CN109112161A - The method for preparing the animal of ROSA26 gene mutation and intramuscular fat raising - Google Patents

Abstract

The invention discloses a kind of methods of animal for preparing ROSA26 gene mutation and intramuscular fat raising.The present invention provides the method for mutation ROSA26 gene, the method is carried out using CRISPR/Cas9 system, and the CRISPR/Cas9 system includes the sgRNA for targeting ROSA26 gene, and the target sequence of the sgRNA is SEQ ID No.1.Donor dna of this method by importing sgRNA and Cas9 into recipient cell and containing PPAR γ expression casette realizes the site-directed integration of ROSA26 gene.It is demonstrated experimentally that can succeed using method of the invention in 2 gene of ROSA26 gene loci site-directed integration PPAR γ, the animal of expression PPAR γ 2 is finally successfully obtained.

Description

The method for preparing the animal of ROSA26 gene mutation and intramuscular fat raising

Technical field

The present invention relates to field of biotechnology, and in particular to a kind of to prepare what ROSA26 gene mutation and intramuscular fat improved The method of animal.

Background technique

For most countries in the world, pork is the main source of animal protein.With meat quality increasingly by To the concern of people, meat improvement has become the emphasis of current pig breeding work.There are many factor for influencing pig flesh characters, but Intramuscular fat (IMF) content is presently believed to be one of most important meat quality characteristic index, the tenderness and flavor of it and meat There is direct relation, is very important economic characters.

Peroxisome proliferators activated receptor γ (peroxisome proliferator-activated Receptors γ, PPAR γ) be nuclear hormone receptor family a kind of active ligand transcription factor, mainly at fatty group It knits, expressed in vascular smooth muscle and macrophage, wherein adipose tissue expresses highest, research shows that PPAR γ is fatty thin The key factor of born of the same parents' differentiation and mature, fatty acid absorption storage and fat deposition.

A large number of studies show that PPAR γ is the great influence factor that fat deposition and meat are formed in animal productiong, PPARG is related to animal intramuscular fat content, is the main effect candidate gene for influencing fat content in livestock and poultry muscle, pig PPAR γ Gene encodes 1.8Kb mRNA, has high homology with people, ox and mouse PPAR γ, similitude is respectively 99%, 97 and 96%.The high duroc intramuscular fat content of PPAR γ expression quantity is higher than Landrace.Therefore PPAR γ is to improve intramuscular fat One of main candidate of content.Pig PPAR γ gene polynorphisms influence significantly, in mRNA water intramuscular fat content It is flat that intramuscular fat content is influenced significantly.

CRISPR/Cas9 system is found in bacterium earliest as third generation gene editing tool, is to be widely present in Bacterium and a kind of intracorporal acquired immune system of archeobacteria.Cas9 albumen is a kind of more knots being made of 1409 amino acid Structure domain albumen, there are two structural domain, the RuvC structural domain of N-terminal and the HNH nuclease domains among albumen.HNH nucleic acid The template strand of enzyme domains cutting and crRNA complementary pairing, cleavage site are located at prototype intervening sequence and adjoin on motif (PAM) It swims at 3nt, RuvC cuts another chain, and cleavage site is located at the 3-8nt of the upstream PAM, so that it is disconnected to form double-stranded DNA It splits, generates a DSB.Improved Cas9 system is mainly made of guide RNA and Cas9 albumen, and guide RNA is by one section of 20bp The identification sequence of size and one section of repetitive sequence that can form hairpin structure form.Identify that sequence by base complementrity, is targeted to The corresponding position of genome, repetitive sequence and Cas9 albumen form complex and play digestion effect, cut off the double of target area Nucleotide chain.Non-homologous end joining repair mechanism (NHEJ) or homologous recombination repair in the DNA active cell of double-strand break Mechanism (HDR) is to realize quickly knocking out and knock in for gene.

Currently, CRISPR/Cas9 system has been applied to bacterium, zebra fish, in mammalian cell.2013 first Head is born by the clone pig that CRISPR/cas9 technology knocks out DAZL and apc gene.Utilization has been successfully realized on pig at present CRISPR/Cas9 technology carries out breeding for disease resistance and improves the breedings such as lean meat percentage.2015, Sino-Korean scientist was utilizing CRISPR/ Cas9 technology will inhibit gene myostatin (MSTN) successful knockout of muscle development, cultivate " double flesh pigs "；2016, The team that American scientist professor Prather leads successfully utilizes CRISPR/CAS9 technology by porcine reproductive and respiratory syndrome The receptor CD163 of (Porcine reproductive and respiratory Syndrome, PRRS) cell entry cell It knocks out, virus infection experiment shows CD163 knock-out pig, and without any clinical symptoms, such as fever, breathing is tired within 35 days after virus infection Difficulty, pulmonary abnormalities etc..These successful examples realize the rearing new variety of big domestic animal for us using CRISPR/Cas9 technology The open world newly.

In gene knock-in, external source or endogenous gene knock in site, particularly significant for Transgene-safty, at these The foreign gene in site should stability and high efficiency expression, while again to cell and tissue without known side effect.

Summary of the invention

The object of the present invention is to provide a kind of methods of animal for preparing ROSA26 gene mutation and intramuscular fat raising.

The present invention provides a kind of methods for being mutated ROSA26 gene.

The method of mutation ROSA26 gene provided by the present invention, is carried out using CRISPR/Cas9 system, described CRISPR/Cas9 system includes the sgRNA for targeting ROSA26 gene, and the target sequence of the sgRNA is SEQ ID No.1.

Further, the method may include following steps: importing the sgRNA and Cas9 into recipient cell, realizes The mutation of ROSA26 gene.

Further, described " sgRNA and Cas9 is imported into recipient cell " can be described by that will contain The expression vector of sgRNA and expression vector containing Cas9, which import in the recipient cell, to be realized.

More specifically, described " expression vector containing the sgRNA " and " expression vector containing Cas9 " can For identical carrier, which is named as carrier 1, the carrier 1 concretely passes through BbsI restriction enzyme site to CRISPR/ After being inserted into DNA fragmentation shown in SEQ ID No.1 in Cas9 carrier (such as pSpCas9 (BB) -2A-Puro (PX459) V2.0 carrier) Obtained recombinant vector, the carrier 1 can express previously described sgRNA and Cas9.

As needed, the method may also include the step of donor dna is imported into the recipient cell；The donor DNA contains the two segment DNA segments for being located at the target sequence upstream and downstream in ROSA26 gene.

Further, the sequence positioned at the DNA fragmentation (referred to as left homology arm) of the target sequence upstream is SEQ ID No.2 825-1810；Sequence positioned at the DNA fragmentation (referred to as right homology arm) in the target sequence downstream is SEQ ID No.2 6514-8076.

Wherein, the donor dna can also contain 2 encoding gene expression cassette of 2 encoding gene of PPAR γ or PPAR γ.

The amino acid sequence of the PPAR γ 2 can be as shown in SEQ ID No.3.

The nucleotide sequence of 2 encoding gene of PPAR γ can be as shown in 2839-4353 of SEQ ID No.2.

Further, the 2 encoding gene expression cassette of PPAR γ is from 5 ' ends to 3 ' ends successively including MCK promoter, PPAR 2 encoding gene of γ and polyA termination signal sequence.

Further, the sequence of the MCK promoter can be as shown in 1824-2838 of SEQ ID No.2；Institute The sequence for stating polyA termination signal sequence can be as shown in 4354-4593 of SEQ ID No.2.

More specifically, the sequence of the 2 encoding gene expression cassette of PPAR γ can be such as the 1824- of SEQ ID No.2 Shown in 4593.

As needed, the donor is also containing marker gene (such as neo gene) and/or for eliminating marker gene Recombinate the site loxP.In a specific embodiment of the invention, the sequence of the donor dna is specially SEQ ID No.2.Wherein The 817-824 identification sequences for Not I, 825-1810 are left homology arm sequence, and 1816-1823 are known for Swa I Other sequence, 1824-2838 are MCK promoter sequence, the 2839-4353 gene orders for PPAR γ, 4354- 4593 are polyA termination signal sequence, and 4594-4599 identify sequence for sal I, and 4600-4632 are loxP sequence Column, 4658-5164 are PGK promoter sequence, and 5175-5978 are NEO coded sequence, and 6468-6502 are LoxP sequence, 6508-6513 identify sequence for sal I, and 6514-8076 are right homology arm sequence, 8077-8082 Position is that EcoR I identifies sequence.

Any one of the present invention also provides following 1) -6):

1) method for preparing ROSA26 Genetic Mutant Cell includes the following steps: to prepare using previously described method The cell of ROSA26 gene mutation；

2) method for preparing ROSA26 gene mutant animals includes the following steps: to prepare using previously described method The zooblast of ROSA26 gene mutation；ROSA26 gene mutation is prepared using the zooblast of the ROSA26 gene mutation Animal；

3) method of preparation expression 2 cell of PPAR γ includes the following steps: to prepare using previously described method The zooblast of ROSA26 gene mutation obtains the cell of expression PPAR γ 2；

4) method of preparation expression 2 animal of PPAR γ includes the following steps: to prepare using previously described method The zooblast of ROSA26 gene mutation obtains the cell of expression PPAR γ 2；It is prepared using the cell of the expression PPAR γ 2 Express the animal of PPAR γ 2；

5) method for preparing the cell of intramuscular fat content raising includes the following steps: to utilize previously described method system The zooblast of standby ROSA26 gene mutation, obtains the cell of intramuscular fat content raising (compared with receptor cell)；

6) method for preparing the animal of intramuscular fat content raising includes the following steps: to utilize previously described method system The zooblast of standby ROSA26 gene mutation, obtains the cell of expression PPAR γ 2；Utilize the cell system of the expression PPAR γ 2 The animal that standby intramuscular fat content improves (compared with receptor).

In above-mentioned each method, the cell can be following a1) or a2) or a3):

A1) zooblast；

A2) mammalian cell；

A3) pig cell.

In the present invention, the pig cell can be pig fibroblast, such as porcine fetus fibroblasts.

In above-mentioned each method, the animal can be b1) or b2):

B1) mammal；

B2) pig.

Protection scope of the present invention is also belonged to using the various cells that method described above are prepared.

Application of the method described above in animal breeding also belongs to protection scope of the present invention.

In the application, the animal can be b1) or b2):

B1) mammal；

B2) pig.

It is demonstrated experimentally that mutation 2 gene of ROSA26 Gene targeting PPAR γ can be successfully obtained using method of the invention High intramuscular fat animal.The method that transgenic breeding is applied successfully in the present invention accelerates the cultivation of high-quality transgene pig new varieties.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of plasmid p-loxp and pLOXP-PPAR γ 2.A is the structural representation of p-loxp plasmid Figure；B is the structural schematic diagram of pLOXP-PPAR γ 2 (being Dondor in figure).

Fig. 2 is at primary 2 transgene pig insertion point of PPAR γ across left homology arm PCR qualification result.First swimming lane from left to right For 10000 DNA Marker；Second to the 7th swimming lane is primary PPAR γ transgene pig genomic DNA testing result, PCR inspection Expected specific fragment is measured, illustration purpose gene success site-directed integration is at Rosa26 fixed position；8th swimming lane is feminine gender Control, using the genomic DNA of pure negative pig as negative control.

Fig. 3 is at primary 2 transgene pig insertion point of PPAR γ across right homology arm PCR qualification result.First swimming lane from left to right For 10000 DNA Marker；Second to the 7th swimming lane is primary PPAR γ transgene pig genomic DNA testing result, PCR inspection Expection specific fragment is measured, illustration purpose gene succeeds site-directed integration at Rosa26 fixed position, wherein #320, # 322, #324 pig is not remove label transgene pig, and #314, #321, #323 are the pig for deleting marker gene expression frame, PCR Result difference is consistent with clip size is deleted；8th swimming lane is negative control, using the genomic DNA of pure negative pig as yin Property control.9th swimming lane is blank control, is detected by template of ddH2O.

Fig. 4 is primary 2 transgene pig marker gene Neo PCR qualification result of PPAR γ.The first swimming lane is 2000 from left to right DNA Marker；Second to the 7th swimming lane is primary PPAR γ transgene pig genomic DNA testing result, wherein #320, # 322, #324 pig is not remove label transgene pig, and #314, #321, #323 are the pig for deleting marker gene expression frame；8th Swimming lane is plasmid positive control；9th swimming lane is negative control, using the genomic DNA of pure negative pig as negative control.

Fig. 5 is #314, #321, #323 transgene pig (TD-6GZX I) sequencing result analysis chart.

Fig. 6 is #320, #322, #324 transgene pig (TD-6GZX II) sequencing result analysis chart.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

In following embodiments, unless otherwise specified, the 1st of each nucleotide sequence is the 5 ' of corresponding DNA in sequence table Terminal nucleotide, last bit are the 3 ' terminal nucleotides of corresponding DNA.

PSpCas9 (BB) -2A-Puro (PX459) V2.0 carrier: Addgene#62988.

P-loxp plasmid: by military science medical college king have it is bright give, the complete sequence of the plasmid such as SEQ ID No.6 institute Show plasmid map as shown in figure 1 shown in A.

2 plasmid of pEGFP-N1L-sMCK-sPPAR γ: it is recorded in " Jinliang Huang, et al.Ectopic overexpression of swine PPARγ2 upregulated adipocyte genes expression and triacylglycerol in skeletal muscle of mice.Transgenic Res(2012)21:1311-1318” One text, the public can obtain from applicant, can only be used to repeat present invention experiment use.

Embodiment 1 turns PPAR γ gene pig using sgRNA1 target sequence building ROSA26 site-directed integration

One, the building of recombinant vector

1, the building of CRISPR/Cas9-sgRNA1 carrier

The target DNA sequence that the sgRNA of ROSA26 gene is targeted in CRISPR/Cas9 technology is designed, such as SEQ ID No.1 institute Show.

Synthesize upstream and downstream oligonucleotides:

5’-CACCAGTTTGCTCCTTCTCGATTATGG- 3 ' (underscore part is SEQ ID No.1)；

5’-AAACCCATAATCGAGAAGGAGCAAACT- 3 ' (underscore part be SEQ ID No.1 reverse complemental Sequence).

Use ddH₂O is dissolved to 10 μM, takes each 3.9 μ L mixing of upstream and downstream complementary series solution respectively, is placed in 95 DEG C of 10min, 65 DEG C of 30min form the sequence with cohesive terminus,cohesive termini, obtain the target DNA double chain oligonucleotide of sgRNA after denaturation annealing. Using pSpCas9 (BB) -2A-Puro (PX459) V2.0 carrier framework, (pSpCas9 (BB) -2A-Puro (PX459) V2.0 is carried Body obtains after I digestion of BBS) it is connected with the target DNA double chain oligonucleotide of sgRNA, obtained sequence is correctly recombinated into load Body is named as CRISPR/Cas9-sgRNA, can express targeting sgRNA and cas9.

2, the building of donor template vector (PPAR γ-ploxp)

The sequence of each 1500bp in ROSA26 gene two sides of sgRNA targeting or so is expanded from pig genome with high fidelity enzyme Column, as homology arm, are respectively designated as left homology arm (LA) and right homology arm (RA).Use Not I (GCGGCCGC) and Xho I (CTCGAG) double digestion p-loxp plasmid and LA segment, connection obtain LA-ploxp plasmid.Use Sal I (GTCGAC) and EcoR I (GAATTC) double digestion LA-ploxp and RA segment, connection obtain LA-ploxp-RA plasmid.Using Swa I (ATTTAAAT) and 2 plasmid of Xho I (CTCGAG) double digestion LA-ploxp-RA and pEGFP-N1L-sMCK-sPPAR γ, connection obtain LA-MCK- The correct recombinant vector of obtained sequence is named as PPAR γ-ploxp (structural schematic diagram by PPAR γ-ploxp-RA plasmid See B in Fig. 1) as donor template vector.

In PPAR γ-ploxp carrier, left and right homology arm and between the two PPAR γ express the sequence such as SEQ ID of frame Shown in No.2.Wherein 817-824 of SEQ ID No.2 are the identification sequence of Not I, and 825-1810 are left homology arm Sequence, 1816-1823 for Swa I identify sequence, 1824-2838 be MCK promoter sequence, 2839-4353 For the gene order of PPAR γ, 4354-4593 are polyA termination signal sequence, and 4594-4599 identify for sal I Sequence, 4600-4632 are loxP sequence, and 4658-5164 are PGK promoter sequence, and 5175-5978 are NEO Coded sequence, 6468-6502 are loxP sequence, and 6508-6513 identify sequence for sal I, and 6514-8076 are Right homology arm sequence, 8077-8082 identify sequence for EcoR I.

Shown in the gene order coding SEQ ID No.3 of PPAR γ shown in 2839-4353 of SEQ ID No.2 PPAR γ protein.

Two, the screening and identification of 2 gene single cell clone of the site ROSA26 site-directed integration PPAR γ

It, will be thin with trypsase after being washed twice with PBS after porcine fetus fibroblasts grow into convergence degree >=80% Born of the same parents digest and carry out accurate metering, and 1500r/min is centrifuged 5min, removes supernatant after centrifugation, and cell is resuspended with PBS and washs 1 It is secondary to be centrifuged again, supernatant is removed completely as far as possible, then presses 200 L/1 × 10 μ⁶The concentration Electroporation of a cell Buffer is resuspended, and 10 μ g carrier (CRISPR/Cas9-sgRNA and the pLOXP-PPAR mixed are previously added in electric revolving cup γ 2 is mixed in the ratio of 1:1 (mass ratio)), the rear 200 μ l of cell liquid being added after being resuspended simultaneously is mixed well, and uses Bio-RAD Electroporation electric shock, electric shock program is 120v, 20ms, after electric shock, by cell inoculation into 6 orifice plates, with containing 20% tire ox The DMEM of serum is cultivated 48 hours, is gone in the DMEM containing 400 μ g/ml G418 and 20% fetal calf serum and is cultivated 48 hours, It shifts in the DMEM containing 400 μ g/ml G418 and 2 μM of Ganciclovir and 20% fetal calf serum and is cultivated to cell clone point again Occur.Clone's point is marked with marking pen, and after PBS is washed, clone's ring is placed on clone's point, 1 drop trypsase is added, disappears Complete medium is added after the completion of changing and terminates digestion, the liquid in ring will be cloned and be all sucked out, be transferred in 48 orifice plates and continue to train It supports, cell takes part for identifying after covering with, remaining cell freezes.

Cell genomic dna is extracted, PCR detection is carried out.It is identified respectively using left homology arm identification primer, right homology arm Primer identifies whether 3 ' ends and 5 ' ends occur homologous recombination, primer sequence respectively are as follows:

LA-F:5 '-TATCGTTTGTTACGCTGGAAGGGGAAGA-3 '

LA-R:5 '-GGGAGTTTATTTTTAGAGCTCGCTACTCG-3 '

RA-F:5 '-GCTGTGAAGTTCAACGCACTGGAATTAG-3 '

RA-R:5 '-GGTACAAGACTCAACAAGAACCTGTGCC-3 '

After correct homologous recombination occurs for left homology arm, can expand to size is 2800bp segment；Right homology arm occurs correct After homologous recombination, it can expand and arrive 2200bp segment.

The positive colony cell that correct homologous recombination occurs is chosen, is recovered into 48 orifice plates, it is normal to cultivate, it is long to cell To when converging rate up to 80%, handled 48 hours with Cre adenovirus.Pancreatin digestion centrifugation, collects cell, a part of cell extraction Micromanipulation liquid is added in cell genomic dna, remaining cell, be placed at room temperature for after resuspension 1h then go to 4 DEG C it is spare.It utilizes Neo identifies primer, to identify whether Neo gene is deleted, primer sequence are as follows:

Neo-F:5 '-TTGTCACTGAAGCGGGAAGGG-3 '

Neo-R:5 '-TCAAGAAGGCGATAGAAGGCG-3 '

Neo gene can't detect aim sequence if being deleted, and deleted can expand to size is 400bp segment. The cell that Neo gene is deleted is used for nuclear transfer as donorcells.

Three, ROSA26 gene loci turns the building and identification of 2 gene pig of PPAR γ

1, the preparation of solution

NCSU-23 culture solution sequentially adds 3.178g NaCl, 1.053g NaHCO in 300mL Milli-Q ultrapure water₃, 0.178g KCl, 0.081g KH₂PO₄, 0.147g MgSO₄·7H₂O, 0.125g CaCl₂·2H₂O, 0.500g D-Glucose (D-Glucose), 0.073g glutamine (Glutamine), 0.438g β-aminoethanesulfonic acid (Taurine), 0.273g ammonia second Base sulfinic acid (Hypotaurine), 0.025g streptomysin, 0.033g penicillin powder, by pH value be adjusted to 7.2~7.4 it Between, it finally is settled to 500mL with ultrapure water, the osmotic pressure after constant volume is 295~310m Osm, with Millipore company After 0.22 μm of filter of SVGP01050 type carries out aseptic filtration, it is stored in 4 DEG C, it is spare.

IVM1 culture solution, into NSCU23 culture solution press 10% pFF, 10IU/mL PMSG, 10ng/mL EGF and The culture solution that the hCG concentration addition respective substance of 10IU/mL obtains, mixes well.

IVM2 maturation liquid is obtained into NSCU23 culture solution by 10% pFF and 10ng/mL EGF addition respective substance Culture solution.

Micromanipulation liquid containing BSA weighs 0.770g NaCl, 0.0356g NaHCO₃, 0.0296g MgSO₄· 7H₂O, 0.0162g KCl, 0.0296g KH₂PO₄, 0.0146g L-Glutamine, 0.1g glucose be dissolved in 60 milliliters it is ultrapure In water, after mixing well dissolution, 0.238g HEPES is added, 0.150g taurine, 0.0065g penicillin, 0.4g ox blood is pure Albumen, 0.005g streptomycin sulphate adjust pH in 7.2-7.4 range after completely dissolution, and constant volume is in 100 milliliters of volumetric flasks, and 0.22 μm filter degerming packing, is placed in 4 DEG C of preservations.

Fusion/activating fluid, solvent are water, and solute and its concentration are respectively 0.1mmol/L MgCl₂, 0.28mol/L sweet dew Alcohol, 0.5mmol/L HEPES, 0.1mmol/L CaCl₂And 0.01%PV (mass percent concentration).

2, operating method

Using 12# needle applicator, well-developed, the liquid in ovarian follicle being of moderate size on pig ovary, injection are drawn In 50ml centrifuge tube.Supernatant is abandoned after 37 DEG C of natural sedimentations, egg liquid is cleaned twice.The densification of picking cytoplasm, ovum under stereomicroscope Wrap up 3 layers or more of egg mother cell in mound.After cleaning 3 times with DMEM, it is transferred to the IVM1 culture for being incubated for 4h in 38 DEG C of incubators In liquid, after maturation in vitro, egg mother cell is transferred in the mature liquid IVM2 of no PMSG and hCG and continues to cultivate, carry out in vitro at It is ripe.By accumulative 40 hours or so maturation in vitro, it was 1mg/ml hyaluronidase that egg mother cell, which is gone to containing concentration, In the culture dish of DMEM, egg mother cell is firmly blown and beaten under the microscope with the pipettor of 200 μ L, is completely fallen off to cumulus cell Afterwards, solid glass needle is used under Stereo microscope, it is egg membrane form is complete, perivitelline size is suitable and has first polar body discharge Mature oocyte, be put into spare in micromanipulation liquid.

Mature egg mother cell is sucked with holding ovum needle under the microscope, then egg mother cell is stirred with injection needle, by ovum Mature first polar body is adjusted to the position at 1 o'clock of clock on mother cell；First polar body is drawn in first polar body with injection needle The cytoplasm of 10%~20% adjacent egg mother cell of side removes ovocyte karyon；20 μm of diameter or so are selected, The nuclear donor cell that round, smooth step two obtains, is put into perivitelline, presses oolemma with injection needle, make donorcells with Receptor ovum after birth contact closely to get arrive reconstructed eggs；Reconstructed eggs are added in the micromanipulation liquid containing BSA and restore 5 points Clock；Then reconstruct egg white is washed three times with fusion/activating fluid, then reconstructed eggs is transferred in batches in fusion/activating fluid, are washed Wash 3 times, each a small amount of being put into has been paved in the 1mm integration slot of fusion liquid, with solid glass needle by reconstructed eggs cell with Integration slot is vertical, then applies the electric pulse induced fusion of one 100 μ s, 140V with BLS cell fusion apparatus, uses later The CHX of 7.5 μ g/ml carries out the chemical Assisted Activation of 4h or so, and the nuclear donor for obtaining replacing with the core of pig egg cell step 2 is thin The reconstruct cell of the core of born of the same parents.

The reconstruct cell of acquisition is cultivated in vitro, Successful development is selected after 12h to 1-2 cell stage and form Good reconstructed embryo carries out embryo transfer.The receptor of embryo transfer is the large white sow of the puberty of spontaneous estrus.Embryo transfer Method be to be pulled out the ovary of receptor sow by modus operandi, check the heat condition of receptor sow, with follicular development compared with It is good, and the sow that will be ovulated is that receptor is transplanted.Fallopian tubal is gently pulled out, embryo is injected to the deep of fallopian tubal, often Head receptor sow transplants 200 pieces or so of reconstructed embryo.After embryo transfer 30 days, ultrasonic wave gestation detector test receptor is utilized Pig pregnancy situation, confirms receptor pig successful pregnancies.

Receptor sow is given a birth transgene pig by the way of spontaneous labor.Be obtained transgenosis piggy 6 (#314, #320, #321, #322, #323, #324), the PCR that birth samples birth piggy ear tissue within latter week carries out transgene pig reflects It is fixed.The piggy of birth is identified respectively using left and right homology arm detection primer and Neo identification primer (sequence is seen above), And compareed with negative pig, qualification result is as shown in Figure 2, Figure 3 and Figure 4.The result shows that utilizing method success of the invention It has obtained ROSA26 gene loci and has turned 2 gene pig of PPAR γ.

Further, 6 primary transgenosis piggys (#314, #320, #321, #322, #323, #324) are sequenced, As a result further confirm that successfully having obtained ROSA26 gene loci using method of the invention turns 2 gene pig of PPAR γ.Wherein, Fig. 5 is #314, #321, #323 transgene pig (TD-6GZX I) sequencing result analysis chart, and SEQ ID No.4 is #314 transgene pig Sequencing result.Fig. 6 is #320, #322, #324 transgene pig (TD-6GZX II) sequencing result analysis chart, and SEQ ID No.5 is # 320 transgene pig sequencing results.

<110>Hua Zhong Agriculture University

<120>method of the animal of ROSA26 gene mutation and intramuscular fat raising is prepared

<130> GNCLN181594

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 23

<212> DNA

<213> Artificial sequence

<400> 1

agtttgctcc ttctcgatta tgg 23

<210> 2

<211> 12750

<212> DNA

<213> Artificial sequence

<400> 2

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 60

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 120

acaccgaact gagataccta cagcgtgagc tatgagagag cgccacgctt cccgaaggga 180

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 240

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 300

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 360

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 420

tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 480

gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 540

gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 600

ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 660

caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 720

aacaatttca cacaggaaac agctatgacc atgattacgc caagctcgaa attaaccctc 780

actaaaggga acaaaagctg gagctccacc gcggtggcgg ccgcgaggca ggcgggagtg 840

cggcccgccc tgcggcaacc ggagggggag ggagaaggga gcggaaaagc ctggaatacg 900

gacggagcca ttgctcccgc agagggagga gcgcttcctg ctcttctctt gtcactgatt 960

ggccgcttct cctcccgccg tgtgtgaaaa cacaaatggc gtgttttggt tggagtaaag 1020

ctcctgtcag ttacagcctc gggagtgcgc agcctcccag gaactctcgc attgccccct 1080

gggtgggtag gtaggtgggg tggagagagc tgcacaggcg ggcgctgtcg gcctcctgcg 1140

gggggagggg agggtcagtg aaagtggctc ccgcgcgggc gtcctgccac cctcccctcc 1200

gggggagtcg gtttacccgc cgcctgctcg gctttggtat ctgattggct gctgaagtcc 1260

tgggaacggc cccttgttat tggcttgggt cccaaatgag cgaaaccact acgcgagtcg 1320

gcagggaggc ggtctttggt acggccctcc ccgaggccag cgccgcagtg tctggcccct 1380

cgcccctgcg caacgtggca ggaagcgcgc gcaggaggcg ggggcgggct gccgggccga 1440

ggcttctggg tggtggtgac tgcggctccg ccctgggcgt ccgccgcctg aaggacgaga 1500

ctagctctct acctgctctc ggacccgtgg gggtgggggg tggaggaagg agtggggggt 1560

cggtcctgct ggcttgtggg tgggaggcgc atgttctcca aaaacccgcg cgagctgcaa 1620

tcctgaggga gctgcagtgg aggaggcgga gagaaggccg cacccttctc cgcaggggga 1680

ggggagtgcc gcaatacctt tatgggagtt ctctgctgcc tccttttcct aaggaccgcc 1740

ctgggcctag aaaaatccct ccctcccccg cgatctcgtc atcgcctcca tgtcagtttg 1800

ctccttctcg ttaacattta aatccacaga gtctcctcaa ccccgagagc cctgtgctct 1860

gacccgagtt gccgcccagc ctcctccccc tgcgggtggg tgtggacgcc tccccagggc 1920

cggggctgtg gctgcccttg taaggaggtg aggcctgggg acaccagaga ggcctggtta 1980

taattaaccg ggacacgtgg ccagcccgcc cccaacacct gcccccgccc ctgcccccat 2040

ccccagcgcc tcgggtctcc cggaggagac agcgagtagc gagctctaaa aataaactcc 2100

cttttctgca agcctgcagg ccctgtcccc tccagcgtgg aatcacccag tgtcactggg 2160

ccctgcgccg cttctggcct ggctttgagt ctgaatggcc cccctgggcc cggcctcgtg 2220

tcccccccac tgccatcaag gagggaaaac ccgctaagca caggcatcag ggatcaggct 2280

gcccagctcc cacctctgcc cgggtcacag gctccctgta gccgggtgac agtaggcaaa 2340

tcacgcagcc tctctgggcc actatttcct cctctggaga accagacact tggtccttct 2400

gggatgatgg cagggtttcc ccagaagcag ggctcaggac tttgctgggg tcaaggccac 2460

cctgggggcc aaggagagac tggcggccta gcggagggcc aggggagggt ggtttctacg 2520

tgcctgggac agcctctgac acagtcccgt ggccccggcg gggggccagc tgtccccgcc 2580

agcccgactc agcacttggt ctggggacca gcttggtttg ggggtggggg gtgggcccag 2640

cccctggggc ggcccataca aggccatggg gctgggcgca aggcacgcct gggttcaggg 2700

tgggcacggt gcccaggcag cgaagcgaga gcgcagctgc cctccacccc cctcctggcc 2760

agcggcccct cctgaccaat agcacaacct gggccccccc tataaaaggc cagggctgca 2820

gtcctgtcct ttgtcgacat gggtgaaact ctgggagatt ctcttattga cccagaaagc 2880

gatgccttcg acacgctgtc tgcaaacatt tcacaagagg tgaccatggt tgacaccgag 2940

atgccgtttt ggcccaccaa ctttgggatc agctctgtgg acctgtcggt gatggacgac 3000

cactcccact cctttgacat caagcccttc accactgttg atttctccag catttccact 3060

ccacactatg aagacatccc attcccgaga gctgatccaa tggttgcaga ttataagtac 3120

gacctgaagc tccaggacta ccaaagtgcc atcaaagtgg agcctgtgtc cccgccttat 3180

tattctgaaa agactcagct gtacaacaaa cctcacgaag agccttccaa ctccctcatg 3240

gcaattgaat gccgtgtctg tggggataaa gcctcggggt tccactatgg agttcatgct 3300

tgtgaaggat gcaagggttt cttccggagg actatcagat tgaagcttat ttatgatagg 3360

tgcgatctta actgtcggat ccacaaaaaa agtagaaata aatgtcagta ctgtcggttt 3420

cagaaatgcc ttgctgtggg gatgtctcat aacgccatca ggtttgggcg gatgccacag 3480

gctgagaagg agaagctgtt ggcagagatc tccagtgata tcgaccagct gaacccagag 3540

tctgctgacc tccgcgccct ggcaaagcac ttgtatgact catacataaa gtccttcccg 3600

ctgaccaaag caaaggcgag ggcgatcttg acaggaaaga ccacagacaa atcacccttt 3660

gtcatctatg acatgaattc cttaatgatg ggagaagata aaatcaagtt caaacacatc 3720

acccccctgc aggagcagag caaagaggtg gccattcgca tctttcaggg gtgtcagttt 3780

cgctccgtgg aggccgtgca ggagatcaca gagtatgcca agaacatccc tgggtttgta 3840

aaccttgacc tgaatgacca agtaactctc ctaaagtatg gcgtccacga gatcatttac 3900

accatgctgg cctccttgat gaataaagac ggggtcctca tctccgaggg ccaaggattc 3960

atgacaaggg agtttctcaa gagcctgaga aagccctttg gtgactttat ggagcccaag 4020

ttcgagtttg ctgtgaagtt caacgcactg gaattagatg acagcgacct ggcgatattt 4080

atagctgtca ttattctcag tggagaccgc ccaggtttgc tgaatgtgaa gcccatcgag 4140

gacattcaag acaatttgct gcaagccttg gagctgcagc tcaagttgaa ccacccagag 4200

tcctcccagc tctttgctaa gctgctccag aaaatgacag acctcagaca gattgtgaca 4260

gagcacgtgc agctgctgca agtaataaag aagacagaga cggacatgag ccttcaccca 4320

ctcctacagg aaatatacaa ggacttgtac taggcggccg cgactctaga tcataatcag 4380

ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa 4440

cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg 4500

ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 4560

tagttgtggt ttgtccaaac tcatcaatgt atctcgagat aacttcgtat aatgtatgct 4620

atacgaagtt atgtcgaggg cccctgcagg tcaattctac cgggtagggg aggcgctttt 4680

cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag 4740

tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt 4800

tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc 4860

gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca 4920

gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca 4980

gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct 5040

caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg 5100

cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc 5160

gacctgcagc caatatggga tcggccattg aacaagatgg attgcacgca ggttctccgg 5220

ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 5280

atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 5340

tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 5400

cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 5460

tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 5520

tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 5580

tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 5640

tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 5700

ggctcaaggc gcgcatgccc gacggcgatg atctcgtcgt gacccatggc gatgcctgct 5760

tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 5820

gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 5880

gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 5940

gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg ggatcgatcc gctgtaagtc 6000

tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa gtttttcctg 6060

tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga aggattggag 6120

ctacgggggt gggggtgggg tgggattaga taaatgcctg ctctttactg aaggctcttt 6180

actattgctt tatgataatg tttcatagtt ggatatcata atttaaacaa gcaaaaccaa 6240

attaagggcc agctcattcc tcccactcat gatctataga tctatagatc tctcgtggga 6300

tcattgtttt tctcttgatt cccactttgt ggttctaagt actgtggttt ccaaatgtgt 6360

cagtttcata gcctgaagaa cgagatcagc agcctctgtt ccacatacac ttcattctca 6420

gtattgtttt gccaagttct aattccatca gaagctgact ctagcaatat aacttcgtat 6480

aatgtatgct atacgaagtt atctagagtc gacattatgg ggcgggattc ttttgccctg 6540

gcttaacctg attcttgggc gttgtcctgc aggggattga gcaggtgtac gaggacgagc 6600

ccaatttctc tatattccca cagtcttgag tttgtgtcac aaaataatta tagtggggtg 6660

gagatgggaa atgagtccag gcaacaccta agcctgattt tatgcattga gactgcgtgt 6720

tattactaaa gatctttgtg tcgcaatttc ctgatgaagg gagataggtt aaaaagcacg 6780

gatctactga gttttacagt catcccattt gtagactttt gctacaccac caaagtatag 6840

catctgagat taaatattaa tctccaaacc ttaggccccc tcacttgcat ccttacggtc 6900

agataactct cactcatact ttaagcccat tttgtttgtt gtacttgctc atccagtccc 6960

agacatagca ttggctttct cctcacctgt tttaggtagc cagcaagtca tgaaatcaga 7020

taagttccac caccaattaa cactacccat cttgagcata ggcccaacag tgcatttatt 7080

cctcatttac tgatgttcgt gaatatttac cttgattttc atttttttct ttttcttaag 7140

ctgggatttt actcctgacc ctattcacag tcagatgatc ttgactacca ctgcgattgg 7200

acctgaggtt cagcaatact cccctttatg tcttttgaat acttttcaat aaatctgttt 7260

gtattttcat tagttagtaa ctgagctcag ttgccgtaat gctaatagct tccaaactag 7320

tgtctctgtc tccagtatct gataaatctt aggtgttgct gggacagttg tcctaaaatt 7380

aagataaagc atgaaaataa ctgacacaac tccattactg gctcctaact acttaaacaa 7440

tgcattctat catcacaaat gtgaaaaagg agttccctca gtggactaac cttatctttt 7500

ctcaacacct ttttctttgc acaattttcc acacatgcct acaaaaagta cttctctgct 7560

caagtcacac tgagttgatt gctatttacc gaaatcaaag taacattatc agatctctgt 7620

agggtggttc cctctggaat gctaccctcc atagtcctta cccttcaagt aaagagcatg 7680

aagactgaaa tatctctgtg atctgtcatc ctttaagcca gaatccccca taaaaaagtt 7740

agtattgctt tctcctgatc ccatagcagg ttgaatcata gcacttatca ggttgttgtc 7800

attgcttgct taaattctcc taactatttg gagcttcttg agggcacagg ttcttgttga 7860

gtcttgtacc taagcaccta gtatagtcct tgatgtctag ccaaccctaa ataaaatgca 7920

gtgagtgaca tgtagatgtc tttataaggt ttgataggtt ggtctctcaa acagttcttt 7980

tgtatgtttg gtagtgctct agattagcac tggccagtat aactctgatg atggaaatgt 8040

tctatagcta tgctgtctaa tatggtaccg agctcgaatt ctaccgggta ggggaggcgc 8100

ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca 8160

caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt 8220

tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg 8280

ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg 8340

tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat 8400

agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg 8460

ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat 8520

tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc 8580

tttcgacctg cagcgacccg cttaacagcg tcaacagcgt gccgcagatc ttggtggcgt 8640

gaaactcccg cacctcttcg gcaagcgcct tgtagaagcg cgtatggctt cgtacccctg 8700

ccatcaacac gcgtctgcgt tcgaccaggc tgcgcgttct cgcggccata gcaaccgacg 8760

tacggcgttg cgccctcgcc ggcagcaaga agccacggaa gtccgcctgg agcagaaaat 8820

gcccacgcta ctgcgggttt atatagacgg tcctcacggg atggggaaaa ccaccaccac 8880

gcaactgctg gtggccctgg gttcgcgcga cgatatcgtc tacgtacccg agccgatgac 8940

ttactggcag gtgctggggg cttccgagac aatcgcgaac atctacacca cacaacaccg 9000

cctcgaccag ggtgagatat cggccgggga cgcggcggtg gtaatgacaa gcgcccagat 9060

aacaatgggc atgccttatg ccgtgaccga cgccgttctg gctcctcata tcggggggga 9120

ggctgggagc tcacatgccc cgcccccggc cctcaccctc atcttcgacc gccatcccat 9180

cgccgccctc ctgtgctacc cggccgcgcg ataccttatg ggcagcatga ccccccaggc 9240

cgtgctggcg ttcgtggccc tcatcccgcc gaccttgccc ggcacaaaca tcgtgttggg 9300

ggcccttccg gaggacagac acatcgaccg cctggccaaa cgccagcgcc ccggcgagcg 9360

gcttgacctg gctatgctgg ccgcgattcg ccgcgtttac gggctgcttg ccaatacggt 9420

gcggtatctg cagggcggcg ggtcgtggcg ggaggattgg ggacagcttt cggggacggc 9480

cgtgccgccc cagggtgccg agccccagag caacgcgggc ccacgacccc atatcgggga 9540

cacgttattt accctgtttc gggcccccga gttgctggcc cccaacggcg acctgtacaa 9600

cgtgtttgcc tgggccttgg acgtcttggc caaacgcctc cgtcccatgc acgtctttat 9660

cctggattac gaccaatcgc ccgccggctg ccgggacgcc ctgctgcaac ttacctccgg 9720

gatggtccag acccacgtca ccacccccgg ctccataccg acgatctgcg acctggcgcg 9780

cacgtttgcc cgggagatgg gggaggctaa ctgaaacacg gaaggagaca ataccggaag 9840

gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt tgggtcgttt 9900

gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc 9960

cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt 10020

gaaggcccag ggctcgcagc caacgtcggg gcggcaagcc ctgccatagc cacgggcccc 10080

gtgggttagg gacggggtcc cccatgggga atggtttatg gttcgtgggg gttattattt 10140

tgggcgttgc gtggggtcag gtccacgact ggactgagca gacagaccca tggtttttgg 10200

atggcctggg catggaccgc atgtactggc gcgacacgaa caccgggcgt ctgtggctgc 10260

caaacacccc cgacccccaa aaaccaccgc gcggatttct ggcgccgccg gacgaactaa 10320

acctgactac ggcatctctg ccccttcttc gctggtacga ggagcgcttt tgttttgtat 10380

tggtcaccac ggccgagttt ccgcgggacc ccggccagga cctgcagaaa ttgatgatct 10440

attaaacaat aaagatgtcc actaaaatgg aagtttttcc tgtcatactt tgttaagaag 10500

ggtgagaaca gagtacctac attttgaatg gaaggattgg agctacgggg gtgggggtgg 10560

ggtgggatta gataaatgcc tgctctttac tgaaggctct ttactattgc tttatgataa 10620

tgtttcatag ttggatatca taatttaaac aagcaaaacc aaattaaggg ccagctcatt 10680

cctcccactc atgatctata gatctataga tctctcgtgg gatcattgtt tttctcttga 10740

ttcccacttt gtggttctaa gtactgtggt ttccaaatgt gtcagtttca tagcctgaag 10800

aacgagatca gcagcctctg ttccacatac acttcattct cagtattgtt ttgccaagtt 10860

ctaattccat cagaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc 10920

ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 10980

gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 11040

gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 11100

ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 11160

ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 11220

ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 11280

gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 11340

agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 11400

aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 11460

attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 11520

cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 11580

aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 11640

ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 11700

gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 11760

attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 11820

tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 11880

tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 11940

atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 12000

agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 12060

aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 12120

caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 12180

ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 12240

gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 12300

tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 12360

atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 12420

tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 12480

accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 12540

gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 12600

caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 12660

tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 12720

ctctgctaat cctgttacca gtggctgctg 12750

<210> 3

<211> 504

<212> PRT

<213> Artificial sequence

<400> 3

Met Gly Glu Thr Leu Gly Asp Ser Leu Ile Asp Pro Glu Ser Asp Ala

1 5 10 15

Phe Asp Thr Leu Ser Ala Asn Ile Ser Gln Glu Val Thr Met Val Asp

20 25 30

Thr Glu Met Pro Phe Trp Pro Thr Asn Phe Gly Ile Ser Ser Val Asp

35 40 45

Leu Ser Val Met Asp Asp His Ser His Ser Phe Asp Ile Lys Pro Phe

50 55 60

Thr Thr Val Asp Phe Ser Ser Ile Ser Thr Pro His Tyr Glu Asp Ile

65 70 75 80

Pro Phe Pro Arg Ala Asp Pro Met Val Ala Asp Tyr Lys Tyr Asp Leu

85 90 95

Lys Leu Gln Asp Tyr Gln Ser Ala Ile Lys Val Glu Pro Val Ser Pro

100 105 110

Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Lys Pro His Glu Glu

115 120 125

Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg Val Cys Gly Asp Lys

130 135 140

Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly

145 150 155 160

Phe Phe Arg Arg Thr Ile Arg Leu Lys Leu Ile Tyr Asp Arg Cys Asp

165 170 175

Leu Asn Cys Arg Ile His Lys Lys Ser Arg Asn Lys Cys Gln Tyr Cys

180 185 190

Arg Phe Gln Lys Cys Leu Ala Val Gly Met Ser His Asn Ala Ile Arg

195 200 205

Phe Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu Ala Glu Ile

210 215 220

Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg Ala

225 230 235 240

Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu Thr

245 250 255

Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr Thr Asp Lys Ser

260 265 270

Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met Met Gly Glu Asp Lys

275 280 285

Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu Val

290 295 300

Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala Val

305 310 315 320

Gln Glu Ile Thr Glu Tyr Ala Lys Asn Ile Pro Gly Phe Val Asn Leu

325 330 335

Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu Ile

340 345 350

Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu Ile

355 360 365

Ser Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu Arg

370 375 380

Lys Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe Ala Val Lys

385 390 395 400

Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile Ala

405 410 415

Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys Pro

420 425 430

Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln Leu

435 440 445

Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys Leu Leu Gln

450 455 460

Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val Gln Leu Leu

465 470 475 480

Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu Leu

485 490 495

Gln Glu Ile Tyr Lys Asp Leu Tyr

500

<210> 4

<211> 3845

<212> DNA

<213> Artificial sequence

<400> 4

ctactacccg cttgggtcca atgagcgaac cactacgcga gtcggcaggg aggcggtctt 60

tggtacggcc ctccccgagg ccagcgccgc agtgtctggc ccctcgcccc tgcgcaacgt 120

ggcaggaagc gcgcgcagga ggcgggggcg ggctgccggg ccgaggcttc tgggtggtgg 180

tgactgcggc tccgccctgg gcgtccgccg cctgaaggac gagactagct ctctacctgc 240

tctcggaccc gtgggggtgg ggggtggagg aaggagtggg gggtcggtcc ctgctggctt 300

gtgggtggga ggcgcatgtt tctccaaaac ccgcgcgagc tgcaatcctg agggagctgc 360

agtggaggag gcggagagaa ggccgcaccc ttctccgcag ggggagggga gtgccgcaat 420

acctttatgg gagttctctg ctgcctcctt ttcctaagga ccgccctggg cctagaaaaa 480

tccctccctc ccccgcgatc tcgtcatcgc ctccatgtca gtttgctcct tctcgttaac 540

atttaaatcc acagagtctc ctcaaccccg agagccctgt gctctgaccc gagttgccgc 600

ccagcctcct ccccctgcgg gtgggtgtgg acgcctcccc agggccgggg ctgtggctgc 660

ccttgtaagg aggtgaggcc tggggacacc agagaggcct ggttataatt aaccgggaca 720

cgtggccagc ccgcccccaa cacctgcccc cgcccctgcc cccatcccca gcgcctcggg 780

tctcccggag gagacagcga gtagcgagct ctaaaaataa actccctttt ctgcaagcct 840

gcaggccctg tcccctccag cgtggaatca cccagtgtca ctgggccctg cgccgcttct 900

ggcctggctt tgagtctgaa tggcccccct gggcccggcc tcgtgtcccc cccactgcca 960

tcaaggaggg aaaacccgct aagcacaggc atcagggatc aggctgccca gctcccacct 1020

ctgcccgggt cacaggctcc ctgtagccgg gtgacagtag gcaaatcacg cagcctctct 1080

gggccactat ttcctcctct ggagaaccag acacttggtc cttctgggat gatggcaggg 1140

tttccccaga agcagggctc aggactttgc tggggtcaag gccaccctgg gggccaagga 1200

gagactggcg gcctagcgga gggccagggg agggtggttt ctacgtgcct gggacagcct 1260

ctgacacagt cccgtggccc cggcgggggg ccagctgtcc ccgccagccc gactcagcac 1320

ttggtctggg gaccagcttg gtttgggggt ggggggtggg cccagcccct ggggcggccc 1380

atacaaggcc atggggctgg gcgcaaggca cgcctgggtt cagggtgggc acggtgccca 1440

ggcagcgaag cgagagcgca gctgccctcc acccccctcc tggccagcgg cccctcctga 1500

ccaatagcac aacctgggcc ccccctataa aaggccaggg ctgcagtcct gtcctttgtc 1560

gacatgggtg aaactctggg agattctctt attgacccag aaagcgatgc cttcgacacg 1620

ctgtctgcaa acatttcaca agaggtgacc atggttgaca ccgagatgcc gttttggccc 1680

accaactttg ggatcagctc tgtggacctg tcggtgatgg acgaccactc ccactccttt 1740

gacatcaagc ccttcaccac tgttgatttc tccagcattt ccactccaca ctatgaagac 1800

atcccattcc cgagagctga tccaatggtt gcagattata agtacgacct gaagctccag 1860

gactaccaaa gtgccatcaa agtggagcct gtgtccccgc cttattattc tgaaaagact 1920

cagctgtaca acaaacctca cgaagagcct tccaactccc tcatggcaat tgaatgccgt 1980

gtctgtgggg ataaagcctc ggggttccac tatggagttc atgcttgtga aggatgcaag 2040

ggtttcttcc ggaggactat cagattgaag cttatttatg ataggtgcga tcttaactgt 2100

cggatccaca aaaaaagtag aaataaatgt cagtactgtc ggtttcagaa atgccttgct 2160

gtggggatgt ctcataacgc catcaggttt gggcggatgc cacaggctga gaaggagaag 2220

ctgttggcag agatctccag tgatatcgac cagctgaacc cagagtctgc tgacctccgc 2280

gccctggcaa agcacttgta tgactcatac ataaagtcct tcccgctgac caaagcaaag 2340

gcgagggcga tcttgacagg aaagaccaca gacaaatcac cctttgtcat ctatgacatg 2400

aattccttaa tgatgggaga agataaaatc aagttcaaac acatcacccc cctgcaggag 2460

cagagcaaag aggtggccat tcgcatcttt caggggtgtc agtttcgctc cgtggaggcc 2520

gtgcaggaga tcacagagta tgccaagaac atccctgggt ttgtaaacct tgacctgaat 2580

gaccaagtaa ctctcctaaa gtatggcgtc cacgagatca tttacaccat gctggcctcc 2640

ttgatgaata aagacggggt cctcatctcc gagggccaag gattcatgac aagggagttt 2700

ctcaagagcc tgagaaagcc ctttggtgac tttatggagc ccaagttcga gtttgctgtg 2760

aagttcaacg cactggaatt agatgacagc gacctggcga tatttatagc tgtcattatt 2820

ctcagtggag accgcccagg tttgctgaat gtgaagccca tcgaggacat tcaagacaat 2880

ttgctgcaag ccttggagct gcagctcaag ttgaaccacc cagagtcctc ccagctcttt 2940

gctaagctgc tccagaaaat gacagacctc agacagattg tgacagagca cgtgcagctg 3000

ctgcaagtaa taaagaagac agagacggac atgagccttc acccactcct acaggaaata 3060

tacaaggact tgtactaggc ggccgcgact ctagatcata atcagccata ccacatttgt 3120

agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat 3180

gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa 3240

tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 3300

caaactcatc aatgtatctc gagataactt cgtataatgt atgctatacg aagttatcta 3360

gagtcgacat tatggggcgg gattcttttg ccctggctta acctgattct tgggcgttgt 3420

cctgcagggg attgagcagg tgtacgagga cgagcccaat ttctctatat tcccacagtc 3480

ttgagtttgt gtcacaaaat aattatagtg gggtggagat gggaaatgag tccaggcaac 3540

acctaagcct gattttatgc attgagactg cgtgttatta ctaaagatct ttgtgtcgca 3600

atttcctgat gaagggagat aggttaaaaa gcacggatct actgagtttt acagtcatcc 3660

catttgtaga cttttgctac accaccaaag tatagcatct gagattaaat attaatctcc 3720

aaaccttagg ccccctcact tgcatcctta cggtcagata actctcactc atactttaag 3780

cccattttgt ttgttgtact tgctcatcca gtcccagaca taaacattgg cagggtgagg 3840

cgggc 3845

<210> 5

<211> 5733

<212> DNA

<213> Artificial sequence

<400> 5

tgattggctg ctgaagtcct gggaacggcc ccttgttatt ggcttgggtc ccaaatgagc 60

gaaaccacta cgcgagtcgg cagggaggcg gtctttggta cggccctccc cgaggccagc 120

gccgcagtgt ctggcccctc gcccctgcgc aacgtggcag gaagcgcgcg caggaggcgg 180

gggcgggctg ccgggccgag gcttctgggt ggtggtgact gcggctccgc cctgggcgtc 240

cgccgcctga aggacgagac tagctctcta cctgctctcg gacccgtggg ggtggggggt 300

ggaggaagga gtggggggtc ggtcctgctg gcttgtgggt gggaggcgca tgttctccaa 360

aaacccgcgc gagctgcaat cctgagggag ctgcagtgga ggaggcggag agaaggccgc 420

acccttctcc gcagggggag gggagtgccg caataccttt atgggagttc tctgctgcct 480

ccttttccta aggaccgccc tgggcctaga aaaatccctc cctcccccgc gatctcgtca 540

tcgcctccat gtcagtttgc tccttctcgt taacatttaa atccacagag tctcctcaac 600

cccgagagcc ctgtgctctg acccgagttg ccgcccagcc tcctccccct gcgggtgggt 660

gtggacgcct ccccagggcc ggggctgtgg ctgcccttgt aaggaggtga ggcctgggga 720

caccagagag gcctggttat aattaaccgg gacacgtggc cagcccgccc ccaacacctg 780

cccccgcccc tgcccccatc cccagcgcct cgggtctccc ggaggagaca gcgagtagcg 840

agctctaaaa ataaactccc ttttctgcaa gcctgcaggc cctgtcccct ccagcgtgga 900

atcacccagt gtcactgggc cctgcgccgc ttctggcctg gctttgagtc tgaatggccc 960

ccctgggccc ggcctcgtgt cccccccact gccatcaagg agggaaaacc cgctaagcac 1020

aggcatcagg gatcaggctg cccagctccc acctctgccc gggtcacagg ctccctgtag 1080

ccgggtgaca gtaggcaaat cacgcagcct ctctgggcca ctatttcctc ctctggagaa 1140

ccagacactt ggtccttctg ggatgatggc agggtttccc cagaagcagg gctcaggact 1200

ttgctggggt caaggccacc ctgggggcca aggagagact ggcggcctag cggagggcca 1260

ggggagggtg gtttctacgt gcctgggaca gcctctgaca cagtcccgtg gccccggcgg 1320

ggggccagct gtccccgcca gcccgactca gcacttggtc tggggaccag cttggtttgg 1380

gggtgggggg tgggcccagc ccctggggcg gcccatacaa ggccatgggg ctgggcgcaa 1440

ggcacgcctg ggttcagggt gggcacggtg cccaggcagc gaagcgagag cgcagctgcc 1500

ctccaccccc ctcctggcca gcggcccctc ctgaccaata gcacaacctg ggccccccct 1560

ataaaaggcc agggctgcag tcctgtcctt tgtcgacatg ggtgaaactc tgggagattc 1620

tcttattgac ccagaaagcg atgccttcga cacgctgtct gcaaacattt cacaagaggt 1680

gaccatggtt gacaccgaga tgccgttttg gcccaccaac tttgggatca gctctgtgga 1740

cctgtcggtg atggacgacc actcccactc ctttgacatc aagcccttca ccactgttga 1800

tttctccagc atttccactc cacactatga agacatccca ttcccgagag ctgatccaat 1860

ggttgcagat tataagtacg acctgaagct ccaggactac caaagtgcca tcaaagtgga 1920

gcctgtgtcc ccgccttatt attctgaaaa gactcagctg tacaacaaac ctcacgaaga 1980

gccttccaac tccctcatgg caattgaatg ccgtgtctgt ggggataaag cctcggggtt 2040

ccactatgga gttcatgctt gtgaaggatg caagggtttc ttccggagga ctatcagatt 2100

gaagcttatt tatgataggt gcgatcttaa ctgtcggatc cacaaaaaaa gtagaaataa 2160

atgtcagtac tgtcggtttc agaaatgcct tgctgtgggg atgtctcata acgccatcag 2220

gtttgggcgg atgccacagg ctgagaagga gaagctgttg gcagagatct ccagtgatat 2280

cgaccagctg aacccagagt ctgctgacct ccgcgccctg gcaaagcact tgtatgactc 2340

atacataaag tccttcccgc tgaccaaagc aaaggcgagg gcgatcttga caggaaagac 2400

cacagacaaa tcaccctttg tcatctatga catgaattcc ttaatgatgg gagaagataa 2460

aatcaagttc aaacacatca cccccctgca ggagcagagc aaagaggtgg ccattcgcat 2520

ctttcagggg tgtcagtttc gctccgtgga ggccgtgcag gagatcacag agtatgccaa 2580

gaacatccct gggtttgtaa accttgacct gaatgaccaa gtaactctcc taaagtatgg 2640

cgtccacgag atcatttaca ccatgctggc ctccttgatg aataaagacg gggtcctcat 2700

ctccgagggc caaggattca tgacaaggga gtttctcaag agcctgagaa agccctttgg 2760

tgactttatg gagcccaagt tcgagtttgc tgtgaagttc aacgcactgg aattagatga 2820

cagcgacctg gcgatattta tagctgtcat tattctcagt ggagaccgcc caggtttgct 2880

gaatgtgaag cccatcgagg acattcaaga caatttgctg caagccttgg agctgcagct 2940

caagttgaac cacccagagt cctcccagct ctttgctaag ctgctccaga aaatgacaga 3000

cctcagacag attgtgacag agcacgtgca gctgctgcaa gtaataaaga agacagagac 3060

ggacatgagc cttcacccac tcctacagga aatatacaag gacttgtact aggcggccgc 3120

gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc 3180

tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt 3240

ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 3300

catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tctcgagata 3360

acttcgtata atgtatgcta tacgaagtta tgtcgagggc ccctgcaggt caattctacc 3420

gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg 3480

gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc 3540

gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca 3600

ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac 3660

gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt 3720

ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg 3780

gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct 3840

ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt 3900

cctcatctcc gggcctttcg acctgcagcc aatatgggat cggccattga acaagatgga 3960

ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 4020

cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 4080

ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg 4140

ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 4200

gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 4260

cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 4320

gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 4380

cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 4440

ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgatga tctcgtcgtg 4500

acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 4560

atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 4620

gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 4680

gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaggg 4740

gatcgatccg ctgtaagtct gcagaaattg atgatctatt aaacaataaa gatgtccact 4800

aaaatggaag tttttcctgt catactttgt taagaagggt gagaacagag tacctacatt 4860

ttgaatggaa ggattggagc tacgggggtg ggggtggggt gggattagat aaatgcctgc 4920

tctttactga aggctcttta ctattgcttt atgataatgt ttcatagttg gatatcataa 4980

tttaaacaag caaaaccaaa ttaagggcca gctcattcct cccactcatg atctatagat 5040

ctatagatct ctcgtgggat cattgttttt ctcttgattc ccactttgtg gttctaagta 5100

ctgtggtttc caaatgtgtc agtttcatag cctgaagaac gagatcagca gcctctgttc 5160

cacatacact tcattctcag tattgttttg ccaagttcta attccatcag aagctgactc 5220

tagcaatata acttcgtata atgtatgcta tacgaagtta tctagagtcg acattatggg 5280

cgggattctt ttgccctggc ttaacctgat tcttgggcgt tgtcctgcag gggattgagc 5340

aggtgtacga ggacgagccc aatttctcta tattcccaca gtcttgagtt tgtgtcacaa 5400

aataattata gtggggtgga gatgggaaat gagtccaggc aacacctaag cctgatttta 5460

tgcattgaga ctgcgtgtta ttactaaaga tctttgtgtc gcaatttcct gatgaaggga 5520

gataggttaa aaagcacgga tctactgagt tttacagtca tcccatttgt agacttttgc 5580

tacaccacca aagtatagca tctgagatta aatattaatc tccaaacctt aggccccctc 5640

acttgcatcc ttacggtcag ataactctca ctcatacttt aagcccattt tgtttgttgt 5700

acttgctcat ccagtcccag acatagcatt ggc 5733

<210> 6

<211> 7459

<212> DNA

<213> Artificial sequence

<400> 6

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 60

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 120

acaccgaact gagataccta cagcgtgagc tatgagagag cgccacgctt cccgaaggga 180

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 240

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 300

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 360

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 420

tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 480

gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 540

gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 600

ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 660

caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 720

aacaatttca cacaggaaac agctatgacc atgattacgc caagctcgaa attaaccctc 780

actaaaggga acaaaagctg gagctccacc gcggtggcgg ccgctcgtta acctactcga 840

gataacttcg tataatgtat gctatacgaa gttatgtcga gggcccctgc aggtcaattc 900

taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg 960

ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt 1020

aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta 1080

gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta 1140

gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc 1200

ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga 1260

aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg 1320

tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc 1380

tcttcctcat ctccgggcct ttcgacctgc agccaatatg ggatcggcca ttgaacaaga 1440

tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 1500

acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 1560

ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc 1620

gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 1680

tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 1740

tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 1800

gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 1860

tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 1920

cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt 1980

cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 2040

attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 2100

ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 2160

tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 2220

aggggatcga tccgctgtaa gtctgcagaa attgatgatc tattaaacaa taaagatgtc 2280

cactaaaatg gaagtttttc ctgtcatact ttgttaagaa gggtgagaac agagtaccta 2340

cattttgaat ggaaggattg gagctacggg ggtgggggtg gggtgggatt agataaatgc 2400

ctgctcttta ctgaaggctc tttactattg ctttatgata atgtttcata gttggatatc 2460

ataatttaaa caagcaaaac caaattaagg gccagctcat tcctcccact catgatctat 2520

agatctatag atctctcgtg ggatcattgt ttttctcttg attcccactt tgtggttcta 2580

agtactgtgg tttccaaatg tgtcagtttc atagcctgaa gaacgagatc agcagcctct 2640

gttccacata cacttcattc tcagtattgt tttgccaagt tctaattcca tcagaagctg 2700

actctagcaa tataacttcg tataatgtat gctatacgaa gttatctaga gtcgaccatc 2760

gatggatccc cgggtaccga gctcgaattc taccgggtag gggaggcgct tttcccaagg 2820

cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc 2880

tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc 2940

cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc 3000

gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac 3060

agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc 3120

tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc 3180

gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt 3240

caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgacctgc 3300

agcgacccgc ttaacagcgt caacagcgtg ccgcagatct tggtggcgtg aaactcccgc 3360

acctcttcgg caagcgcctt gtagaagcgc gtatggcttc gtacccctgc catcaacacg 3420

cgtctgcgtt cgaccaggct gcgcgttctc gcggccatag caaccgacgt acggcgttgc 3480

gccctcgccg gcagcaagaa gccacggaag tccgcctgga gcagaaaatg cccacgctac 3540

tgcgggttta tatagacggt cctcacggga tggggaaaac caccaccacg caactgctgg 3600

tggccctggg ttcgcgcgac gatatcgtct acgtacccga gccgatgact tactggcagg 3660

tgctgggggc ttccgagaca atcgcgaaca tctacaccac acaacaccgc ctcgaccagg 3720

gtgagatatc ggccggggac gcggcggtgg taatgacaag cgcccagata acaatgggca 3780

tgccttatgc cgtgaccgac gccgttctgg ctcctcatat cgggggggag gctgggagct 3840

cacatgcccc gcccccggcc ctcaccctca tcttcgaccg ccatcccatc gccgccctcc 3900

tgtgctaccc ggccgcgcga taccttatgg gcagcatgac cccccaggcc gtgctggcgt 3960

tcgtggccct catcccgccg accttgcccg gcacaaacat cgtgttgggg gcccttccgg 4020

aggacagaca catcgaccgc ctggccaaac gccagcgccc cggcgagcgg cttgacctgg 4080

ctatgctggc cgcgattcgc cgcgtttacg ggctgcttgc caatacggtg cggtatctgc 4140

agggcggcgg gtcgtggcgg gaggattggg gacagctttc ggggacggcc gtgccgcccc 4200

agggtgccga gccccagagc aacgcgggcc cacgacccca tatcggggac acgttattta 4260

ccctgtttcg ggcccccgag ttgctggccc ccaacggcga cctgtacaac gtgtttgcct 4320

gggccttgga cgtcttggcc aaacgcctcc gtcccatgca cgtctttatc ctggattacg 4380

accaatcgcc cgccggctgc cgggacgccc tgctgcaact tacctccggg atggtccaga 4440

cccacgtcac cacccccggc tccataccga cgatctgcga cctggcgcgc acgtttgccc 4500

gggagatggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct 4560

atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttcataaacg 4620

cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca 4680

atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg 4740

gctcgcagcc aacgtcgggg cggcaagccc tgccatagcc acgggccccg tgggttaggg 4800

acggggtccc ccatggggaa tggtttatgg ttcgtggggg ttattatttt gggcgttgcg 4860

tggggtcagg tccacgactg gactgagcag acagacccat ggtttttgga tggcctgggc 4920

atggaccgca tgtactggcg cgacacgaac accgggcgtc tgtggctgcc aaacaccccc 4980

gacccccaaa aaccaccgcg cggatttctg gcgccgccgg acgaactaaa cctgactacg 5040

gcatctctgc cccttcttcg ctggtacgag gagcgctttt gttttgtatt ggtcaccacg 5100

gccgagtttc cgcgggaccc cggccaggac ctgcagaaat tgatgatcta ttaaacaata 5160

aagatgtcca ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag 5220

agtacctaca ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag 5280

ataaatgcct gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt 5340

tggatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca 5400

tgatctatag atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg 5460

tggttctaag tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag 5520

cagcctctgt tccacataca cttcattctc agtattgttt tgccaagttc taattccatc 5580

agaagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 5640

caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 5700

cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg 5760

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca 5820

atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 5880

ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 5940

agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 6000

gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 6060

ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 6120

aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 6180

aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 6240

cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 6300

ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 6360

cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 6420

ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 6480

gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 6540

gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 6600

acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 6660

cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 6720

acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 6780

ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 6840

ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 6900

gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 6960

atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 7020

ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 7080

attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 7140

ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 7200

aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 7260

aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 7320

ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 7380

tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 7440

ctgttaccag tggctgctg 7459

Claims (10)

1. the method for being mutated ROSA26 gene, it is characterised in that: the method is carried out using CRISPR/Cas9 system, described CRISPR/Cas9 system includes the sgRNA for targeting ROSA26 gene, and the target sequence of the sgRNA is SEQ ID No.1.

2. according to the method described in claim 1, it is characterized by: described method includes following steps: being led into recipient cell Enter the sgRNA and Cas9, realizes the mutation of ROSA26 gene.

3. method according to claim 2, it is characterised in that: it is described " imported into recipient cell the sgRNA and Cas9 " is that the expression vector by that will contain the sgRNA and the expression vector containing Cas9 import in the recipient cell in fact Existing.

4. according to the method described in claim 3, it is characterized by: " expression vector containing the sgRNA " and described " expression vector containing Cas9 " is identical carrier, which is named as carrier 1, the carrier 1 is to pass through BbsI digestion position Point is inserted into the recombinant vector obtained after DNA fragmentation shown in SEQ ID No.1 into CRISPR/Cas9 carrier, and the carrier 1 can table Up to sgRNA described in claim 1 and Cas9.

5. according to the method any in claim 2-4, it is characterised in that: the method also includes to the recipient cell The step of middle importing donor dna；The donor dna contains two segment DNAs for being located at the target sequence upstream and downstream in ROSA26 gene Segment；

Further, the sequence positioned at the DNA fragmentation of the target sequence upstream is 825-1810 of SEQ ID No.2；Position In 6514-8076 that the sequence of the DNA fragmentation in the target sequence downstream is SEQ ID No.2.

6. according to the method described in claim 5, it is characterized by: the donor dna also contain 2 encoding gene of PPAR γ or 2 encoding gene expression cassette of PPAR γ；

Further, the 2 encoding gene expression cassette of PPAR γ is successively compiled including MCK promoter, PPAR γ 2 from 5 ' ends to 3 ' ends Code gene and polyA termination signal sequence；

Further, the sequence of the donor dna is SEQ ID No.2.

Any one of 7. following 1) -6):

1) method for preparing ROSA26 Genetic Mutant Cell includes the following steps: to utilize the side any in claim 1-6 Method prepares the cell of ROSA26 gene mutation；

2) method for preparing ROSA26 gene mutant animals includes the following steps: to utilize the side any in claim 1-6 Method prepares the zooblast of ROSA26 gene mutation；ROSA26 gene is prepared using the zooblast of the ROSA26 gene mutation Mutant animals；

3) method of preparation expression 2 cell of PPAR γ includes the following steps: to utilize the method any in claim 1-6 The zooblast for preparing ROSA26 gene mutation obtains the cell of expression PPAR γ 2；

4) method of preparation expression 2 animal of PPAR γ includes the following steps: to utilize the method any in claim 1-6 The zooblast for preparing ROSA26 gene mutation obtains the cell of expression PPAR γ 2；Utilize the cell of the expression PPAR γ 2 The animal of preparation expression PPAR γ 2；

5) method for preparing the cell of intramuscular fat content raising includes the following steps: using any described in claim 1-6 Method prepare the zooblast of ROSA26 gene mutation, obtain the cell of intramuscular fat content raising；

6) method for preparing the animal of intramuscular fat content raising includes the following steps: using any described in claim 1-6 Method prepare the zooblast of ROSA26 gene mutation, obtain the cell of expression PPAR γ 2；Utilize the expression PPAR γ 2 Cell prepare intramuscular fat content raising animal.

8. any the method in -7 according to claim 1, it is characterised in that: the cell is following a1) or a2) or a3):

A1) zooblast；

A2) mammalian cell；

A3) pig cell；

The animal is b1) or b2):

B1) mammal；

B2) pig.

9. cell that the method that ROSA26 Genetic Mutant Cell is prepared according to claim 7 or 8 the methods obtains or The method of cell or the cell for preparing intramuscular fat content raising that the method for preparation expression 2 cell of PPAR γ obtains Obtained cell.

10. application of any the method in animal breeding in claim 1-8.