CN110272900A - It is used to prepare sgRNA and its application of skeleton development exception pig model - Google Patents
It is used to prepare sgRNA and its application of skeleton development exception pig model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention belongs to Animal genome editing technique fields, and in particular to be used to prepare sgRNA and its application of skeleton development exception pig model.The present invention is to solve existing mouse skeleton development exception animal model and the mankind have the technical issues of larger difference in terms of skeletal structure, function.Technical measure is to provide a kind of sgRNA for preparing skeleton development exception pig model.SgRNA identification is located at the target site that the nucleotides sequence on pig fibroblast growth factor receptor3 editor's gene is classified as 5 '-GAAAGCCCAGCCCGTAGCTGAGG-3 '.On the basis of obtaining above-mentioned sgRNA, design the donor dna of cooperation, can efficiently prepare Fgfr3 c.1132G > A is precisely mutated pig model, the FGFR3 p.G378R mutation pig system of dyschondroplasia is cultivated, the clinical treatment for skeleton development research and skeletal diseases provides and the closer big animal model of people.
Description
Technical field
The invention belongs to Animal genome editing technique fields, and in particular to one kind is used to prepare skeleton development exception pig mould
The sgRNA of type and its application.
Background technique
Bone is one of developmental deformity, the organ of genetic disease most species, current online mankind's Mendelian inheritance disease number
Having included single-gene bone hereditary disease according to library (OMIM) has more than 400.Common skeletal dysplasia includes dwarf, vertebral column development
Bad, osteogenesis imperfecta, craniofacial deformity, heredity bone metabolic disease (osteosclerosis, rickets) etc..Currently, being used for skeleton development
The most common animal model of disease research is genetic engineering mice, is opened as model animal around skeleton development, metabolic disease
More research is opened up.But mouse belongs to rodent, has larger difference in terms of skeletal structure, function with the mankind, such as
Early stage mode development of the mouse bone without Haversian system, the bone-free property soleplate of backbone, and bone is different from morphogenesis mode, raw
Long slab is not closed all the life.In addition, because small, it is difficult to realize sampling (blood, urine) repeatedly for related-metabolism index detection, power
Learn performance measurement and surgical procedure (such as orthomorphia, suboffice and tissue engineering material implantation).
Pig, especially miniature pig, in weight, the dissection of bone, institutional framework, (bone trabecula thickness, collagen arrangement, mineralising are heavy
Product speed, joint size and weight bearing mechanics, articular cartilage thickness, the structure of interverbebral disc and component, lumbar vertebrae rotation angle etc.) and
Growth course etc. is almost the same with the mankind.Currently, pig is primarily used to during the kinematic systems injury repair such as Bones and joints
The assessment of biomaterial, stem-cell therapy and surgical instrument, modus operandi curative effect etc., also lacks using genetic modification pig in bone
The research of aspect.
Fgfr3 (fibroblast growth factor receptor3) is the key molecule of skeleton development regulation, the mutation meeting of the molecule
Cause skeleton development abnormal.Wherein, can lead to the 378th glycine mutation of FGFR3 albumen is arginic point mutation Fgfr3
C.1132G > A is most common dyschondroplasia mutation in people's clinic, but there is no at present heredity skeleton development exception pig and
The report of other big animal models.
Summary of the invention
The technical problem to be solved by the present invention is to existing mouse skeleton development exception animal model and the mankind skeletal structure,
There is the defect of larger difference in terms of function.
The technological means that the present invention solves technical problem is to provide a kind of sgRNA for preparing skeleton development exception pig model.
The nucleotides sequence that sgRNA identification is located on pig fibroblast growth factor receptor3 encoding gene is classified as 5 '-GAAAGCC
The target site of CAGCCCGTAGCTGAGG-3 ' (SEQ ID NO.l).
Further, the boot sequence of target site is identified in the sgRNA are as follows:
5’-GAAAGCCCAGCCCGTAGCTG-3’(SEQ ID NO.2)。
Further, the nucleotide sequence of the sgRNA are as follows:
5’-GAAAGCCCAGCCCGTAGCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTA GTCCGTT
ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3’(SEQ ID NO.3)。
Invention also uses a kind of for being used cooperatively with the above-mentioned sgRNA for preparing skeleton development exception pig model,
It is arginine by the codon mutation for encoding the 378th glycine on the fibroblast growth factor receptor3 encoding gene of pig
The donor dna of codon.Further, is encoded on the fibroblast growth factor receptor3 encoding gene by pig
The codon mutation of 378 glycine is that arginine codon refers to that pig Fgfr3c.1132G > A is mutated.
Wherein, the donor dna is double stranded donor DNA or single donor DNA.
Preferably, include following core sequence 5 '-TTATA-3 ' (SEQ ID NO.4) in the donor dna or it is mutual
Complementary series.
Therein, the core sequence two sides in the donor dna include the homologous sequence of 50~70 bases.
Preferably, the nucleotide sequence of the donor dna are as follows:
5’-CCGAGGAGGAGCTGGTGGAGGCTGGTGAGGCTGGCAGTGTGTACGCGGGGGTCCTCAGTTATA G
GCTGGGCTTTCTCCTCTTCATCCTGGTGGTGGCCACCGTGACACTCTGCCGCCTGCGCAGCCCCCC -3’(SEQ ID
It NO.5) or is its complementary series.
On this basis, the present invention also provides a kind of kit for preparing skeleton development exception pig model, the kits
Including following component: above-mentioned sgRNA and/or above-mentioned donor dna.Further, the kit may also include coding
MRNA the and/or spCas9 recombinant protein of spCas9 albumen.Further, each main component in kit is independent packaging.
Present invention provides a kind of methods for preparing skeleton development exception pig model.Method includes the following steps:
A, by mRNA the and/or spCas9 purification of recombinant proteins of above-mentioned sgRNA above-mentioned donor dna and coding spCas9
After being sufficiently mixed, mixture is imported by microinjection in the cytoplasm of pig body early embryo；
B, by step a, treated that pig body early embryo migrates in the fallopian tubal of receptor sow, and receptor sow is made to become pregnant；
C, the receptor sow become pregnant gives birth to piglet after full-term, obtains skeleton development exception pig model.
Wherein, the mRNA and/or spCas9 of sgRNA described in the above method, donor dna and coding spCas9 are pure
Change recombinant protein between with ratio be by the mixture 10~15ng/uL of final concentration sgRNA, spCas9mRNA and/
Or 5~10ng/uL of 20~25ng/uL of spCas9 purification of recombinant proteins and donor dna.
Further, the embryo of pig body early embryo described in the above method for 4 cell stages and before.
Further, further include having detection verification step in step c described in the above method: using pFgfr3-E9-
F1/R1 (table 1) primer amplification piglet genome DNA sample determines whether according to amplification sequencing containing expected gene editing
Genotype (pFgfr3c.1132G > A), if showing to model successfully containing the genotype.
The beneficial effects of the present invention are: the present invention is directed to the most common mutational site of human cartilage developmental disorder
Fgfr3c.1132G, for the first time in the corresponding site for having found pig Fgfr3 gene, and nearby devise one it is sgRNA efficient
Point, and construct one for the site, can effectively pass through DNA " homologous guidance reparation " (homology-directed repair,
HDR) mechanism realizes the donor dna that Fgfr3c.1132G > A mutation fixed point is knocked in, and it is accurate efficiently to prepare Fgfr3c.1132G > A
It is mutated pig model.Experiment shows that the first mutation efficiency for building pig may be up to 100%, wherein realized by HDR mechanism
The accurate mutation rate of FGFR3p.G378R may be up to 80%.The traversed by breeding that pig is built by head, can effectively train that educate cartilage development different
Normal FGFR3 p.G378R mutation pig system；Meanwhile using the site sgRNA of the present invention, pig Fgfr3 can be effectively induced
Frameshift mutation occurs for gene, can cultivate Fgfr3 gene by the traversed by breeding that head builds pig and the mutation pig knocked out completely occurs, this is prominent
Become the big animal model that pig can be used as bone undue growth and lateral bending occurs for backbone.The present invention can efficiently grind for skeleton development
Study carefully and the clinical treatment of skeletal diseases provides and the closer big animal model of people, there is good application value.
Detailed description of the invention
Fig. 1 experimental design.A, people, mouse and pig FGFR3 encode protein amino acid sequence comparison result, black box
Homologous amino acid site is mutated for people FGFR3p.G380R.B, pig FGFR3 gene composition, homologous site are located at 9 exons,
Lower section block arrow is sgRNA, and grey horizontal line is ssODN template, and left side block arrow indicates the transcriptional orientation of gene.C,FGFR3-
G378R-ssODN structure chart.The A base of scribing line is targeted mutagenesis base pFgfr3c.1132G > A；The T base of two grey is
Two same sense mutation bases (pFgfr3c.1131C > T and pFgfr3c.1128C > T)；Black letters of mutating alkali yl two sides and black
Lines are the homologous sequence of mutating alkali yl two sides.D, sgRNA of the invention (being named as Fgfr3-G378-sgRNA herein) and-confession
Body DNA (being named as Fgfr3-G378R-ssODN herein) mediates the working principle diagram of expected gene mutation.Arrow institute below sequence
It is shown as sgRNA identification region, position shown in scissors symbol is spCas9 protein cleavage site；Identify 3 alkali of 3 ' end of sequence
Base AGG is PAM structure；The GGG base that underscore indicates in target gene recognition site is the password for encoding the 378th glycine
Son, wherein the G base of grey mark is quasi- mutation target spot；The base A to cross in donor dna and edited gene order is mesh
Mutating alkali yl is marked, the T base of two grey is same sense mutation base.
Fig. 2 head builds pig genetic analysis result.A, number pF1-1~pF1-5 (from left to right) ear tissue genome amplification
The 9th exon PCR product gel electrophoresis result (Marker 100bp) of pFgfr3.B, number pF1-1~pF1-5 gene are compiled
Collect location proximate sequence to analyze as a result, in bracket: number is to contain corresponding gene type clone number/always effective sequencing to clone
Number；KI: it is expected that gene knock-in genotype pFgfr3c.1132G > A；The international naming rule of other gene mutation types
Mark；" PCR product " indicates that the genotype is the result of PCR product direct Sequencing.Peak figure, black is sequenced in C, each genotype of individual
Base in box is the expection mutational site based on donor dna homologous recombination, wherein 2 T bases are same sense mutation base, A
Base is targeted mutagenesis base (Fgfr3c.1132G > A)；Black arrow indicates base deletion mutated site (pFgfr3
c.1124_1130delCAGCT)；Base shown in circular frame is the base of insertion mutation.
The living body phenotype of Fig. 3 Fgfr3 frameshift mutation pig and Fgfr3c.1132G > A heterozygous mutant pig.A, 5 head of birth
Build the scoliosis of photo (wherein white arrow show the pF1-3 individual containing frameshift mutation) and pF1-3 individual of piglet
Phenotype；The photo of B, Fgfr3c.1132G > A heterozygous mutant pig and wild type pig.
Fig. 4 is mutated pig long bone relative length (* indicates P < 0.05).Femur: femur；Tibia: shin bone；Humerus: the upper arm
Bone；WT: wild type pig；MT: mutation pig.
Fig. 5 wild type (WT) and mutation pig (MT) x-ray detection (left: upper limb is right: lower limb).
Fig. 6 X-ray takes the photograph piece observation pig's head cranium form result.MT: mutation pig；WT: wild type pig.
(* indicates P < 0.05 for Fig. 7 wild type (WT) and mutation pig (MT) femur x-ray dual intensity bone densitometry；* expression P <
0.01).Relative BMD: opposite bone density；Total Femur: full femur；Midshaft: femur middle section；Distal: stock
Bone end；WT: wild type pig；MT: mutation pig.
Tu8Zhu metaphysis Histological section figure.WT: wild type pig；MT: mutation pig.
Tu9Zhu growth plate Histological section figure.WT: wild type pig；MT: mutation pig.
Specific embodiment
Introduction below by way of specific embodiment carries out further description to the present invention.
The present invention passes through to dyschondroplasia mutational site (coding the 380th, FGFR3 albumen most common in people's clinic
First bases G of the codon of glycine) sequence research, searched out the homologous position of corresponding pig Fgfr3 gene
Point.By further studying, an efficient sgRNA target site is had chosen on the antisense strand near the homologous site
(being named as pFgfr3-G378-sgRNA), referring to Fig. 1,
It identifies the boot sequence of target site are as follows: 5-GAAAGCCCAGCCCGTAGCTG-3；
The target site sequence of its identification on pig fibroblast growth factor receptor3 gene are as follows:
Wherein 3 bases of black overstriking are PAM structure, and dashed part is the codon GGG for encoding the 378th glycine
Complementary base.
The present invention also devises a preferred sgRNA, nucleotide sequence are as follows:
5’-GAAAGCCCAGCCCGTAGCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTA GTCCGTT
ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3’。
For the recognition site of the sgRNA, design can efficiently be realized most common soft in people's clinic in pig Fgfr3 gene
The DNA homologous recombination template (donor dna) of dysostosis homeotic mutation Fgfr3c.1132G > A.The donor dna need to comprising with
Lower core sequence:Wherein scribing line base is mutating alkali yl, wherein the T base of 2 scribing line is same sense mutation alkali
Base, the A base of scribing line are targeted mutagenesis base.In general, in actual use, donor dna is needed in core above-mentioned
The homologous sequence of 50~70 bases is arranged in sequence two sides.
Further, the present invention has further preferably obtained a donor dna (name are as follows: Fgfr3-G378R-ssODN), core
Thuja acid are as follows:
Wherein the base sequence of black overstriking is core sequence, and the T base of 2 scribing line is same sense mutation base, the A of scribing line
Base is targeted mutagenesis base；The base of italic mark is homology arm sequence.The donor dna, which contains, can lead to pig FGFR3 egg
White 378th glycine mutation is arginic Fgfr3c.1132G > A mutation, while at coding the 377th, pig FGFR3 albumen
2 synonymous base mutations are directed respectively into the codon (TAC and AGC) of tyrosine and the 376th serine, respectively
Fgfr3c.1131C > T and Fgfr3c.1128C > T, to inhibit pFgfr3-G378-sgRNA/spCas9 protein complexes to homologous
It is cut again in mutational site after recombination；Homologous sequence in the two sides of above-mentioned mutating alkali yl, respectively containing 50~70 bases
Column, to realize knocking in for homologous recombination and mutational site.
In an example of the present invention, the site pFgfr3-G378-sgRNA designed of the present invention and donor dna are utilized
On the basis of, those skilled in the art can be by the genome editing system and microinjection technique of CRISPR/Cas9, will be upper
After mRNA the and/or spCas9 purification of recombinant proteins of the sgRNA, donor dna and the coding spCas9 that state are sufficiently mixed, by mixture
It is imported by microinjection in the cytoplasm of pig body early embryo；Treated pig body early embryo is migrated into receptor sow again
Fallopian tubal in, so that receptor sow is become pregnant；The receptor sow become pregnant gives birth to piglet after full-term, obtains skeleton development exception
Pig model.
Wherein, above-mentioned sgRNA, donor dna and encode spCas9 mRNA and/or spCas9 purification of recombinant proteins it
Between with ratio be by the mixture 10~15ng/uL of final concentration sgRNA, spCas9mRNA and/or spCas9 purify
5~10ng/uL of 20~25ng/uL of recombinant protein and donor dna.
Further, the embryo of pig body early embryo described in the above method for 4 cell stages and before.It is above-mentioned in order to detect
Whether Method Modeling succeeds, and can also use primer amplification piglet genome DNA sample, is determined whether according to amplification sequencing
Containing expected gene editing genotype (pFgfr3c.1132G > A), if showing to model successfully containing the genotype.The present invention
Provide effective detection primer pFgfr3-E9-F1/R1 (referring to table 2).
The site pFgfr3-G378-sgRNA designed in an example of the present invention using the present invention and Fgfr3-
G378R-ssODN template, by the sgRNA being transcribed in vitro accordingly and artificial synthesized single-stranded DNA templates and spCas9mRNA
Pass through microinjection to import in the fertilized eggs of 1~2 cell stage of pig simultaneously, and by the zygote transplation after injection in being in heat
Adult sow fallopian tubal in, efficiently obtain the 378th glycine mutation of FGFR3 albumen be arginic mutation pig
(FGFR3p.G378R is mutated pig), the mutation efficiency that head builds pig may be up to 100%, wherein realized by HDR mechanism
The accurate mutation rate of FGFR3p.G378R may be up to 80%.The traversed by breeding that pig is built by head, has effectively trained and has educated dyschondroplasia
FGFR3p.G378R be mutated pig.And the site sgRNA of the present invention is utilized, it can effectively induce pig Fgfr3 gene to occur
Frameshift mutation can cultivate Fgfr3 gene by the traversed by breeding that head builds pig and the mutation pig knocked out completely occurs, which can make
For bone undue growth and the big animal model of lateral bending occurs for backbone.
Below by way of to embodiment, the present invention will be described in more detail.
The preparation of one FGFR3p.G378R of embodiment mutation pig
One, experimental program:
1, the preparation of homologous recombination template
According to the sequence of the homologous recombination template of design, artificial synthesized single strain oligonucleotide (ssODN ,≤150bp) (on
Hai Shenggong)；The ssODN freeze-dried powder of synthesis is dissolved and dispensed with the concentration of 500ng/uL with RNase-free ultrapure water, is saved
It is spare in -80 degrees Celsius.
2, the preparation of sgRNA
1) two complementary oligonucleotide single stranded DNAs (oligo DNA) containing sgRNA identification sequence, justice are synthesized
Chain (SEQ ID NO.6): 5-TAGGAAGCCCAGCCCGTAGCTG-3；Antisense strand (SEQ ID NO.7): 5-
AAACCAGCTACGGGCTGGGCTT-3.Two complementary single-stranded dna renaturation is after double-strand, matter will be transcribed in vitro with sgRNA in end
Subcloning sites after digestion on grain are complementary；It is pUC57kan-T7- that plasmid, which is transcribed in vitro, in sgRNA used in this example
GRNA-U6V2 (Addgene#115520, y, the restriction enzyme site for subclone are BsaI)；
2) the single-stranded oligo DNA of every synthesis takes 1OD, is dissolved in deionized water with the concentration of 0.2ug/uL, and with
It is saved backup after the packing of 20uL/ pipe；
3) the single-stranded oligo DNA solution for respectively taking the complementation of 1 pipe after mixing and mix well in equal volume, is containing 1L originally
In the water-bath of water, in 95 degrees Celsius of incubation 10min, so that two complementary single-stranded oligo DNA are sufficiently denaturalized as single-stranded shape
State；
4) turn off water-bath power supply, make its natural cooling, complementary single-stranded oligo DNA renaturation is allowed to be double-stranded state；
5) vector plasmid pUC57Kan-T7-gRNA-U6V2, exhausting line property is transcribed in vitro with the abundant digestion sgRNA of BsaI
Grain segment；
6) the linear plasmid segment that the double-stranded DNA and step 5) obtained step 4) obtains, is connected with T4DNA ligase,
Obtain recombinant plasmid；
7) the connection product transformed competence colibacillus bacterium of step 6), picking kalamycin resistance bacterium colony, and amplification cultivation obtain
Resistance bacterium solution；
8) recombinant plasmid, and sequence verification are extracted using resistance bacterium solution；
9) plasmid obtained with the correct step 8) of the abundant digestion sequence verification of restriction endonuclease DraI realizes that recombinant plasmid is complete
Entirely, it adequately linearizes；
10) purified linear Plasmid DNA and remove RNA enzyme pollution；
11) DNA precipitating is sufficiently washed with 70% ethyl alcohol of no RNA enzyme, later with the deionized water dissolving DNA of no RNA enzyme
Precipitating；
12) linear plasmid for the purifying for taking 1ug step 11) to obtain is template, uses in-vitro transcription kit
MEGAshortscriptTM T7Transciption Kit (invitrogen, the U.S., catalog number (Cat.No.) AM1354) is turned in vitro
Record obtains sgRNA (operating procedure is detailed in kit specification)；
13) MEGAClear Transcription Clean-Up Kit (invitrogen, the U.S., catalog number (Cat.No.) are utilized
AM1908), purification step 12) obtain in-vitro transcription product, according to concentration be packed as 5-10uL/ pipe save backup (behaviour
Kit specification is detailed in as step).
3, the preparation of Cas9mRNA
1) preparation of transcription templates:
With AgeI restriction endonuclease sufficiently (overnight) digested plasmid pST1374-N-NLS-flag-linker-Cas9 (Addgene,
The U.S., catalog number (Cat.No.) #44758), the preparation of template is transcribed in vitro with sgRNA for remaining step.
2) it is transcribed in vitro:
It is transcribed using mMESSAGE mMACHINE T7Ultra kit (invitrogen, the U.S., catalog number (Cat.No.) AM1345)
Cas9mRNA, experimental procedure are detailed in kit specification.It transcribes and 1ul Turbo DNase is added after completing into system, 37 DEG C
It is incubated for 15min, to remove remaining template DNA.
3) tailing:
(1) following reagent is sequentially added into T7Ultra Reaction system:
1 reagent system of table
(2) after mixing, 2.5ul mix is drawn as control；4ul E-PAP enzyme is added, 37 DEG C of incubations after mixing
45min。
4) mRNA purification and recovery:
Utilize the RNAeasy kit of Qiagen company, purification and recovery mRNA:
(1) template is adjusted to 100ul with NFW water；
(2) the RLT Buffer that 350ul is added is sufficiently mixed uniformly；
(3) dehydrated alcohol of 250ul is added, mixes；
(4) it is transferred in the included pillar of kit, >=8000g is centrifuged 15s；
(5) waste liquid in collecting pipe is abandoned, the Buffer RPE of 500ul, >=8000g is added, is centrifuged 15s；
(6) it repeats the above steps；
(7) pillar is transferred to new 2ml collecting pipe, dally 1min；
(8) pillar is put in 1.5ml centrifuge tube, the RFW of 40ul is added, >=8000g, 1min；
(9) it takes 2ul to survey concentration, dispenses mRNA solution to the EP of no RNA enzyme according to the volume of 1-5uL/ pipe according to concentration
MRNA solution after packing is placed in -80 degrees Celsius of refrigerators and frozen by Guan Zhong.
(10) 1 pipe mRNA electrophoresis is taken, is control with the mRNA before tailing, detects the quality and tailing effect (tailing of mRNA
The pillar location of mRNA afterwards should slightly lag behind the mRNA before tailing).Cas9mRNA is diluted to 20ng/ul using preceding.
4, the acquisition of pig body early embryo, the microinjection of CRISPR reagent and transplanting:
1) acquisition of pig body early embryo
(1) the 3-5 healthy sexal maturity sows being in heat are chosen, are used as embryo after breeding with healthy sexal maturity boar
Donor sow, and an another standby healthy sexal maturity sow being in heat is as receptor sow；
(2) 24-36h after donor insemination of sows, the Nembutal sodium solution for being 3% by auricular vein implantation quality score
10-15mL or the general anesthesia for realizing donor pig by ventilator using isoflurane, and sow is bound on V-arrangement operating table；
(3) last and third is to nipple in sow along abdomen median line after conventional cleaning, disinfection sow abdomen
Between, hara kiri skin, fascia, muscle and peritonaeum, size incision 5-8cm；
(4) sow ovary, fallopian tubal and part uterus are taken out, visible sow Ovarian surface haemorrhagia ovulations point, shows at this time
Sow has ovulated；
(5) it is the grass tube of the both ends 4-6mm passivation by internal diameter, passes through fimbriae tubae portion and be inserted into fallopian tubal；
(6) embryo washing water (PBS+1% fetal calf serum) the about 20mL sufficiently incubated in 38 DEG C of water-baths is extracted with syringe, led to
It crosses intravenous infusion needle and the syringe containing embryo washing water is connected to (its one end containing syringe needle passes through fallopian tubal and uterus with fallopian tube lumen
Fallopian tube lumen is inserted into engaging portion, and the other end is connected with syringe)；
(7) embryo washing water is injected by fallopian tubal by syringe, and with the collection of the 50mL centrifuge tube of sterilizing flow through fallopian tubal, from
It is inserted into the embryo washing water flowed out in the grass tube in fimbriae tubae portion；
(8) embryo washing water of collection is transferred in the sterile petri dish that diameter is 9cm, picks embryo under stereomicroscope,
The embryo picked at this time is generally 1-cell the or 2-cell phase, as shown in Figure 7；
(9) Pig embryos picked are placed in the culture solution drop for covering paraffin oil, sufficiently having incubated balance in the incubator
Middle culture is spare, and (Pig embryos culture solution used in present case is PZM-3, and formula is shown in document Biology of
Reproduction,2002, 66:112)；
2) microinjection of pig body early embryo:
By the sgRNA of the above-mentioned preparation of step, spCas9mRNA and homologous recombination template DNA (donor dna), respectively with dense eventually
Degree sgRNA 10ng/uL, spCas9mRNA 20ng/uL and donor dna 10ng/uL are sufficiently mixed, and later lead to mixed solution
Cross the cytoplasm of microinjection injection pig body early embryo.Hogan et al.Manipulating Mouse is shown in operation in detail
Embryo Manipulation Manual,Cold Spring Harbor Laboratory Press,1994,Second
Edition。
3) Pig embryos are transplanted
(1) it anaesthetizes and Baoding receptor sow, cuts abdominal cavity, takes out ovary, defeated ovum according to obtaining identical mode with embryo
Pipe and part uterus；
(2) embryo suction pipe (U.S., Agtech company) will be picked up to be connected with 1mL syringe, and inhaled in advance in picking up embryo suction pipe
Enter one section of air；
(3) under stereomicroscope, embryo's sucking after 20-30 pieces of injection in culture solution is picked up by the syringe of connection
(note in embryo suction pipe: sucking one section of air before liquid section where embryo, and suck one section of liquid again before air, with antifouling
Dye)；
(4) the embryo suction pipe of picking up equipped with embryo is inserted into receptor pig fallopian tubal by umbrella portion, syringe is pushed to import embryo
In fallopian tubal；
(5) receptor pig uterus, fallopian tubal and ovary are put back in abdomen, successively peritoneal suture, muscle, fascia and skin, and
Routine disinfection processing is done to wound；The receptor sow become pregnant can give birth to piglet after full-term.
5, the genetic analysis of gene mutation pig
1) clip is mutated pig otic tissues, extracts genomic DNA；
2) primer is chosen in the exon upstream and downstream where being mutated target site, is obtained near target site by PCR amplification
Amplified production；
3) the direct sample presentation of PCR product is sequenced, if sequencing result is single sequencing peak, illustrates that mutated individual is homozygote；
If sequencing result contains set peak, illustrate that mutated individual is heterozygote；
4) pcr amplification product of heterozygote individual is subjected to TA clone, obtains resistant transformants bacterium after transformed competence colibacillus bacterium
It falls, each random about 20 conversion bacterium colony sample presentation sequencings of picking of individual determine the genotype of mutated individual.
Two, experimental result
1, the result of sequence analysis and design
By comparing people, pig and mouse Fgfr3 gene coded protein amino acid sequence on NCBI, finder's cartilage development is not
Complete the 380th, mutational site glycine is the 378th glycine in the corresponding site of pig.Compare pig Fgfr3 reference on NCBI
Gene order determines that the site is located at the 9th exon (Figure 1B) of pig Fgfr3 (pFgfr3).It is designed according to reference gene order
The sequencing of the 9th exon of primer amplification obtains the accurate base sequence of Ba-Ma mini pig, finds Ba-Ma mini pig pFgfr3 gene
Homeotic mutation site be c.1132G > A, cause encode amino acid codes GGG sport AGG, so as to cause glycine mutation
For arginine.Have chosen the efficient site sgRNA (being named as pFgfr3-G378-sgRNA), sequence are as follows:Wherein the AGG base of overstriking label is PAM structure, dashed part
For the complementary base of the codon GGG of the 378th glycine of coding.
For the site sgRNA, designing and screening to have obtained one can efficiently be realized in pFgfr3 gene in people's clinic
Most common dyschondroplasia homeotic mutation pFgfr3c.1132G > A DNA homologous recombination template (donor dna, name are as follows:
PFgfr3-G378R-ssODN): it is arginic that the template, which contains and can lead to the 378th glycine mutation of pig FGFR3 albumen,
PFgfr3c.1132G > A mutation, while in the codon of coding pig FGFR3 albumen the 377th tyrosine and the 376th serine
2 synonymous base mutations are directed respectively into (TAC and AGC), respectively pFgfr3c.1131C > T and pFgfr3 are c.1128C
> T, to inhibit pFgfr3-G378-sgRNA/Cas9 protein complexes to cut the mutational site after homologous recombination again.For
It realizes knocking in for homologous recombination and mutational site, in the two sides of above-mentioned mutating alkali yl, is added to the homologous sequence of 65 bases respectively
Column.The sequencing results and experimental design of the present embodiment are shown in Figure 1.
2, gene mutation head builds the preparation and genetic analysis of pig
By embryo transfer, obtains 5 tau gene editor head and build pig.Clip ear tissue extracts genomic DNA, uses pFgfr3
- E9-F1/R1 (table 2) primer amplification target fragment obtains PCR product (Fig. 2A).Directly PCR product is sent to survey PCR product
Sequence is then homozygote for mutated individual, directly determines its genotype if single sequencing peak；Peak is covered if sequencing, then by PCR
Product carries out TA clone, and after transformed competence colibacillus bacterium obtains conversion resistant clones, each individual is selected~20 bacterium colonies at random and sent
Sample carries out Sanger sequencing.Analysis is found: building in pig in 5 (number pF1-1~pF1-5) head, there is 4 individual (number pF1-
1, pF1-2, pF1-4 and pF1-5) gene editing site PCR product sequencing result be it is unimodal, by analyse and compare be found to be
It is expected that gene editing genotype (pFgfr3c.1132G > A) genotype, illustrates that this 4 individuals are expected gene editing genotype
Homozygote (see Fig. 2 B, C)；The PCR product sequencing of number pF1-3 is set peak, send sequencing to be sequenced through TA clone, effectively surveys at 12
Sequence clone in: pFgfr3c.1132G > A mutated-genotype 1, pFgfr3c.1127_1128insTA genotype 6,
PFgfr3c.1127_1128insG genotype 4, pFgfr3c.1124_1130delCAGCT genotype 1, illustrate the individual
For the chimeric individual (Fig. 2 B, C) of hereditary height.The genotype statistics that 5 head build pig individual is shown in Table 3.
2 genetic analysis amplimer of table.
3 head of table builds pig genotype statistics
It was found from above-mentioned data: using the donor dna in the site sgRNA and design that the present invention selects, head, which is built in pig, to be had
80% individual is the expected homozygous mutation individual for knocking in genotype (pFgfr3c.1132G > A), is shown used in the present invention
The site sgRNA (pFgfr3-G378-sgRNA) and donor dna (pFgfr3-G378-ssODN) are living with extremely strong gene editing
Property；Traversed by breeding is carried out by the way that said gene mutation head is built pig, the FGFR3p.G378R that can cultivate dyschondroplasia is pure
Conjunction or heterozygous mutant pig, i.e. Ba-Ma mini pig achondroplasia (ACH) model.Meanwhile being selected and being designed using the present invention
Gene editing reagent also can get the genotype that frameshift mutation occurs for Fgfr3 gene, can be cultivated by the traversed by breeding that head builds pig
The mutation pig knocked out completely occurs for Fgfr3 gene, which can be used as bone undue growth and the animal of lateral bending occurs for backbone
Model.
3, the phenotypic analysis of gene mutation pig
It is built in pig in 5 head of acquisition, mainly the pF1-3 individual based on frameshift mutation, is presented significant four limbs and repairs
Long phenotype (Fig. 3 A)；PF1-3 individual is performed an autopsy on sb. discovery: apparent lateral bending phenotype (Fig. 3 A) is presented in its backbone, this meets
Fgfr3 gene knocks out the clinic of mutation and the phenotype of animal model (mouse).Show not only make using sgRNA of the present invention
Standby pFgfr3c.1132G > A point mutation model, can also efficiently prepare the scoliosis animal model that pFgfr3 is knocked out.
In order to exclude the first influence for building animal genetic mosaic that may be present, otic tissues are chosen in homozygous expected clpp gene
The male head for entering mutation (pFgfr3c.1132G > A) builds pig pF1-5 individual and wild type insemination of sows, obtains F1 generation
PFgfr3 c.1132G > A heterozygous mutant individual and brood wild type individual.It was found that: compared with brood normal individual, F1 generation
PFgfr3 c.1132G > symptom (Fig. 3 B) of apparent four limbs tubbiness is presented in A heterozygous mutant individual, this people's clinic with early period and
The result of study of mouse model is consistent.
The length of the further skeleton development phenotype of analysis mutation pig, the long bones such as discovery shin bone, femur and humerus is wilder
Raw obvious cripetura, 84.4%, 84.5% and 90.8% (see the Fig. 4) respectively compareed.Piece observation, hair are further taken the photograph by X-ray
Under the conditions of identical throwing is shone now, the light transmittance at the long epiphysis end of ACH pig obviously lowers (Fig. 5) compared with wild type pig；Head is wilder
Live pig round blunt, cranial cavity projected area obviously become smaller, and length shortens trend, and width obviously narrows (Fig. 6).
X-ray dual intensity bone densitometry has been carried out due to the not energetic bone amount of x-ray photo, and to femur, has been mutated as the result is shown
The full femur of pig, femur middle section bone density and end metaphysis bone density respectively compared with wild type pig reduce 7.2%, 8.4% with
9.8% (Fig. 7).The enhancing mutation of its function can lead to mutation pig bone amount reduction after the above results illustrate FGFR3 mutation.
The expression of FGFR3 mainly influences entochondrostosis process.Growth plate is the motive power of long bone uptake.Pass through the dry epiphysis of pig
The discovery of Histological section's preliminary observation is held, the hypertrophic chondrocyte that mutation pig matrix contaminates deeply reduces (Fig. 8)；Further analysis growth
The discovery of plate phenotype, the growth plate of wild pig is orderly to be divided into tranquillization band, is proliferated band and loose band, and is mutated the growth plate column of pig
Structural arrangement disorder prompts its growth plate development in visible small and circle the tranquillization sample cartilage cell being dispersed on a small quantity of each band
Process is significantly affected (Fig. 9).
Sequence table
<110>army medical university, ground force, the Chinese People's Liberation Army
<120>sgRNA and its application of skeleton development exception pig model are used to prepare
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gaaagcccag cccgtagctg agg 23
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gaaagcccag cccgtagctg 20
<210> 3
<211> 96
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gaaagcccag cccgtagctg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 96
<210> 4
<211> 5
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ttata 5
<210> 5
<211> 130
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ccgaggagga gctggtggag gctggtgagg ctggcagtgt gtacgcgggg gtcctcagtt 60
ataggctggg ctttctcctc ttcatcctgg tggtggccac cgtgacactc tgccgcctgc 120
gcagcccccc 130
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
taggaagccc agcccgtagc tg 22
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
aaaccagcta cgggctgggc tt 22
<210> 8
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ccgaggagga gctggtggag gctggt 26
<210> 9
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ctgtcgcttg agcgggaagc ggga 24
Claims (10)
1. preparing the sgRNA of skeleton development exception pig model, identification is located at pig fibroblast growth factor receptor3 and encodes base
Because upper, nucleotides sequence is classified as the target site of 5 '-GAAAGCCCAGCCCGTAGCTGAGG-3 '.
2. sgRNA according to claim 1, it is characterised in that it identifies the boot sequence of target site are as follows:
5’-GAAAGCCCAGCCCGTAGCTG-3’。
3. sgRNA according to claim 2, it is characterised in that nucleotide sequence are as follows:
5’-GAAAGCCCAGCCCGTAGCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA
CTTGAAAAAGTGGCACCGAGTCGGTGC-3’。
4. for being used cooperatively with the sgRNA of any of claims 1 or 2 for preparing skeleton development exception pig model, by pig at
The codon mutation that the 378th glycine is encoded on 3 encoding gene of bfgf receptor is arginine codon
Donor dna.
5. donor dna according to claim 4, it is characterised in that: encode the fibroblast growth factor receptor3 of pig
The codon mutation that the 378th glycine is encoded on gene is arginine codon (pig Fgfr3c.1132G > A mutation)；It is preferred that
, the donor dna are as follows: double stranded donor DNA or single donor DNA；It preferably, include following core in the donor dna
Heart sequence: 5 '-TTATA-3 ' or its complementary series；Preferably, the core sequence two sides in the donor dna also include 50~
The homologous sequence of 70 bases；Preferably, the nucleotide sequence of the donor dna are as follows:
5’-CCGAGGAGGAGCTGGTGGAGGCTGGTGAGGCTGGCAGTGTGTACGCGGGGGTCCTCAGTTATAGGCTGG
GCTTTCTCCTCTTCATCCTGGTGGTGGCCACCGTGACACTCTGCCGCCTGCGCAGC CCCCC-3 ' is its complementary sequence
Column.
6. preparing the kit of skeleton development exception pig model, including following component: claims 1 to 3 is described in any item
SgRNA and/or the described in any item donor dnas of claim 4~9；Preferably, the kit further includes coding spCas9 egg
White mRNA and/or spCas9 recombinant protein.
7. the method for preparing skeleton development exception pig model, it is characterised in that the following steps are included:
A, by sgRNA described in claim 1, the described in any item donor dnas of claim 4~9, the mRNA for encoding spCas9
And/or after spCas9 purification of recombinant proteins is sufficiently mixed, mixture is imported to the cytoplasm of pig body early embryo by microinjection
In；
B, by step a, treated that pig body early embryo migrates in the fallopian tubal of receptor sow, and receptor sow is made to become pregnant；
C, the receptor sow become pregnant gives birth to piglet after full-term, obtains skeleton development exception pig model.
8. method according to claim 11, it is characterised in that: sgRNA, donor dna and the mRNA for encoding spCas9
And/or between spCas9 purification of recombinant proteins with ratio be by the mixture 10~15ng/uL of final concentration sgRNA,
SpCas9 20~25ng/uL of mRNA and/or spCas9 purification of recombinant proteins and 5~10ng/uL of donor dna.
9. method according to claim 12, it is characterised in that: embryo of the pig body early embryo for 4 cell stages and before.
10. according to the method for claim 13, it is characterised in that further include having detection verification step in the step c: using
PFgfr3-E9-F1/R1 (table 1) primer amplification piglet genome DNA sample determines whether according to amplification sequencing containing pre-
Phase gene editing genotype (pFgfr3 c.1132G > A), if showing to model successfully containing the genotype.
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