CN107699571A - A kind of porcine somatostatin gene editing site and its application - Google Patents

Abstract

The invention discloses a kind of porcine somatostatin gene editing site and its application, its nucleotide sequence such as SEQ ID NO:Shown in 1, the present invention injects the sgRNA containing the coded sequence and Cas9mRNA in Pig embryos by kytoplasm microinjection, realizes the rite-directed mutagenesis to pig SST genes.The present invention enters edlin in embryo's level by CRISPR Cas9 technologies to pig endogenous gene SST.Statistical result shows that it is 65% that external digestion activity is detected in the site by SSA luciferase, and can form SST point mutation in the cell in embryo's horizontal upper knock-out pig SST genes.The present invention is to promoting the application of endogenous gene knockout or fixed point integration of foreign gene technology in transgene pig is studied and is produced to have a very important role.

Description

A kind of porcine somatostatin gene editing site and its application

Technical field

The invention belongs to biomedicine technical field, is related to a kind of porcine somatostatin gene editing site and its application, tool Say body, be related to a kind of porcine somatostatin gene SST editing sites (30-52) and its application.

Background technology

Foreign gene is precisely knocked in pig genome, is always extremely difficult.Common microinjection, electroporation, The methods of calcium phosphate precipitation, retroviral vector infection, can not realize foreign gene in genome certain bits due to its randomness The site-directed integration put；Although the gene targeting dependent on homologous recombination can import foreign gene the certain bits of target cell Put, but after its gene targeting efficiency is low, operation difficulty is big, and the marker gene introduced during cell screening can also arrive The bio-safety hidden danger of phase, these problems all significantly limit the development and application of the technology.Until in recent years in artificial nucleic acid The appearance of enzyme cutting (engineered endonuclease, EEN), just revolutionizes this present situation.

Artificial endonucleases' technology, including zinc finger endonuclease (zinc finger endonuclease, ZFN), Class activating transcription factor effector nuclease (transcription activator-like effctor nuclease, ) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats TALEN (CRISPR)/CRISPR-associated (Cas)) technology.Wherein, the technology using ZFN and TALEN as representative can realize base Because of a group pointed decoration, but it is high with certain cytotoxicity, operating process complexity, miss rate, in mammalian genome editor Middle application is less.CRISPR/Cas technologies since the advent of the world is liked that the technology is repaiied in genome fixed point by researcher deeply More other technologies have unrivaled advantage, simple to operate, targeting precision height, wide application and low-cost, warp in terms of decorations Cross and technology is updated over 3 years, the technology has been listed in living cells at present and individual level is most effective, most easily base Because of a group pointed decoration technology.

Porcine somatostatin growth hormone release inhibiting hormone (somatostatin, SST) is a kind of nerve modulation for suppressing growth hormone secretion Peptide, mainly secreted by hypothalamus, have the function that to suppress growth hormone release, while to insulin, hyperglycemic factor, rush first shape The secretion of glandular hormone etc. also has inhibitory action.Pig is inseparable with human lives, is one of most important butcher's beast of the mankind, it In anatomic tissue, physiological metabolism etc. with people more closely, being the ideal animals model for studying human diseases.Therefore utilize CRISPR/Cas9 genetic modification technology knock-out pig somatostatin genes, not only contribute to study SST gene pairs pig growth and development, Metabolism and the influence of expression characteristic etc., and then breeding is improved on Animal husbandry production, pig growth rate is improved, can be also used as real Test the research that animal model carries out the relevant diseases such as growth inhibition.

The content of the invention

It is an object of the invention to provide a kind of porcine somatostatin gene editing site and its application, there is provided one can be efficient The editing sites (30-52) of knock-out pig flesh chalone gene (SST).The editing sites can be used for the pig SST genes of Cas9 mediations to beat Target, by somatostatin gene functionally inactive, the present invention screens the sgRNA of high efficiency cutting for the design of SST genes, and it is prominent to analyze it Become efficiency, knocked out for structure SST genes endogenous or fixed point knocks in the transgene pig of foreign gene and provides new way.

A kind of porcine somatostatin gene editing site, is one section of DNA sequence dna comprising 23 deoxyribonucleotides, its core Nucleotide sequence such as SEQ ID NO:Shown in 1, the site is No. 13 chromosome SST code areas first in being accurately positioned for pig genome On extron, chromosome coordinate is 125337589-125337611, and code area coordinate is 30-52.

A kind of application of porcine somatostatin gene editing site during Cas9 endonuclease specific recognitions.

Further, following steps are specifically included：

(1) suitable sgRNA target sites are found by PAM from objective gene sequence, target site sequence is：5’- GGCCGCGCTCTCCATCGTCCTGG-3’。

(2) sgRNA expression vector of the structure containing editor's target site (30-52)；

(3) above-mentioned carrier is subjected to in-vitro transcription and obtains sgRNA；

(4) it is co-injected into after mixing above-mentioned sgRNA and Cas9mRNA by a certain percentage in the parthenogenetic embryo after pig activation；

(5) confirm that SST genes are modified using T7NE1 digestions detection and TA cloning and sequencings.

Compared with prior art, beneficial effects of the present invention are：

, can be under endonuclease Cas9 mediation with more efficient using editing sites provided by the invention (30-52) Rate carries out double-strand break, then points out deletion in cleavage by way of non-homogeneous restructuring or increase base reaches gene site-directed The purpose of modification.To promoting, endogenous gene is knocked out the present invention or fixed point integration of foreign gene technology is studied and produced in transgene pig In application have a very important role.

Statistical result shows, it is 65% that external digestion activity is detected in the site by SSA luciferase, and can be Embryo's horizontal upper knock-out pig SST genes, SST point mutation is formed in the cell.Statistical result shows that the invention provides pin To the sgRNA of porcine somatostatin gene, foundation is provided to carry out effectively editor to the gene, to develop the live pig of high lean meat percentage New varieties provide new strategy, also provide reliable means and element to verify the molecular mechanism of growth hormone release inhibiting hormone and signal path Material.

Brief description of the drawings

Fig. 1 pDR274-sgRNA expression vector schematic diagrams；

Using pDR274 carriers as skeleton, pDR274 plasmids are linearized with restriction enzyme Bsa I, with sgRNA oligonucleotides Double-strand connects to form specific expression vector pDR274-sgRNA；

Fig. 2 pDR274-sgRNA plasmid order-checking results；

From the sequencing result of pDR274-sgRNA plasmids, the position and direction of sgRNA oligonucleotides double-strand insertion are equal Correctly, namely pDR274-sgRNA plasmid constructions are successful；

Fig. 3 sgRNA and Cas9 in-vitro transcription；

A figures are SST-sgRNA in-vitro transcription figures, 100bp DNA ladder (TaKaRa)；

B figures are Cas9mRNA in-vitro transcription figures, 5000bp DNA ladder (TaKaRa)；

Fig. 4 T7EN1 digestion results；

The egg mother cell in each period under Fig. 5 microscopes

A：The egg mother cell do not cultivated under 50 power microscopes；B：48h after ripening egg mother cell under 50 power microscopes；C：400 There is the egg mother cell of first polar body under power microscope；

Fig. 6 is each period embryo figure under microscope；

A in figure：Two cells；B：Four cells；C：Eight cells.Egg mother cell after activation is transferred to after NCSU-23 is washed 3 times In embryo medium covered with paraffin oil, culture dish is placed in 39 DEG C, 5%CO₂, in the constant incubator of 100% humidity.Culture After 2 days, cell spilting of an egg situation is observed and recorded under stereomicroscope, statistics is respectively at embryo's number of different development stage.Point It is not two cells and four cells and eight cells after 3 days after lonely female activation culture 2 days.

Embodiment

Technical scheme is described in more detail with specific embodiment below in conjunction with the accompanying drawings.

It is used for the sgRNA's of selectively targeted SST genes in embodiment 1CRISPR/Cas9 specific knockdown pig SST genes Design and synthesis

1. target the sgRNA of pig SST genes design

Using website design SST-sgRNA, concretely comprise the following steps：1. logging in GenBank websites, pig SST gene orders are searched for And download, its nucleotide sequence such as SEQ ID NO:Shown in 2.2. open target spot prediction website (http:// Crispr.mit.edu/), using target site Photographing On-line instrument, " CRISPR design tool ", input base in text box Because of sequence, species are selected, you can draw all target sites of the gene order, several groups of target position are chosen after considering parameters Point.3. long 20nt oligonucleotides sgRNA core sequences are designed by GG (N) 19NGG sequences.

2. build sgRNA oligonucleotide

According to the sgRNA of selection, AAACACCG is added at the 5' ends of positive-sense strand template, it is mutual with vector plasmid cohesive terminus,cohesive termini Mend；The 5' ends addition CTCTAAAAC of antisense strand template, with the complementation of vector plasmid cohesive terminus,cohesive termini.It is respectively synthesized the above-mentioned few core of forward direction Thuja acid and reverse oligonucleotide, the sgRNA oligonucleotides of synthesis are denatured in pairs, annealed, being formed after annealing to be connected into

In this experiment, 1 sgRNAs action target spot is designed for SST genes, its nucleotide sequence is shown in Table 1.

Table 1

The preparation of embodiment 2sgRNA, Cas9mRNA

1. main agents：

PmLM3613 (Addgene No.42251), BsaI, PmeI enzyme are purchased from NEB companies；MEGA shortscript Kit (AM1354) sgRNA in-vitro transcription kits, mMESSAGE mMACHINE Kit (AM1344) Cas9 in-vitro transcription reagents Box, Poly (A) Tailing Kit are purchased from Ambion companies；PDR274 (Addgene No.42250) plasmid is by animal embryo Engineering and key lab of molecular breeding Hubei Province provide；Other common agents are that domestic analysis is pure；Primer is synthesized and DNA is surveyed Sequence is completed by Shenzhen Huada gene company.

2. operating procedure：

(1) in-vitro transcription obtains sgRNA：

1) sgRNA plasmid pDR274 structures (as shown in Figure 2) are expressed.

1. synthesize oligonucleotide chain double-strand：The high sgRNA-g1 sites of target active are respectively in 5 ＇ of its positive-sense strand template End addition TAGG, the 5 ＇ ends addition CAAA of antisense strand template, with vector plasmid cohesive terminus,cohesive termini complementary with vector plasmid cohesive terminus,cohesive termini It is complementary.The single-stranded annealed formation double-strand of oligonucleotide chain of synthesis.

2. linearize pDR274 plasmids：With the digestion cyclic plasmid pDR274 of restriction enzyme Bsa I, digestion system is：10 × NEB Buffer4,5uL；100 × BSA, 0.5uL；Bsa I, 2uL；PDR274 cyclic plasmids, 25uL；Add ddH₂O water arrives 50uL.37 DEG C of digestions 2-3h, 65 DEG C of inactivation 20min.

3. DNA digestion purifies：Plasmid after digestion is utilized into High pure PCR product Purification Kit are purified.

4. connect：The pDR274 plasmids of linearisation are connected to form specific targeting vector with sgRNA oligonucleotides double-strands pDR274-sgRNA.Linked system：10 × Buffer, 2uL；T4 ligases, 0.5uL；SgRNA oligonucleotides double-strands, 1uL；Line Property pDR274 plasmids, 20ng；Add ddH₂O water is to 20uL.Overnight, 65 DEG C inactivate 20min for connection in 22 DEG C of constant incubators.

5. conversion, monoclonal are selected, plasmid extraction.

6. pDR274-sgRNA plasmid constructions well after sequence verification, select the plasmid correctly cloned and largely extract for external Transcription.

2) acquisition of sgRNA-PCR products

Enter performing PCR amplification by template of pDR274-sgRNA plasmids, obtain sgRNA-PCR products.PCR reaction systems 50uL：PDR274 plasmids, 10ng；T7-sgRNA-FPg (10uM), 1.5uL；SgRNA-RP (10uM), 1.5uL；2×Pfu Mix, 25uL add DEPC H₂O to 50uL.PCR programs are：95 DEG C, 3min；35cycles×(94℃ 30s；58℃ 30s；72 ℃ 30s)；72 DEG C, 10min；4 DEG C ,+∞.1% agarose gel electrophoresis detection PCR primer accuracy, the purified recovery of product, DEPC water back dissolvings are finally used, more than 500ng/uL need to be reached by surveying concentration.

3) sgRNA in-vitro transcription, recovery, purifying.

In-vitro transcription is carried out by template of the sgRNA-PCR products of acquisition, transcription system is 10 × Transcription Buffer, 2uL；RNTP, 2uL；T7RNA polymerase Mix, 2uL；SgRNA PCR primers, 1ug；Add DEPC H₂O is extremely 20uL.37 DEG C of constant incubator culture 2h, reaction add 2uL DNase I, 37 DEG C of reaction 30min after terminating.Take 2uL transcription productions Thing carries out 2% agarose gel electrophoresis detection.SgRNA is reclaimed and that extracts concretely comprises the following steps：1. add into the sgRNA transcribed Enter 115uL DEPC water, after 15uL 3M sodium acetates (pH 5.2) mix, the 1 of addition isometric (130uL):1 phenol/isoamyl alcohol Mixture, after being sufficiently mixed uniformly, 4 DEG C of centrifugation 15min of 12000-15000r/min, supernatant is transferred in new EP pipes. 2. adding isometric chloroform into supernatant, fully mix, 12000-15000r/min4 DEG C of centrifugation 15min, Aspirate supernatant Into new EP pipes.3. isometric isopropanol precipitating RNA is added, after -80 DEG C stand 30min, 4 DEG C of 12000-15000r/min Centrifuge 20-30min and collect precipitation.4. removing supernatant, the 70% cold ethanol for adding 500uL washes precipitation.After ethanol dries, according to institute SgRNA concentration is needed, adds respective volume DEPC water dissolving RNA precipitate.Notice that above-mentioned Overall Steps need to be in the environment of without RNase Carry out, prevent the sgRNA of extraction to be degraded.(2) in-vitro transcription obtains Cas9mRNA：

1) pmLM3613 plasmid linearizations：Cyclic plasmid forms wire plasmid through PmeI digestions, and digestion system is：10× NEB 4Buffer, 10uL；100 × BSA, 1uL；PmeI, 5uL；Plasmid pmLM3613,10ug；Add ddH₂O to 100uL；37℃ Digestion is stayed overnight, and 65 DEG C of inactivation 20min, runs 1% agarose gel electrophoresis detection digestion integrality.Then reclaimed with Ago-Gel Kit reclaims wire pmLM3613 plasmids, and Cas9mRNA template is formed as lower step in-vitro transcription.

2) in-vitro transcription forms Cas9mRNA：In-vitro transcription is carried out using kit mMESSAGE mMACHINE Kit, is turned Record system is：10 × Reaction Buffer, 2uL；2 × NTP/CAP, 10uL；Enzyme Mix, 2uL；Nuclease-free Water, 1uL；Wire pmLM3613 plasmids, 3ug；2uL Dnase enzymes are added after 37 DEG C of water-bath 2h.1% agarose is run afterwards to coagulate Gel electrophoresis detect.

3) Cas9mRNA adds PolyA tails：Because Cas9mRNA needs to be transcribed into albumen and functioned, PolyA need to be added Tail, make its complete.Reaction system is：5 × E-PAP Buffer, 20uL；25mM；Mncl 210uL；10mM ATP, 10uL； Cas9 (- PolyA), 20uL；Nuclease-free water, 36uL, 0.5uL is taken to add 4uL E- as control after mixing PAP；37 DEG C of water-bath 2h, run the detection of 1% agarose gel electrophoresis.

4) Cas9mRNA recovery, extraction：Ibid test.

The collection of the egg mother cell of embodiment 3 and In-vitro maturation

1. main solution is prepared

(1) preparation of oocyte in vitro maturation culture solution：Maturation culture solution is made up of the TCM-199 improved,

Namely added into TCM-199 culture mediums 3.05mmol/L glucose, 1mmol/L Glus, 0.91mmol/L Sodium Pyruvates, 0.57mmol/L Cys, 75ug/mL penicillin, 50ug/mL streptomycin sulphates.According to After above component prepares, pH to 7.2-7.4 is adjusted, after being filtered on superclean bench, 4 DEG C save backup filter.Test 2h Preceding that 10IU/mL hCG, 10IU/mL PMSG, 10%pFF are added on this medium base, preheating is stand-by in constant incubator.

(2) preparation of embryo medium：It is made up of NCSU-23 basal medium+4mg/mL BSA.NCSU-23 components are： 108.73mmol/L NaCl、1.19mmol/L MgSO₄、25.07mmol/L NaHCO₃、4.78mmol/L KCl、1.70mmol/ L CaCl₂, 5.55mmol/L glucose, 1.19mmol/L KH₂PO₄, 1.00mmol/L glutamine, 7.00mmol/L ox sulphurs Acid, 5.00mmol/L aminoethane sulfinic acids, 65ug/mL penicillin, 50ug/mL streptomycin sulphates.The nutrient solution regulation pH prepared It is worth neutrality and is filtered with filter, 4 DEG C of preservations is stand-by.

(3) preparation of DPBS egg-cleaning liquids：0.1mg/mL PVA and dual anti-(75ug/mL moulds are added into DPBS buffer solutions Element and 50ug/mL streptomycin sulphates).

(4) preparation of pig follicle liquid (pFF)：The liquor folliculi that a diameter of 2-6mm ovarian follicles are aspirated with No. 16 needle applicators arrives In sterile centrifugation tube, 2000 × g centrifugation 30min, into new sterile centrifugation tube, filter is collected by filtration Aspirate supernatant by several times Supernatant, be dispensed into 2mL centrifuge tubes, sealed membrane sealing centrifuge tube, carry out mark after -20 DEG C of preservations it is stand-by.

(5) preparation of ovum liquid is taken off：1mg/mL hyaluronidases are added into the TCM199 of improvement.

(6) preparation of liquid is electrically activated：Component is 0.5mmol/L calcium chloride, 0.5mmol/L HEPES, 0.1mmol/L MgSO₄, 0.28mol/L mannitol and 0.1%BSA.

2. operating procedure

Fresh pig ovary is collected from Jiangxia China Oil and Food Import and Export Corporation slaughterhouse, is put in 37 DEG C of physiological saline containing penicillin and streptomysin, Laboratory is delivered to as early as possible.The ovary adopted back is again with normal saline flushing 3-5 all over until clean up, by ovarian metastasis to sterilizing burning It is placed in cup in 37 DEG C of thermostat water baths.With connection No. 16 syringe needles disposable syringe, extract ovary on 3-6mm ovum Bubble, liquor folliculi is collected into 50mL centrifuge tubes.After egg mother cell sedimentation, absorption, which is deposited under stereomicroscope, selects ovarian cumulus cause Close complete, the parcel number of plies is more and the uniform cumulus cell-oocyte complex (COCs) of kytoplasm.With addition PVA's The COCs that DPBS will be singled out is cleaned 3 times, then with advance in incubator inner equilibrium 2h or so porcine oocytes maturation culture solution Cleaning 3 times.To ensure the vigor of egg mother cell, cleaning process need to operate in 37 DEG C of thermal station.The COCs cleaned up is put in 39 DEG C, culture 48h or so in 5%CO2 constant incubators, wherein in the rear 20h of culture, egg mother cell are transferred to and is not added with HCG Cultivated with PMSG maturation medium.Cell is observed after 48h, it is female to spread preferable ovum with 0.1% hyaluronic acid enzymic digestion The granular cell of cell peripheral, then cleaned 2-3 times with DPBS egg-cleaning liquids, finally egg mother cell is cleaned up with mTCM199.Choose Select the obvious clear, cell membrane in cell peripheral gap completely and the egg mother cell of discharge first polar body is used for kytoplasm microinjection.

The selection of embodiment 4RNA kytoplasm microinjection porcine oocytes and RNA optimal injection concentration

After in-vitro transcription good sgRNA and Cas9mRNA are determined into concentration, 250ng/uL and 1ug/uL are diluted to respectively.Examine The later stage parthenogenetic development of egg mother cell of In-vitro maturation may be influenced whether by considering RNA injection concentrations, be set in being tested for this Determine different RNA concentration, the cleavage rates developed by observing statistics parthenogenetic embryo, find out optimal injection concentration.In experiment, control group Mature oocyte without RNA microinjections, the sgRNA concentration that experimental group setting mixes two-by-two is constant, is 12.5ng/ UL, Cas9mRNA concentration set tetra- gradients of 100ng/uL, 150ng/uL, 200ng/uL and 300ng/uL.After microinjection RNA Carry out external lonely female activation immediately, experimental group and cellular control unit after activation are placed in 39 DEG C, in 5%CO2 constant incubators Culture.Select egg mother cell that is well-developed, having discharged first polar body and carry out RNA kytoplasm microinjections.

Concretely comprise the following steps：

1. in the RNA parenteral solutions that injection needle insertion is mixed, mixed liquor is sucked in pin using capillary attraction, pin Load in microinjector.

2. in concave slide center drop 20uL DPBS, paraffin oil is covered, the egg mother cell reached maturity is moved in drop. Zona-free oocytes are arranged under low-powered microscope into a line, regulation injection needle and hold ovum pin, made itself and parthenogenetic embryo while focus on In visual field center.

3. being held under low power lens after ovum pin handles 1 piece of ovum, microinjection under high power lens is gone to.Under microscope (>=400x), With holding ovum pin pressure-vaccum egg mother cell repeatedly, untill cell is adjusted into clear, position suitable, then sticking embryo.

4. the injection needle that has RNA solution will have been inhaled quickly to be pierced into oocyte cytoplasm, note is exited after injection rapidly Penetrate pin.The ovum injected is moved on one side, continues to inject next piece of ovum.

5. being moved into zona-free oocytes in new TCM-199 culture mediums after the completion of injection, media surface is covered with mineral oil. Culture dish is placed in 39 DEG C, the interior culture of 5%C02 insulating boxs.

The parthenogenetic activation of porcine oocytes of embodiment 5

By above-mentioned each group after kytoplasm microinjection RNA egg mother cell electricity consumption transactivation liquid cleans 3 times, mouth suction pipe is successively Cell is drawn, cell is uniformly placed in the electric shock tank for having added activation liquid.Adjust fusion instrument voltage and electric shock time (1.3kv/cm, 30us), in vitro lonely female activation egg mother cell.Egg mother cell after activation is transferred to after NCSU-23 is washed 3 times In embryo medium covered with paraffin oil, culture dish is placed in 39 DEG C, 5%CO2, in the constant incubator of 100% humidity.Culture After 2 days, cell spilting of an egg situation is observed and recorded under stereomicroscope, statistics is respectively at embryo's number of different development stage.

The detection of the mutation efficiency of embodiment 6

(1) preparation of the mono- blastaea lysates of NP-40 (exemplified by preparing 1mL)：

Concretely comprise the following steps：

1. it is 1 × Tag Buffer to dilute 10 × Tag Buffer：10 × Tag of 100uL Buffer are taken to add 900uL ddH₂O, common 1mL, takes 657.6uL；

2. dilute NP-40 (10 times)：NP-40 original liquid concentrations are very high, take 20uL stoste+180uL 1 × Tag Buffer, inhale Taking will gently inhale during stoste, then by its 1 × Tag of hanging instillation Buffer, mix.The NP-40 after 42.4uL dilutions is taken to add Into 1 × Tag of 657.6uL Buffer centrifuge tube；

3. 300uL Proteinase Ks are taken into above-mentioned centrifuge tube.Three is mixed, you can use.

(2) spilting of an egg egg mother cell DNA extraction：

1. ectogenesis 48h after the lonely female activation of egg mother cell, observes each group parthenogenetic embryo developmental state under stereomicroscope, Statistics is respectively at the cell number of different development stage, by each group spilting of an egg situation, selects the cleavage-cell under optimal injection concentration It is cleaned 3 times by individual cells with DPBS；

2. single egg mother cell is picked up equipped with 5uL NP-40 lysates successively with mouth suction pipe under stereomicroscope, 1uL paraffin oil is added dropwise on lysate surface.

3. by the lysate equipped with cell according to 37 DEG C of 60min；55℃ 60min；95 DEG C of 30min program is in PCR instrument Interior cracking.It is stand-by that the cell cracked puts 4 DEG C of preservations.

(3) T7EN1 digestions detection mutation efficiency

1. choosing each 200bp-400bp sequences of target site upstream and downstream, primer is designed, enters performing PCR amplification, PCR amplification programs For：95 DEG C, 5min；(95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 30s) × 30cycles；72 DEG C, 5min；4 DEG C of preservations.Purifying obtains PCR recovery products are obtained, 20ng/ μ l is uniformly diluted to and carries out digestion identification.Identify that the primer sequence of SST mutation effects is as follows, piece Duan great little 621bp.

SST-PL1：5’-CTTCAGGAGTCGCGAGGTTC-3’

SST-PR1：5’-TGGGTGGGAGGTACTGCTTA-3’

(2) the μ l of 10 × Buffer 1.5 μ l, T7 endonucleases 0.5 are added in 15 μ l systems, substrate 12.5 μ l, 37 DEG C Digestion 30min, 2 μ l 10 × Loading buffer are added, detected with 2% agarose gel electrophoresis.As 2 different RNA of table are noted Penetrate in the influence that concentration is developed to porcine oocytes zona-free oocytes shown in sg1 samples, by agarose gel electrophoresis it can be found that Occur broken ends connection repair genome can because with protogene group Incomplete matching and cut by T7EN1, show compared with Small band.

Table 2

The egg mother cell of In-vitro maturation, each group is through RNA microinjections, and observation spilting of an egg situation shows after lonely female activation 48h Show：Experimental group each group embryo cleavage rates (65.87%, 63.43%, 65.88% and 56.43%) are substantially less than control group (83.41%) (P ＜ 0.05)；Without significant difference (P ＞ between 100ng/uL groups cleavage rates and 150ng/uL groups and 200ng/uL groups 0.05) significant difference (P ＜ 0.05), but between 100ng/uL groups and 150ng/uL groups, 200ng/uL groups cleavage rates and 300ng/uL. Thus show that the microinjection of RNA kytoplasms can influence the ectogenesis potentiality of parthenogenetic embryo, and with concentration increase to a certain extent When, cleavage rates can significantly reduce, it can thus be appreciated that Cas9mRNA can obtain the higher spilting of an egg in 100-200ng/uL concentration ranges The zona-free oocytes of rate, beneficial to follow-up abrupt climatic change；

(3) TA cloning and sequencings

1. T7EN1 digestions are detected to the PCR primer obtained to take 1 μ l to be connected with pMD18-T carriers and convert DH5 α competence Cell (TransGen, CD201), picking monoclonal bacterium colony are sequenced, and are found according to sequencing result (such as sequence table), target gene SST has lacked one section of target sequence, gene knockout success.

The foregoing is only a preferred embodiment of the present invention, protection scope of the present invention not limited to this, any ripe Those skilled in the art are known in the technical scope of present disclosure, the letter for the technical scheme that can be become apparent to Altered or equivalence replacement are each fallen within protection scope of the present invention.

Sequence table

<110>Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences

<120>A kind of porcine somatostatin gene editing site and its application

<141> 2017-09-06

<160> 2

<170> SIPOSequenceListing 1.0

<210> 2

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

ggccgcgctc tccatcgtcc tgg 23

<210> 2

<211> 1183

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

atgctgtcct gccgcctcca gtgcgcgctg gccgcgctct ccatcgtcct ggctctgggc 60

ggtgtcactg gcgcgccctc ggatccccga ctccgtcagt ttctgcagaa gtccctggct 120

gctgccgctg ggaagcaggt aaggagactc cctcgacgcc ttctttcccc tctcgcgaat 180

cccctaacct taccttagcc ttgcccctcc tcccttgggt ggacttagga ggtggtccca 240

aagagtatcg gtgcttttct gggtccctta ggcaccaaat ctctcaggaa aactttcaaa 300

gtccagaatt cctttttacc tctttgtttt ttccctcttt gatcagcgca gtaggtcaca 360

gttcaggtga gttctttggc tttcaagaaa attctaagat ctggggaact gagctcgagg 420

ggatgatggc atctatccgc ggtgctgacc atgggaggtg ctgacccagg tgctgaaagc 480

gcggacctct gaagcttcct aagcagtacc tcccacccat gcagcagggc tgggggctga 540

agggcacaac agctagaaca caatatgttt acgactgtga aaagtcttgt ttcccacagt 600

tgatttagta aaaatggtaa gaacaattct attttgtagc tcatgatatg gaaattgagt 660

taaataaaat gttggcatat attttacctt tgctaactaa tgatgttcta tttctttcta 720

tgtgtgagtt ctaaacctgt agacactaaa accttgcaga aacatttgag tttttaaagc 780

ttccttgtgt aacttggtaa attataacaa cagccctgtt tttatatcct taacctattt 840

taagcattac acaaagtgca catagaaaat tgggggttgg atgtgattaa atctgttttt 900

taaaccaagc ttttccattt ctttttcact atcctcattc tcatccccct ccccctccca 960

ttccacacag gaactggcca agtacttctt ggcggagctg ctctctgaac ccaaccagac 1020

agagaacgat gccctggagc ctgaagattt gtcccaggct gctgagcagg atgaaatgag 1080

gctggagctg cagagatcag ctaactcaaa cccggccatg gcaccccgag aacgcaaagc 1140

tggctgcaag aatttcttct ggaagacttt cacatcctgt tag 1183

Claims (3)

1. a kind of porcine somatostatin gene editing site, it is characterised in that be one section of DNA for including 23 deoxyribonucleotides Sequence, its nucleotide sequence such as SEQ ID NO:Shown in 1, the site is No. 13 chromosome SST in being accurately positioned for pig genome On the First Exon of code area, chromosome coordinate is 125337589-125337611, and code area coordinate is 30-52.

A kind of 2. application of porcine somatostatin gene editing site during Cas9 endonuclease specific recognitions.

3. porcine somatostatin gene editing site according to claim 2 is during Cas9 endonuclease specific recognitions Application, it is characterised in that specifically include following steps：

(1) suitable sgRNA target sites are found by PAM from objective gene sequence, target site sequence is：5’- GGCCGCGCTCTCCATCGTCCTGG-3’；

(2) sgRNA expression vector of the structure containing editor's target site 30-52；

(3) above-mentioned carrier is subjected to in-vitro transcription and obtains sgRNA；

(4) it is co-injected into after mixing above-mentioned sgRNA and Cas9mRNA by a certain percentage in the parthenogenetic embryo after pig activation；

(5) confirm that SST genes are modified using T7NE1 digestions detection and TA cloning and sequencings.