nucleotides sequence is classified as: 5 '-CAAAGATGCTATAAGACAATTCAAGAGATTGTCTTATAGCATCTTTGTTTTTTGGA ACG-3 ' 3 '-GTTTCTACGATATTCTGTTAAGTTCTCTAACAGAATATCGTAGAAACAAAAAACCT TGC-5 '.
2. a method of utilizing the RNA perturbation technique to make up the reticent Transgenic Sheep of myostatin gene is characterized in that, comprises following key step:
(1), identifies the shRNA fragment that can efficiently suppress myostatin genetic expression a: shMSTN3 according to the sequence of sheep myostatin gene;
(2) made up the shRNA expression vector of target myostatin gene;
(3) transfection sheep inoblast, acquisition is integrated with the sheep inoblast of shRNA expression vector;
(4) body-cell neucleus transplanting and embryo transfer obtain to be integrated with the Transgenic Sheep of shRNA expression vector;
(5) utilize PCR, fluorescence quantitative RT-RCR confirms that the myostatin gene expression amount of Transgenic Sheep significantly descends.
3. method according to claim 2 is characterized in that: in said step (1), and the said shRNA fragment that can efficiently suppress myostatin genetic expression: shMSTN3, it is positioned at the 219-237bp position of myostatin gene, and target site length is 19bp.
4. method according to claim 2 is characterized in that: in said step (2), clone U6 promotor makes up the shRNA expression vector with this promotor, realizes the shRNA specifically expressing of target myostatin gene.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104232670A (en) * | 2014-08-25 | 2014-12-24 | 中国水产科学研究院黑龙江水产研究所 | Carp-MSTN (myostatin)-based interfere vector constructed by adopting fish transgenic method and construction method of carp-MSTN-based interfere vector |
| CN104894266A (en) * | 2015-06-08 | 2015-09-09 | 中国农业科学院兰州畜牧与兽药研究所 | Specific primer for detecting mRNA expression levels of MSTN genes of cows and fluorescent quantitative detecting kit |
| WO2016013956A1 (en) * | 2014-07-21 | 2016-01-28 | Освилт Корпорейшн Лимитед | Method of correcting pathological human skin conditions related to aging |
| RU2574905C1 (en) * | 2014-07-21 | 2016-02-10 | Освилт Корпорейшн Лимитед | Method for correction of age-related pathological skin conditions |
| CN112913778A (en) * | 2021-01-29 | 2021-06-08 | 石河子大学 | Construction method of sheep chronic inflammation model |
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| CN102021201A (en) * | 2010-09-06 | 2011-04-20 | 南京农业大学 | Preparation method for myostatin knock-out pig |
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| CN102021201A (en) * | 2010-09-06 | 2011-04-20 | 南京农业大学 | Preparation method for myostatin knock-out pig |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016013956A1 (en) * | 2014-07-21 | 2016-01-28 | Освилт Корпорейшн Лимитед | Method of correcting pathological human skin conditions related to aging |
| RU2574905C1 (en) * | 2014-07-21 | 2016-02-10 | Освилт Корпорейшн Лимитед | Method for correction of age-related pathological skin conditions |
| CN104232670A (en) * | 2014-08-25 | 2014-12-24 | 中国水产科学研究院黑龙江水产研究所 | Carp-MSTN (myostatin)-based interfere vector constructed by adopting fish transgenic method and construction method of carp-MSTN-based interfere vector |
| CN104894266A (en) * | 2015-06-08 | 2015-09-09 | 中国农业科学院兰州畜牧与兽药研究所 | Specific primer for detecting mRNA expression levels of MSTN genes of cows and fluorescent quantitative detecting kit |
| CN104894266B (en) * | 2015-06-08 | 2018-03-06 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of specific primer and fluorescence quantitative detection kit for detecting ox MSTN mrna expressions |
| CN112913778A (en) * | 2021-01-29 | 2021-06-08 | 石河子大学 | Construction method of sheep chronic inflammation model |
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Application publication date: 20120321 |