CN105821116A - Directional knockout on sheep MSTN gene and detection method for influence thereof on myogenic differentiation - Google Patents
Directional knockout on sheep MSTN gene and detection method for influence thereof on myogenic differentiation Download PDFInfo
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Abstract
The invention discloses directional knockout on sheep MSTN gene by applying the CRISPR/Cas9 technology, and a method for verifying the impact effect of directional knockout on skeletal muscle satellite cell differentiation. The concrete contents are as follows: firstly, target gene cloning; secondly, gRBA design and synthesis; thirdly, CRISPR/Cas9 gene knockout vector construction; fourthly, CRISPR/Cas9 gene knockout vector exogenous activity detection; fifthly, CRISPR/Cas9 gene knockout vector endogenous activity detection; sixthly, CRISPR/Cas9 gene knockout vector knockout effect detection. The method has the following advantages: firstly, the experimental period is shorter; secondly, the method is simple and practicable, has high repeatability, and can be performed in common laboratories; thirdly, active vector constructed through the method is small in toxic and side effects, and can be applied to the preparation and production of transgenic animals; fourthly, the method is small in non-specific shear, and obviously improves the gene targeting knockout efficiency; fifthly, the method is high in applicability.
Description
Technical field:
The present invention relates to a kind of sheep MSTN gene orientation and knock out and affect the detection method of myogenic differentiation, belong to the fields such as molecular biology, genetic engineering and transgenic technology.
Technical background:
The function of research negative-feedback regu-lation gene mainly uses artificial endonucleases (Engineeredendonuclease at present, EEN) technology carries out targeting and knocks out and carry out functional authorization gene, Application comparison ENN widely mainly has ZFN (Zinc-fingernuclease at present, and TALEN (Transcriptionactivator-likeeffectornuclease ZFN), TALEN), however ZFN technology shears because having relatively low specific homodimer form that specificity is poor, cytotoxicity is big.TALEN improves in terms of specific cleavage as second filial generation EEN technology, but still has relatively maxicell toxicity.The experimentation of both technology is loaded down with trivial details, design the most complicated, novel C RISPR/Cas9 system is to imitate antibacterial for resisting the genic system of virus attack/hide mammalian immune reaction and being born as third generation ENN technology, Cas9 system as third generation ENN technology is then a kind of quickly targeted gene disruption technology risen for these 2 years, it is simple that it has experimentation, the advantages such as the shortest and workload is little, and be easier to obtain homozygous mutation body, and multiple sudden change can be simultaneously introduced in different sites, there is good development prospect, therefore, Cas9 system the most little by little replaces ZFN and TALEN technology.Research worker has utilized Cas9 system successfully to construct gene knockout model in the animals such as mice, Brachydanio rerio and fruit bat at present, and in the cells such as HEK293 and iPS, achieve gene knock out and create stable cell line, it is achieved that the pointed decoration of gene and functional study thereof.
Summary of the invention:
Shortcoming loaded down with trivial details for traditional E NN technology experiment, that efficiency is low, toxicity is big, it is provided that a kind of sheep MSTN gene orientation knocks out and affect the detection method of myogenic differentiation.
The present invention comprises the following steps:
(1) genes of interest clone
Inquiry ncbi database, obtain the CDS sequence (serial number: NM_001009428) of sheep genes of interest, design specific primer, clone for template with the cDNA that sheep muscular tissue total serum IgE reverse transcription is formed and obtain genes of interest whole CDS sequence, purification reclaims the order-checking of Hou Song company, obtains complete genes of interest exon sequence and mutational site information by comparison；
(2) gRNA design and synthesis
Avoiding mutational site, find PAM sequence in exon 1, and about 20bp designs target sequence before PAM sequence, all of target sequence and synthesizes as gRNA at full-length genome comparison, the target site without homology；
(3) CRISPR/Cas9 gene knockout carrier builds
GRNA being connected into carrier is carrier VK001-02, starts the expression of gRNA with people source U6 promoter, T7 promoter starts the expression of Cas9 enzyme, and insertion GFP green fluorescence gene is as reporter gene, and puro gene sieves gene as resistance medicine.
(4) CRISPR/Cas9 gene knockout carrier external source Activity determination
It is building up to gRNA sequence (including PAM sequence) in PCR primer framework synthesize enzyme action DNA by the way of bridging PCR, in vitro transcription gRNA adds reaction system, and with standard substance active for known SSA for comparison, Cas9 enzyme In vitro digestion reaction detection luciferase signal, obtaining luciferase activity by comparison enzyme action band gray scale, luciferase activity the highest then gRNA external Cas9 digestion activity is the highest；
(5) CRISPR/Cas9 gene knockout carrier endogenous activity detection
Select the sheep ear fibroblast that the transfection of shear active higher CRISPR/Cas9 knockout carrier is in good condition, positive cell is filtered out with puromycin after 24h, extract genomic DNA, before and after cloning target site, (target spot is not positioned at middle to about 1000bp fragment, it is easy to differentiate enzyme action band), T7E1 enzyme carries out Activity determination, knocks out activity if existing, non-matching DNA fragmentation will be produced, can be by non-matching restriction endonuclease--T7 Cobra venom endonuclease I shears；If not undergoing mutation, by generation pairing DNA fragmentation, and cannot be by non-matching restriction endonuclease--T7 Cobra venom endonuclease I shears；
(6) CRISPR/Cas9 gene knockout carrier knocks out effect detection
Select to have concurrently the carrier of outer source activity and endogenous activity, liposome mediated-method transfection sheep skeletal muscle satellite cell, positive cell is filtered out with puromycin after 48h, add proliferated culture medium normally to cultivate to cell density and reach 40-50%, reduce serum-concentration and induce satellite cell myogenic differentiation to 1% hunger, induce as compareing the same period using the normal cell cultivated simultaneously.Induction 72h, statistics myotube forms number.
The invention discloses a kind of application CRISPR/Cas9 technology sheep MSTN gene is oriented to knock out, and verify its method to skeletal muscle satellite cell differentiation impact effect, particular content is as follows: (1) vector construction: inquire about the CDS sequence of sheep MSTN gene on NCBI, clone and check order to obtain SNP site, avoid mutational site design CRISPR/Cas9 and knock out target site as gRNA, vector construction CRISPR/Cas9 double-promoter knockout carrier based on VK001-02, and insert GFP albumen and sieve gene as reporter gene and puro gene as medicine；(2) Carriers Active detection: be building up to gRNA sequence (including PAM sequence) in PCR primer framework synthesize enzyme action DNA by the way of bridging PCR, with standard substance active for known SSA for comparison, after Cas9 endonuclease reaction, comparison band gray scale obtains luciferase signal, and the gRNA choosing external Cas9 digestion activity the highest carries out endogenous activity checking；The vector plasmid screened is transfected sheep ear fibroblast in good condition with liposome mediated-method, puromycin screening positive cell after 24h, extract genomic DNA, clone about 1000bp fragment before and after target site, T7E1 enzyme carries out shear active detection, knocking out activity if existing, non-matching DNA fragmentation will be produced；(3) myogenic differentiation checking: select to have concurrently the carrier of outer source activity and endogenous activity, liposome mediated-method transfection sheep skeletal muscle satellite cell, positive cell is filtered out with puromycin after 48h, add proliferated culture medium normally to cultivate to cell density and reach 40-50%, 1% serum-concentration hunger induced muscle satellite cell myogenic differentiation, simultaneously using the normal cell induction same period cultivated as comparison.Induction 72h, indirect IF staining statistics myotube forms number.
Traditional ZFN technology is because having the false palindrome that relatively low specific homodimer form can be cut in genome, and single ZFN unit is incorporated into DNA will also result in the shearing of DNA, these non-specific cleavage all can affect gene knockout efficiency, in some instances it may even be possible to causes cytotoxicity.TALEN is greatly improved in terms of specific cleavage as second filial generation EEN technology, but still has certain cytotoxicity, and module assembled process is loaded down with trivial details, needs more manpower and materials.Novel C RISPR/Cas9 system is imitated antibacterial and is used for resisting the genic system of virus attack/hide mammalian immune reaction, it is physically easier to perform, in hgher efficiency, it is easier to obtain homozygous mutation body, and multiple sudden change can be simultaneously introduced in different sites, there is good development prospect.
The present invention uses novel C RISPR/Cas9 technology that sheep MSTN gene is implemented fixed point and knocks out, and assessment knocks out efficiency, and verifies that MSTN gene delection expresses the impact for skeletal muscle satellite cell myogenic differentiation.Using this method, this method is simple, and experimenter can complete structure and the activity checking of genes of interest Cas9 knockout carrier in two months, and targeting knocks out efficiency and is up to more than 90% apparently higher than traditional E NN technology, the external Cas9 digestion activity of MSTN gene.In addition this method is the least to the toxic action of cell, provides strong technical support for late stage functionality checking and the preparation of transgenic animal individuality.
The present invention is advantageous in that:
1, shorter speed experimental period；
2, method is simple, repeatable strong, can carry out in common lab；
3, using the active carrier that the method builds, toxic and side effects is little, can be used for preparation and the production of transgenic animal；
4, the non-specific shearing of the method is less, significantly improves gene target and knocks out efficiency height.
5, the method suitability is high, can be by knocking out negative regulator gene for improving the breeding character of various domestic animal.
Accompanying drawing explanation
Fig. 1 is PCR amplification in vitro MSTN gene map of the present invention.
Fig. 2 is that CRISPR/Cas9-gRNA knockout carrier of the present invention builds collection of illustrative plates.
Fig. 3 is that Cas9 enzyme In vitro digestion experimental verification gRNA target sequence of the present invention knocks out activity figure.
Fig. 4 is that the present invention knocks out plasmid transfected fibroblast checking endogenous activity figure.
Fig. 5 is that MSTN gene ectopic expression of the present invention affects figure to skeletal muscle satellite cell myogenic differentiation.
The gene order that this material relates to is shown in " description nucleotide and aminoacid sequence table ".
Detailed description of the invention
1.CRISPR/Cas9 gene knockout carrier builds
(1) genes of interest clone: inquiry ncbi database, obtain the CDS sequence (ID:NM_001009428) of sheep genes of interest, application Primer5.0 software design specific primer, the cDNA formed with sheep muscular tissue total serum IgE reverse transcription clone for template PCR and checks order and obtains genes of interest whole CDS sequence, purification reclaims the order-checking of Hou Song company, obtains complete genes of interest exon sequence and mutational site information by comparison；
(2) gRNA design and synthesis: avoid mutational site, finds PAM sequence, and about 20bp designs target sequence before PAM sequence in exon 1, and all of target sequence and synthesizes as gRNA at full-length genome comparison, the target site without homology；
(3) CRISPR/Cas9 gene knockout carrier builds: the gRNA of synthesis is connected into carrier is carrier VK001-02, the expression of gRNA is started with people source U6 promoter, T7 promoter starts the expression of Cas9 enzyme, insertion GFP green fluorescence gene is as reporter gene, and puro gene sieves gene as resistance medicine.
The Activity determination of 2.CRISPR/Cas9 gene knockout carrier
(1) external source Activity determination: by the way of bridging PCR, it is building up to gRNA sequence (including PAM sequence) in PCR primer framework synthesize enzyme action DNA, in vitro transcription gRNA adds reaction system, and with standard substance active for known SSA for comparison, Cas9 endonuclease reaction detection luciferase signal, obtaining luciferase activity by comparison enzyme action band gray scale, luciferase activity the highest then gRNA external Cas9 digestion activity is the highest；
(2) endogenous activity detection: select the sheep ear fibroblast that the transfection of shear active higher CRISPR/Cas9 knockout carrier is in good condition, i.e., select outer source activity higher CRISPR/Cas9 knockout carrier, use FuGene transfection reagent in transfection reagent: plasmid is that the ratio of 1 μ l:3 μ g transfects sheep ear fibroblast in good condition, positive cell is filtered out with puromycin after 24h, extract genomic DNA, clone about 1000bp fragment before and after target site, target site is not placed on center, it is to avoid enzyme action band overlaps.T7E1 enzyme carries out Activity determination, knocks out activity if existing, and will produce non-matching DNA fragmentation, can be by non-matching restriction endonuclease--and T7 Cobra venom endonuclease I shears；If not undergoing mutation, by generation pairing DNA fragmentation, and cannot be by non-matching restriction endonuclease--T7 Cobra venom endonuclease I shears；
The most specific, select the sheep ear fibroblast that the transfection of Cas9 digestion activity the highest CRISPR/Cas9 knockout carrier plasmid is in good condition, use FuGene transfection reagent in transfection reagent: plasmid is that the ratio of 1 μ l:3 μ g is carried out, 24h observes fluorescence, determine transfection efficiency, and change the Screening of Media positive cell containing 10 μ g/ml puromycins, liquid is changed every 24h, after step sizing 72h, change proliferated culture medium normally to cultivate and collect cell when reaching 70% to degree of converging, extract genomic DNA, clone about 1000bp fragment before and after target site, target site is not placed on center, enzyme action band is avoided to overlap.T7E1 enzyme carries out Activity determination, knocks out activity if existing, and will produce non-matching DNA fragmentation, can be by non-matching restriction endonuclease--and T7 Cobra venom endonuclease I shears；If not undergoing mutation, by generation pairing DNA fragmentation, and cannot be by non-matching restriction endonuclease--T7 Cobra venom endonuclease I shears；
3.CRISPR/Cas9 gene knockout carrier knocks out effect detection
Select to have concurrently the carrier of outer source activity and endogenous activity, liposome mediated-method transfection sheep skeletal muscle satellite cell (sSMSCs), after filtering out positive cell 3d with the puromycin of working concentration 9 μ g/ml after transfection 48h, add proliferated culture medium normally to cultivate to cell density and reach 40-50%, myoblast differentiation is induced to be myotube by the DMEM/F12 culture medium containing 1% serum, reduce serum-concentration starvation method induction myoblast differentiation, simultaneously using the normal cell induction same period cultivated as comparison.Induction 72h, indirect immunofluorescence qualification myogenic differentiation labelled protein Desmin, observation fluorescence, statistics myotube forms number, the impact effect after significance analysis Data Summary MSTN gene knockout, and the trans genie individual for the later stage cultivates offer support.
Sheep MSTN gene is oriented and knocks out by this method application CRISPR/Cas9 technology, and verifies its impact on skeletal muscle satellite cell differentiation effect.First the CDS sequence of sheep MSTN gene is cloned, order-checking aligned sequences are to obtain SNP site, avoid mutational site design CRISPR/Cas9 and knock out target site as gRNA, carrier reconstruction CRISPR/Cas9 double-promoter knockout carrier based on VK001-02, and insert GFP albumen and sieve gene as reporter gene and puro gene as medicine；Then carrier is carried out external source and endogenous activity detection, external source Activity determination uses Cas9 enzyme that the DNA sequence comprising gRNA (including PAM sequence) is carried out enzyme action experiment, with standard substance active for known SSA for comparison, luciferase signal is obtained by comparison band gray scale, choose the highest gRNA vector plasmid of external Cas9 digestion activity and carry out endogenous activity checking with liposome mediated-method transfection sheep ear fibroblast, puromycin screening positive cell after 24h, extract genomic DNA, clone about 1000bp fragment before and after target site, T7E1 enzyme carries out shear active detection；Finally, the carrier that screening In vitro and in vivo activity is higher carries out the checking of myogenic differentiation, liposome mediated-method transfection sheep skeletal muscle satellite cell, positive cell is filtered out with puromycin after 48h, add proliferated culture medium normally to cultivate to cell density and reach 70%, 1% serum-concentration starvation method induction satellite cell myogenic differentiation, simultaneously using the normal cell induction same period cultivated as comparison.Induction 72h, indirect IF staining statistics myotube forms number.The effect of myoblast differentiation is affected after summing up MSTN gene knockout eventually through statistic analysis result.
In accompanying drawing, Fig. 1 is PCR amplification in vitro MSTN gene map of the present invention, and amplified fragments size is 1309bp.
Fig. 2 is that CRISPR/Cas9-gRNA knockout carrier of the present invention builds collection of illustrative plates.
Fig. 3 is that Cas9 enzyme In vitro digestion experimental verification gRNA target sequence of the present invention knocks out activity figure.NC is matched group, 1,2,3,4 it is respectively the gRNA target spot of the most pre-designed four, mark g1 and mark g2 is respectively through the standard substance that SSAluciferase detection activity is 3 and 10, with standard substance for obtaining the digestion activity of four target spots with reference to comparison band gray scale, the results are shown in Table 1.
Table 1Cas9 enzyme In vitro digestion target site Activity determination
| Sample name | Target sequence (gray area is PAM sequence) | External digestion activity |
| MSTN-g1 | ATTTATGCTGCTTGTTGCTGG | 50% |
| MSTN-g4 | TAAGACAACTTTTGCCCAAGG | 65% |
| MSTN-g6 | AACACAATAAAGTAGTAAAGG | 5% |
| MSTN-g7 | ATGGGTTTGATGAGTCTCAGG | 95% |
Fig. 4 knocks out plasmid transfected fibroblast checking endogenous activity: component A is not for fluorescent dark field, light and shade boundary, observation of cell transfection under the conditions of light field；Figure B is T7E1 enzyme action effect, and W is wild type, and M is saltant type.
Fig. 5 MSTN gene ectopic expression impact on skeletal muscle satellite cell myogenic differentiation: indirect immunofluorescene assay myogenic differentiation early sign Desmin albumen (100 ×), result shows that knocking out group (Treatment) myotubes formation number is significantly higher than matched group (Control).
SEQUENCELISTING
<110>Yangzhou University
<120>a kind of sheep MSTN gene orientation knocks out and affects the detection method of myogenic differentiation
<130>2015
<160>1
<170>PatentInversion3.3
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agcagcgacggctccttggaagacgatgactaccacgttacgacggaaacggtcattacc360
atgcccacggagtctgatcttctagcagaagtgcaagaaaaacccaaatgttgcttcttt420
aaatttagctctaagatacaacacaataaagtagtaaaggcccaactgtggatatatctg480
agacctgtcaagactcctacaacagtgtttgtgcaaatcctgagactcatcaaacccatg540
aaagacggtacaaggtatactggaatccgatctctgaaacttgacatgaacccaggcact600
ggtatttggcagagcattgatgtgaagacagtgttgcaaaactggctcaaacaacctgaa660
tccaacttaggcattgaaatcaaagctttagatgagaatggtcatgatcttgctgtaacc720
ttcccagaaccaggagaagaaggactgaatccttttttagaagtcaaggtaacagacaca780
ccaaaaagatctaggagagattttgggcttgattgtgatgagcactccacagaatctcga840
tgctgtcgttaccctctaactgtggattttgaagcttttggatgggattggattattgca900
cctaaaagatataaggccaattactgctctggagaatgtgaatttttatttttgcaaaag960
tatcctcatacccatcttgtgcaccaagcaaaccccaaaggttcagccggcccttgctgt1020
actcctacaaagatgtctccaattaatatgctatattttaatggcaaagaacaaataata1080
tatgggaagattccaggcatggtagtagatcgctgtgggtgctcatga1128
Claims (2)
1. a sheep MSTN gene orients the detection method knocking out and affecting myogenic differentiation, it is characterised in that comprise the steps:
(1) genes of interest clone
Inquiry ncbi database, obtain the CDS sequence of sheep genes of interest, Serial No. NM_001009428 of the CDS sequence of sheep genes of interest, design specific primer, clone for template with the cDNA that sheep muscular tissue total serum IgE reverse transcription is formed and obtain genes of interest whole CDS sequence, purification reclaims the order-checking of Hou Song company, obtains complete genes of interest exon sequence and mutational site information by comparison；
(2) gRNA design and synthesis
Avoiding mutational site, find PAM sequence in exon 1, and about 20bp designs target sequence before PAM sequence, all of target sequence and synthesizes as gRNA at full-length genome comparison, the target site without homology；
(3) CRISPR/Cas9 gene knockout carrier builds
GRNA being connected into carrier is carrier VK001-02, starts the expression of gRNA with people source U6 promoter, T7 promoter starts the expression of Cas9 enzyme, and insertion GFP green fluorescence gene is as reporter gene, and puro gene sieves gene as resistance medicine；
(4) CRISPR/Cas9 gene knockout carrier external source Activity determination
By gRNA sequence, including PAM sequence, it is building up in PCR primer framework synthesize enzyme action DNA by the way of bridging PCR, in vitro transcription gRNA adds reaction system, and with standard substance active for known SSA for comparison, Cas9 endonuclease reaction detection luciferase signal, obtains luciferase activity by comparison enzyme action band gray scale, and luciferase activity the highest then gRNA external Cas9 digestion activity is the highest；
(5) CRISPR/Cas9 gene knockout carrier endogenous activity detection
Select the sheep ear fibroblast that the transfection of shear active higher CRISPR/Cas9 knockout carrier is in good condition, filter out positive cell with puromycin after 24h, extract genomic DNA, clone about 1000bp fragment before and after target site, target site is not placed on center, it is to avoid enzyme action band overlaps；T7E1 enzyme carries out Activity determination, knocks out activity if existing, and will produce non-matching DNA fragmentation, can be by non-matching restriction endonuclease--and T7 Cobra venom endonuclease I shears；If not undergoing mutation, by generation pairing DNA fragmentation, and cannot be by non-matching restriction endonuclease--T7 Cobra venom endonuclease I shears；
(6) CRISPR/Cas9 gene knockout carrier knocks out effect detection
Select to have concurrently the carrier of outer source activity and endogenous activity, liposome mediated-method transfection sheep skeletal muscle satellite cell, with puromycin step sizing 72h after 48h, add proliferated culture medium normally to cultivate to cell density and reach 40-50%, 1% serum-concentration starvation method induction satellite cell myogenic differentiation, simultaneously using the normal cell induction same period cultivated as comparison, 72h, indirect IF staining statistics myotube is induced to form number.
A kind of sheep MSTN gene the most according to claim 1 orientation knocks out and affects the detection method of myogenic differentiation, it is characterized in that, in step (1), applies Primer5.0 software design specific primer.
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