CN104263754A - Reconstructed ovum of albinism model pig and construction method thereof, and construction method of model pig - Google Patents
Reconstructed ovum of albinism model pig and construction method thereof, and construction method of model pig Download PDFInfo
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Abstract
The invention relates to a reconstructed ovum of an albinism model pig and a construction method thereof, and a construction method of the model pig. According to the construction method of the reconstructed ovum, tyrosinase is knocked out to obtain the reconstructed ovum of the albinism model pig. The combination of a CRISPP/Cas9 gene knock-out technique and a somatic nucleus transplantation technique proves that the method for constructing the gene modified pig has high feasibility. The obtained TYR gene knock-out pig has the typical characteristics of albinism, can provide a reliable animal model for researching human albinism, can be applied to toxicity and anaphylaxis detection and many other aspects, and is hopeful to become a standardized experimental animal for albinism-like mice.
Description
Technical field
The present invention relates to genetically engineered field, especially relate to a kind of reconstructed eggs of albinism swine model and the construction process of construction process and swine model thereof.
Background technology
Research and the treatment of human diseases be unable to do without experimental animal model.Rodent differs greatly with the mankind in genetic expression and every physical signs, though and non-human primate is laboratory animal closest to the mankind, expensive, and there is the obstacle of ethics aspect.Pig is similar to the mankind in body size, physiological condition, allelotaxis and disease progression etc., and therefore, pig is considered to a kind of suitable experimental animal model.Genetic modification pig model is expected to play an important role in human diseases study of pathogenesis, therapeutic strategy research.Embryonic cell is only had to be fitted together to method in the method for current making genetically modified animal and transgene clone method can realize mammiferous gene targeting.Relatively easily obtain genetic modification mouse by genetic modification embryonic stem cell and embeding technique, but also have no idea so far to obtain and have the Pig embryos cell that reproductive tract is fitted together to ability, thus the preparation of genetic modification pig is more a lot of than mouse difficulty.
Researchist is devoted to develop novel method always, realizes high efficiency gene site-directed modification.The zinc of nearest rise refers to that technology (Zinc Finger Nucleases, ZFNs) and TALENs (TRNAscription activator-like (TAL) effector nucleases) technology are that gene targeting creates new approach.Research display is had to utilize ZFNs technology that gene targeting efficiency can be made in porcine somatic cell from 10
-6bring up to more than 4% (Yang D, Yang H, Li W, et al.Production of PPAR-gamma mono-allelic knockout pigs via zinc-finger nucleases and nuclear tRNAsfer cloning.Cell Research, 2011,21 (6): 979-82.).TALENs technology is utilized also successfully to obtain the several genes target practice animals such as rat, zebra fish, Xenopus laevis, mouse, rabbit expeditiously.ZFNs and TALENs technology drastically increases gene targeting efficiency, but ZFNs and TALENs technical project and component composition still need the special requirement of context, through a large amount of optimal conditions to obtain specific gene targeting, acquired, handiness, expense is all restricted (Juillerat, A., Dubois, G., Valton, J., et al.Comprehensive analysis of the specificity of tRNAscription activator-like effector nucleases.Nucleic Acids Res., 2014, 42, 5390 – 5402.).
CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease (Cas9)) technology mainly forms based on a kind of acquired immune system transformation of bacterium.This system is made up of two portions: one is the Cas9 albumen that mammalian codons optimizes version, and it is with a nuclear localization signal, to guarantee to express in the core of mammalian cell; Two is that guiding nucleus ribosomal ribonucleic acid (Single-guide RNA, gRNAs) guides the protein of Cas9 to cut target dna with carrying out sequence-specific.Comparatively ZFNs or TALENS, CRISPR/Cas9 system has extensibility and reusability, multiple target site (Wang H can be acted on simultaneously, Yang H, Shivalila C S, et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell, 2013, 153:910 – 918) etc. advantage, be considered to a kind of genome fixed point transformation molecular tool (Mussolino C with broad prospect of application, Cathomen T.RNA guides genome engineering.NatBiotechnol, 2013, 31 (3): 208-209).CRISPR/Cas9 is utilized to obtain several genes target practice animal, as rat, mouse, rabbit, monkey etc., but be all obtained to the method for a cell stage by injection Cas9 mRNA and gRNA mRNA, mostly the cloned animal that this method obtains is the mosaic with various mutations type, need carry out multiple mating and the animal selecting just can obtain single mutated-genotype.
Albinism is a kind of common disease of humans and animals circle.Current Albino mice oneself to become in biological medicine research conventional laboratory animal, burn, dermal toxicity, irritated to detect etc. study in use more, but do not have the report of genetic modification type albefaction pig up to now yet.Skin and the hair of current wild-type miniature pig mostly are black or pattern, are unfavorable under study for action observing, and are more common in the large-scale pig kind of meat sold on the market in wild-type white pig, and build is unfavorable for greatly experimental implementation, is not suitable for doing laboratory animal.
Summary of the invention
Based on this, be necessary to provide a kind of reconstructed eggs of albinism swine model and the construction process of construction process and swine model thereof.
A construction process for the reconstructed eggs of albinism swine model, comprises the steps:
Step one: meet G (N) in the normal chain of the First Exon of the tyrosinase cdna of pig species and complementary strand
19the Sequence of NGG sequence pattern designs the recognition sequence of gRNA respectively, is designated as a gRNA recognition sequence and the 2nd gRNA recognition sequence respectively, wherein, and sequence G (N) in the normal chain of a described gRNA recognition sequence and described First Exon
19unanimously, sequence G (N) on the complementary strand of described 2nd gRNA recognition sequence and described First Exon
19unanimously, N is A, T, C or G, and subscript 19 represents the number of N;
Step 2: respectively to a described gRNA recognition sequence and the 2nd gRNA recognition sequence design complementary sequence, to build the first double-stranded DNA containing a described gRNA recognition sequence and the second double-stranded DNA containing the 2nd gRNA recognition sequence;
Step 3: respectively according to described first double-stranded DNA and described second double-stranded DNA design gRNA expression vector, be designated as a gRNA carrier and the 2nd gRNA carrier, wherein, containing described first double-stranded DNA in a described gRNA carrier, containing described second double-stranded DNA in described 2nd gRNA carrier;
Step 4: a described gRNA carrier, described 2nd gRNA carrier and the expression vector transfection containing Cas9 nickase gene are entered in miniature porcine fetus fibroblasts, filters out the positive colony cell that tyrosinase cdna knocks out;
Step 5: the enucleation oocyte described positive colony cell being injected sow, forms reconstructed eggs.
Wherein in an embodiment, in described step one, the sequence of the tyrosinase cdna of described pig species is numbered the sequence shown in 407745 for gene in ncbi database; The normal chain of described First Exon meets G (N)
19the sequence of NGG sequence pattern, as shown in SEQ ID No.1, the complementary strand of described First Exon meets G (N)
19the sequence of NGG sequence pattern is as shown in SEQ ID No.2; A described gRNA recognition sequence is as shown in SEQ ID No.3, and described 2nd gRNA recognition sequence is as shown in SEQ ID No.4.
Wherein in an embodiment, described step 2 specifically comprises the steps:
Respectively to a described gRNA recognition sequence and the 2nd gRNA recognition sequence design complementary sequence;
CACC sequence fragment is added at 5 ' end of a described gRNA recognition sequence, form the sequence fragment as shown in SEQ ID No.7, and add AAAC sequence fragment at 5 ' end of the complementary sequence of a described gRNA recognition sequence, form the sequence fragment as shown in SEQID No.8, the gRNA recognition sequence being added with sticky end and the complementary sequence that is added with sticky end are carried out anneal, obtains described first double-stranded DNA with sticky end;
CACC sequence fragment is added at 5 ' end of described 2nd gRNA recognition sequence, form the sequence fragment as shown in SEQ ID No.9, and add AAAC sequence fragment at 5 ' end of the complementary sequence of described 2nd gRNA recognition sequence, form the sequence fragment as shown in SEQID No.10, the 2nd gRNA recognition sequence being added with sticky end and the complementary sequence that is added with sticky end are carried out anneal, obtains described second double-stranded DNA with sticky end.
Wherein in an embodiment, described step 3 specifically comprises the steps:
In gRNA-GFP-T1 plasmid vector, introduce two BbsI restriction enzyme sites, obtain intermediate plasmid;
Restriction enzyme BbsI enzyme is used to cut to described intermediate plasmid, and by with sticky end described first double-stranded DNA and described second double-stranded DNA correspondence be connected to enzyme cut after intermediate plasmid on, obtain a described gRNA carrier and described 2nd gRNA carrier respectively.
Wherein in an embodiment, in described step 4, before miniature porcine fetus fibroblasts is entered in a described gRNA carrier, described 2nd gRNA carrier and the expression vector transfection containing Cas9 nickase gene, also comprise respectively to a described gRNA carrier, described 2nd gRNA carrier and the expression vector enlarged culturing after conversion processing containing Cas9 nickase gene, then extract the step of a described gRNA carrier, described 2nd gRNA carrier and the expression vector containing Cas9 nickase gene.
Wherein in an embodiment, the construction process of described reconstructed eggs also comprises the step merging the reconstructed eggs obtained and activate, specific as follows:
Described reconstructed eggs is gone in embryo medium to be fused with activation from stoning operation liquid;
Described reconstructed eggs is transferred to fusion liquid activation solution to balance, the reconstructed eggs balanced is moved in converged container, stir reconstructed eggs gently, the contact surface of the cell of ovocyte and injection is made to be parallel to two strip electrodes, 1mm is spaced apart between two strip electrodes, then carry out electric pulse stimulation, electrofusion parameter is: 120volts/mm, 30 μ s, 2 times;
After electric pulse stimulation, reconstructed eggs is moved in embryo operation liquid, filter out and merge successful reconstructed eggs.
A kind of construction process of the reconstructed eggs of the albinism swine model described in above-mentioned any embodiment that adopts builds the reconstructed eggs obtained.
A construction process for albinism swine model, comprises the steps:
Reconstructed eggs is built according to the construction process of the reconstructed eggs of the albinism swine model described in above-mentioned any embodiment;
Cytogamy and activation are carried out to described reconstructed eggs, obtains the reconstructed eggs activated;
By in the uterine tube of the reconstructed eggs of described activation as replace-conceive sow, or the reconstructed eggs of described activation is cultivated in vitro or in body, form reconstructed embryo, and then described reconstructed embryo is transplanted to the intrauterine of replace-conceive sow;
Raise described replace-conceive sow, produce albinism swine model.
Wherein in an embodiment, described cytogamy and activation are carried out to described reconstructed eggs, obtain the reconstructed eggs activated, specifically comprise the steps:
Described reconstructed eggs is gone in embryo medium to be fused with activation from stoning operation liquid;
Described reconstructed eggs is transferred to fusion liquid activation solution to balance, the reconstructed eggs balanced is moved in converged container, stir reconstructed eggs gently, the contact surface of the cell of ovocyte and injection is made to be parallel to two strip electrodes, 1mm is spaced apart between two strip electrodes, then carry out electric pulse stimulation, electrofusion parameter is: 120 volts/mm, 30 μ s, 2 times;
After electric pulse stimulation, reconstructed eggs is moved in embryo operation liquid, filter out and merge successful reconstructed eggs.
The above-mentioned reconstructed eggs of albinism swine model and the construction process of swine model, albefaction miniature pig is obtained by knocking out tyrosinase cdna (TYR gene), and CRISPR/Cas9 gene Knockout is combined with somatic cell nuclear transfer technique, prove that the method is for building the efficient feasibility of genetic modification pig.Obtain TYR gene knock-out pig and there is typical albefaction feature, can be mankind's albefaction disease research and a kind of animal model is reliably provided, and the many-side such as toxicity and anaphylactoid detection can be applied to, be expected to a kind of standardized laboratory animals becoming similar Albino mice.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet of the construction process of the reconstructed eggs of the albinism swine model of an embodiment;
Fig. 2 is profile and the eyes comparison diagram of albinism swine model and black pig;
Fig. 3 is the albinism swine model at another visual angle and the profile comparison diagram of black pig;
Fig. 4 is the skin basic unit comparison diagram of albinism swine model and black pig, and wherein A represents black pig, and B represents albinism swine model;
Fig. 5 is the iris tissue comparison diagram of albinism swine model and black pig, and wherein C represents black pig, and D represents albinism swine model.
Embodiment
It is main below that the reconstructed eggs of whitening disease model pig and the construction process of construction process and swine model thereof are described in further detail in conjunction with the drawings and the specific embodiments.
The construction process of the albinism swine model of one embodiment comprise build swine model reconstructed eggs, this reconstructed eggs activated and the reconstructed eggs of activation imported the process grown in replace-conceive sow body.
As shown in Figure 1, the construction process of the reconstructed eggs of the albinism swine model of present embodiment comprises the steps:
Step S110, meets G (N) in the normal chain of the First Exon of the tyrosinase cdna of pig species and complementary strand
19the Sequence of NGG sequence pattern designs the recognition sequence of gRNA respectively, is designated as a gRNA recognition sequence and the 2nd gRNA recognition sequence respectively.Wherein, sequence G (N) in the normal chain of a described gRNA recognition sequence and described First Exon
19unanimously, sequence G (N) on the complementary strand of described 2nd gRNA recognition sequence and described First Exon
19unanimously, N is A, T, C or G, and subscript 19 represents the number of N.
Preferably but be not limited to mountain pig (formal name used at school: Sus scrofa), the sequence of its tyrosinase cdna is numbered the sequence shown in 407745 (Gene ID:407745) for gene in ncbi database to above-mentioned pig species.The partial sequence (5 '-GCACCCCTGGGACCTCAGTTCCCCTTCACCGGGGTGGATGAACGGGAGTCTTGGCC CT-3 ') of the normal chain of the First Exon that present embodiment is used is as shown in SEQ ID No.1, and the partial sequence (3 '-CGTGGGGACCCTGGAGTCAAGGGGAAGTGGCCCCACCTACTTGCCCTCAGAACCGG GA-5 ') of the complementary strand of First Exon is as shown in SEQ ID No.2.For the gRNA recognition sequence (5 '-GGGTGGATGA ACGGGAGTCT-3 ') of the partial sequence design of this normal chain and complementary strand as shown in SEQ ID No.3, the 2nd gRNA recognition sequence (5 '-GAAGGGGAAC TGAGGTCCCA-3 ') is as shown in SEQ ID No.4.Can understand, in other embodiments, a sgRNA recognition sequence and the 2nd gRNA recognition sequence can also be other sequential structures, meet G (N) as long as exist in the normal chain of this First Exon and on complementary strand
19the sequence of the sequence pattern of NGG just may correspond to design the one gRNA recognition sequence and the 2nd gRNA recognition sequence.
Step S120: respectively to a gRNA recognition sequence and the 2nd gRNA recognition sequence design complementary sequence, to build the first double-stranded DNA containing a gRNA recognition sequence and the second double-stranded DNA containing the 2nd gRNA recognition sequence.
Specifically in the present embodiment, the building process of the first double-stranded DNA and the second double-stranded DNA comprises the steps:
Respectively complementary sequence is designed to a described gRNA recognition sequence (GGGTGGATGA ACGGGAGTCT) and the 2nd gRNA recognition sequence (GAAGGGGAAC TGAGGTCCCA);
CACC sequence fragment is added at 5 ' end of a described gRNA recognition sequence, form the sequence fragment (CACCGGGTGG ATGAACGGGA GTCT) as shown in SEQ ID No.7, and add AAAC sequence fragment at 5 ' end of the complementary sequence of a described gRNA recognition sequence, form the sequence fragment (AAACAGACTCCCGTTCATCC ACCC) as shown in SEQ ID No.8, the gRNA recognition sequence being added with sticky end and the complementary sequence that is added with sticky end are carried out anneal, obtains described first double-stranded DNA with sticky end;
CACC sequence fragment is added at 5 ' end of described 2nd gRNA recognition sequence, form the sequence fragment (CACCGAAGGG GAACTGAGGT CCCA) as shown in SEQ ID No.9, and add AAAC sequence fragment at 5 ' end of the complementary sequence of described 2nd gRNA recognition sequence, form the sequence fragment (AAACTGGGACCTCAGTTCCC CTTC) as shown in SEQ ID No.10, the 2nd gRNA recognition sequence being added with sticky end and the complementary sequence that is added with sticky end are carried out anneal, obtains described second double-stranded DNA with sticky end.
The first double-stranded DNA formed and the second double-stranded DNA all have " CACC " and " AAAC " sticky end, and this sticky end can be used for being connected on corresponding carrier.
This adds that the process of sticky end can be used but not limited to the method for full genome synthesis or designs suitable primer and obtain through pcr amplification.
Step S130: respectively according to the first double-stranded DNA and the second double-stranded DNA design gRNA expression vector, be designated as a gRNA carrier and the 2nd gRNA carrier.Wherein, containing described first double-stranded DNA in a described gRNA carrier, containing described second double-stranded DNA in described 2nd gRNA carrier.
This step is connected in the plasmid containing gRNA encoding sequence by the first double-stranded DNA and the second double-stranded DNA correspondence, as gRNA-GFP-T1 plasmid vector, this process specifically comprises the steps: to introduce corresponding restriction enzyme site in the plasmid vector containing gRNA encoding sequence, as BbsI restriction enzyme site, obtain intermediate plasmid, then by the first double-stranded DNA and the second double-stranded DNA by the restriction enzyme site (i.e. sticky end) introduced be connected to enzyme cut after the correspondence position of intermediate plasmid obtain required plasmid.Wherein, restriction enzyme site can be, but not limited to BbsI restriction enzyme site, and when restriction enzyme site is BbsI restriction enzyme site, this first double-stranded DNA and this second double-stranded DNA are all connected with above-mentioned " CACC " and " AAAC " sticky end, and cloning vector cuts process through BbsI enzyme.
Step S140: a gRNA carrier, the 2nd gRNA carrier and the expression vector transfection containing Cas9 nickase gene are entered in miniature porcine fetus fibroblasts, filters out the positive colony cell of tyrosine gene knockout.
The positive colony cell filtering out tyrosine gene knockout comprises the steps:
Cracking is carried out to the clone cell that part is cultivated, extracts lysate;
Carry out PCR process to lysate, wherein, the primer sequence of PCR is respectively as shown in SEQ ID No.5 and SEQ ID No.6, and PCR condition is: 95 DEG C of denaturations 5 minutes, 94 DEG C of distortion 20 seconds, 60 DEG C of annealing 30 seconds, and 72 DEG C extend 30 seconds, totally 35 circulations;
Get PCR primer and carry out 1% agarose gel electrophoresis detection, select positive colony cell.
In addition, in the present embodiment, before porcine fetus fibroblasts is entered in a gRNA carrier, the 2nd gRNA carrier and the expression vector transfection containing Cas9 nickase gene, also comprise respectively to a gRNA carrier, the 2nd gRNA carrier and the expression vector enlarged culturing after conversion processing containing Cas9 nickase gene, then extract the step of a gRNA carrier, the 2nd gRNA carrier and the expression vector containing Cas9 nickase gene respectively.
Step S150: enucleation oocyte positive colony cell being injected sow, forms reconstructed eggs.
Specifically in the present embodiment, following steps can be adopted to carry out:
Carry out centrifugal treating after being digested by positive colony cell use trypsinase, abandon supernatant, re-suspended cell;
In stoning operation liquid to ovocyte with the stoning of blind suction method after, absorptions walks resuspended cell direct injection in all gaps of ovum of non-nucleus egg mother cell, extrudes ovocyte gently, make the cell membrane contact of the cytolemma of ovocyte and the cell of injection.
Step S160, merges the reconstructed eggs obtained and activates.
Specifically in the present embodiment, undertaken by following steps:
Reconstructed eggs is gone in embryo medium to be fused with activation from stoning operation liquid;
Reconstructed eggs is transferred to fusion activation solution to balance, and the reconstructed eggs balanced is moved in converged container, stir reconstructed eggs gently, make the contact surface of the cell of ovocyte and injection be parallel to two strip electrodes, then carry out electric pulse stimulation fusion;
After electric pulse stimulation, reconstructed eggs is moved in embryo operation liquid, filter out and merge successful reconstructed eggs.
The process of will carry out growing in the reconstructed eggs of activation importing replace-conceive sow body described in present embodiment, can be by the uterine tube of reconstructed eggs as replace-conceive sow that activate, or the reconstructed eggs of activation is cultivated in vitro or in body, form reconstructed embryo, and then reconstructed embryo is transplanted to the intrauterine of replace-conceive sow, raise replace-conceive sow again, produce albinism swine model.
The reconstructed eggs of this albinism swine model and the construction process of swine model, albefaction miniature pig is obtained by knocking out tyrosinase cdna (TYR gene), and CRISPR/Cas9 gene Knockout is combined with somatic cell nuclear transfer technique, prove that the method is for building the efficient feasibility of genetic modification pig.Obtain TYR gene knock-out pig and there is typical albefaction feature, can be mankind's albefaction disease research and a kind of animal model is reliably provided, and the many-side such as toxicity and anaphylactoid detection can be applied to, be expected to a kind of standardized laboratory animals becoming similar Albino mice.
Be below specific embodiment part:
Following reagent, unless stated otherwise, equal available from Sigma is corresponding Chinese and/or cat. no after parantheses.
The present embodiment mainly comprises the following steps: the structure of (1) CRISPR/Cas9 targeting system; (2) cell transfecting and screening, and the acquisition of TYR Knockout cells clone; (3) body-cell neucleus transplanting; (4) genotype of genetic modification pig and phenotypic evaluation.
(1) structure of CRISPR/Cas9 targeting system:
According to the sequence (Gene ID:407745) of the TYR gene of pig species Sus scrofa in ncbi database, according to G (N) in First Exon sequence
19nGG principle, respectively normal chain (partial sequence is as shown in SEQ ID No.1) and complementary strand (partial sequence is as shown in SEQ ID No.2) are designed to the recognition sequence of a gRNA, be designated as a gRNA recognition sequence (as shown in SEQ ID No.3) and the 2nd gRNA recognition sequence (as shown in SEQ ID No.4) respectively.
Extract the genome being used for cell to be transfected, at the target sequence two ends of the recognition sequence identification of gRNA design pair of primers, wherein upstream primer sequence is 5 '-CCTGATGGAGAAGGAAT GCTGC-3 ' (as shown in SEQ ID No.5), downstream primer sequence is 5 '-TTGGCCATAGGTGCCTGTG-3 ' (as shown in SEQ ID No.6), pcr amplification contains the DNA fragmentation of target sequence, and clip size is 388bp.Pcr amplification condition is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations.Amplification target sequence fragment through order-checking after with the sequence alignment on GenBank, determine that sequence is correct.
Introduce 2 Bbs I restriction enzyme sites at plasmid gRNA-GFP-T1 (purchased from Addgene company, catalog number is 41819) plasmid, obtain U6-gRNA cloning vector.Respectively to a gRNA recognition sequence and the 2nd gRNA recognition sequence design complementary sequence, and add sticky end at the complementary sequence two ends of a gRNA recognition sequence and the 2nd gRNA recognition sequence and correspondence, then anneal forms the first double-stranded DNA and the second double-stranded DNA.First double-stranded DNA and the second double-stranded DNA all include sticky end " CACC " and " AAAC ", wherein, in first double-stranded DNA, the sequence of two chains is respectively as shown in SEQID No.7 and SEQ ID No.8, and in the second double-stranded DNA, the sequence of two chains is respectively as shown in SEQ ID No.9 and SEQ IDNo.10.
Respectively the first double-stranded DNA and the second double-stranded DNA correspondence are connected in the U6-gRNA cloning vector cut through BbsI enzyme, obtain a gRNA carrier and the 2nd gRNA carrier, determine that sequence connects through order-checking correct.
(2) cell transfecting and screening:
By the plasmid vector (Cas9-nickase containing Cas9 gene, purchased from Addgene company, catalog number is 41816) and the gRNA carrier that obtains and the 2nd gRNA carrier enlarged culturing after transforming, large upgrading grain, three kinds of plasmids respectively prepare 20 μ g.
Day before transfection recovery version receives miniature porcine fetus fibroblasts (derive from version receive miniature pig 37 age in days fetus), adds 10mL 15%FBS DMEM substratum, be placed in 37 DEG C of incubators and cultivate in 10cm Tissue Culture Dish; Cultivate electricity after 24 hours to turn, proceed to each 20 μ g of Cas9 plasmid vector, a gRNA carrier and the 2nd gRNA carrier, electricity turns parameter: voltage 230V electric capacity 500 μ F, and instrument is U.S. Bole electric perforating system (Bio-Rad Gene Pulser Xcell).
After electrotransfection, cell is divided into 25 large culture dish cultivations of 10cm; Change liquid every other day, change the 15%FBS DMEM culture medium culturing containing G418 (concentration is 200 μ g/mL) into; Liquid is changed every 2 days, rear clone growth in about 10 days, bottom large culture dish, clone is drawn with marking pen, wash once by PBS solution remove substratum in super clean bench after, live with clone's ring the clone that marking pen draws, add 50-100 μ l 0.05% trysinization 5-8min, add substratum and stop, cell in clone's ring is all transferred in 48 orifice plates as far as possible, and washes twice with substratum.The cell getting 1/10 when going down to posterity for the identification of.
Collected by centrifugation take out for the identification of monoclonal cell, add 15 μ l NP40 lysates (containing 0.45%NP-40 and 0.6% Proteinase K), successively at 56 DEG C of cracking 1h, at 95 DEG C of cracking 10min, the lysate obtained saves backup in-20 DEG C.
The lysate obtained is utilized to the primer pair of above-mentioned design, wherein upstream primer sequence is 5 '-CCTGATGGAGAAGGAAT GCTGC-3 ' (as shown in SEQ ID No.5), downstream primer sequence is 5 '-TTGGCCATAGGTGCCTGTG-3 ' (as shown in SEQ ID No.6), adopt PCR program to increase, PCR condition is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations.Get 3 μ l PCR primer and carry out 1% agarose gel electrophoresis detection analysis.The PCR primer obtaining a single bright band send Shenzhen Hua Da gene sequencing with forward primer.Sequencing result and former sequence alignment, part sequencing result is the PCR primer of rear bimodal collection of illustrative plates, clones be connected after reclaiming purifying with pMD18T carrier through TA, transforms upgrading grain afterwards with universal primer M13 order-checking, obtains sequence.The target site sequence sequencing result of some positive clone is as shown in table 1.
Table 1
Picking 89 cell clones altogether, effectively order-checking 87, knocking out ratio is 49.4%, and wherein two ratio that strikes is 20.7%, and singly striking ratio is 28.7%.Select 6 strain cell clones (T 5, T 12, T 47, T 65, T 70, T 79) to be nuclear transfer donor cell, carry out body-cell neucleus transplanting.
(3) body-cell neucleus transplanting:
The maturation in vitro of porcine oocytes
From slaughterhouse collect pig ovary be placed in 39 DEG C be added with penicillin, Streptomycin sulphate 0.9% physiological saline.With the 10mL syringe with No. 12 syringe needles by the liquor folliculi sucking-off in folliculus ovarii in the centrifuge tube of 50mL, 39 DEG C of water bath heat preservations.Abandon supernatant after leaving standstill 4min, add egg-cleaning liquid PVA-TL-HEPES and (take 6.6633g NaCl, 0.2386g KCl, 0.1680g NaHCO
3, 0.0408g NaH
2pO4,0.1017g MgCl
26H
2o, 2.3830g Hepes (4-hydroxyethyl piperazine ethanesulfonic acid, H3784), 0.0650g Penicillin (penicillin, P3032), 0.0100g pHenol Red (phenolsulfonphthalein, 5530), 0.2940g CaCl
22H
2o, 0.1000g Polyvinyl alcohol (PVA, polyvinyl alcohol P8136), 2.1860g Sorbitol (Sorbitol Powder, S1876), 0.0250gGentamicin (gentamicin), 0.0220g Sodium pyruvate (Sodium.alpha.-ketopropionate, P4562), 998.132mL Milli Q H is added
21.868mL Na Lactate (Sodium.alpha.-hydroxypropionate is added again after O (ultrapure water), L7900), pH is regulated to be 7.2-7.4 after dissolving, osmotic pressure is 295-310mOsm) picking cumulus oocyte mixture, (TCM-199 (Gibco company) adds 3.05mM D-glucose (D-Glucose to be transferred to the ripe liquid balanced in advance, G7021), 0.91mM Sodium pyruvate (Sodium.alpha.-ketopropionate, P4562), 0.1%PVA (Sigma, P8136), 75 μ g/mL Penicillin (Sigma, P3032), 50 μ g/mLStreptomycin (Streptomycin sulphates, S1277), 0.5 μ g/mL Luteinizing hormone (LH, metakentrin, L5269), 10 ng/mL Epidermal growth factor (EGF, Urogastron, S4127), 0.5 μ g/mL Follicle stimulating hormone (FSH, follicle stimulating hormone, F2293), 0.57mM Cysteine (halfcystine, C8152), 10% liquor folliculi.) in 5%CO
2, saturated humidity, to cultivate under 39 DEG C of conditions.12 orifice plates every empty placement 40-70 piece of cumulus oocyte mixture.After In-vitro maturation 42-44h, cumulus oocyte mixture being transferred to 39 DEG C goes ovarian cumulus to operate liquid (0.030gHyaluronidase (Unidasa, H3506), 5.46g Mannitol (N.F,USP MANNITOL, M9647), 0.001g BSA (bovine serum albumin, A8022), 5mL PVA-TL-Hepes egg-cleaning liquid, 95mL Milli Q H
2o), vortex concussion 5min, comes off to cumulus cell.Postdigestive ovocyte is transferred to and fills embryo operation liquid (9.500g TCM-199 (Gibco company), 0.050gNaHCO
3, 0.750g Hepes (H3784), 0.050g Penicillin (P3032), 0.060g Streptomycin (S1277), 1.755g NaCl, 3.00g BSA, 1000mL Milli Q H
2o, dissolve after regulate pH be 7.2-7.4 dissolve after regulate pH be 7.2-7.4, osmotic pressure is 295-310 mOsm) 35mm culture dish in, under body formula mirror picking discharge first polar body ovocyte fill in the 35mm culture dish of embryo operation liquid in another, 39 DEG C of preservations, stand-by.
The positive colony cell cultivated carries out digestion process 3 minutes with 0.05% trypsinase, with the speed of 1000 revs/min centrifugal 3 minutes afterwards, abandons supernatant, with DMEM substratum re-suspended cell.Ovocyte, with after the stoning of blind suction method, is drawn a Donor cell injection in all gaps of ovum of enucleation oocyte, and is extruded ovocyte gently, oocyte membrane is contacted with donorcells film.Reconstructed eggs is put into the embryo medium PZM3 balanced, place in 38.5 DEG C of incubators, to be fused with activation.
Reconstructed eggs is gone to from embryo medium and merges activation solution (0.3M Mannitol (M9647), 1.0mM CaCl
22H
2o, 0.1mM MgCl
26H
2o, 0.5mM Hepes (H3784)) in be fused with activation, before merging, reconstructed eggs is transferred to fusion activation solution to balance, the reconstructed eggs balanced is moved in integration slot, stir reconstruct embryo gently with capillary glass pin, make the contact surface of ovocyte and donorcells be parallel to two strip electrodes, between two strip electrodes, be spaced apart 1mm, then carry out electric pulse stimulation, electrofusion parameter is: 120 volts/mm, 30 μ s, 2 times.After electric pulse stimulation, reconstructed eggs is moved into embryo operation liquid (9.500gTCM-199 (Gibco company), 0.050g NaHCO
3, 0.750g Hepes (H3784), 0.050g Penicillin, 0.060gStreptomycin (S1277), 1.755g NaCl, 3.00g BSA, 1000mL Milli Q H
2o, pH is regulated to regulate pH to be 7.2-7.4 after 7.2-7.4 dissolves after dissolving, osmotic pressure is 295-310 mOsm) in, after putting 39 DEG C of half an hour, reconstructed eggs is stirred with capillary glass pin under body formula mirror, check whether donorcells has merged into ovocyte, is abandoned by the reconstructed eggs do not merged, statistics fusion rate.Namely reconstructed eggs activates while merging.The reconstructed eggs merged with activating is washed three times in embryo medium PZM-3, and puts into PZM-3 and be placed in 5%CO
2, saturated humidity, to cultivate under 39 DEG C of conditions.
Embryo transfer
Within after nuclear transplantation second day, carry out embryo transfer.The Taihu pigs selecting the embryo transfer same day or the day before yesterday to oestrus is surrogate recipient.Embryo in cultivating is transferred to the 1.5mL EP pipe filling operation liquid from embryo medium, puts into 38.5 DEG C of constant temperature embryo carrying cases for subsequent use.Carry out induced anesthesia to acceptor pig injection ketamine, isoflurane breathes anesthesia maintenance.Last to and second from the bottom to nipple between opening about 8cm, find ovary along uterus and fallopian tube direction after finding uterus, ovary uterine tube slowly involved abdominal cavity, observe ovary ovulation situation, and find oviducal umbrella portion tweezers to be fixed.By the embryo transplantation tube installed, from fimbriae tubae, portion slowly stretches into uterine tube, and embryo is pushed uterine tube.After embryo transfer completes, fimbriae tubae is wrapped back ovary again, send ovary uterine tube back to abdominal cavity.To sew up after normal saline flushing wound, first peritoneal suture and muscle layer, after muscle layer has been sewed up, is coated with and spreads mycillin, then skin suture layer.Post operation intramuscular injection mycillin prevents traumatic infection inflammation, and carries out postoperative care.Within after embryo transfer 24-26 days, carry out B ultrasonic monitoring Pregnancy, and a B ultrasonic monitoring is carried out week about to pregnant recipient, follow the trail of clone embryos developmental state.
As shown in table 2, the present embodiment co-transplantation 1705 reconstructed eggs are in 7 acceptor sow bodies, wherein 4 pregnancies 18 clone pigs of being born, numbering #T1-18 respectively, 15 are wherein had to have typical I type oculocutaneous albimism (OCA1) feature, skin and hair are white, the transparent pink of eyes, as shown in Figures 2 and 3.Genotype identification 15 albinism pigs are TYR gene knock-out pig.
Table 2
(4) genotype of genetic modification pig and phenotypic evaluation
Ear edge scissor along clone pig is organized on a small quantity, extract genome with TIANamp Genomic DNA Kit test kit, with the above-mentioned primer pair of identification of cell clone, comprise the sequence at target site two ends with same condition pcr amplification, compare with former sequence after order-checking, determine genotype.
Get skin and the ear tissue of genetic modification pig, 4% paraformaldehyde fixes 48h, transparent through dewatering, and after waxdip embedding, makes section.Hematoxylin-eosin staining is carried out to section, to be presented at skin base layer and whether iris tissue has melanochrome to exist.
Genotype identification 15 pigs are TYR gene knock-out pig, and the mode of knocking out lacks 17 base to 60 bases, or insert 1 base to 188 base, as shown in table 3.As shown in Figure 4 and Figure 5, phenotypic evaluation exists without melanochrome at the skin base layer of TYR gene knock-out pig and iris tissue, shows that TYR gene expression product and downstream product thereof do not have completely.
Table 3
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. a construction process for the reconstructed eggs of albinism swine model, is characterized in that, comprises the steps:
Step one: meet G (N) in the normal chain of the First Exon of the tyrosinase cdna of pig species and complementary strand
19the Sequence of NGG sequence pattern designs the recognition sequence of gRNA respectively, is designated as a gRNA recognition sequence and the 2nd gRNA recognition sequence respectively, wherein, and sequence G (N) in the normal chain of a described gRNA recognition sequence and described First Exon
19unanimously, sequence G (N) on the complementary strand of described 2nd gRNA recognition sequence and described First Exon
19unanimously, N is A, T, C or G, and subscript 19 represents the number of N;
Step 2: respectively to a described gRNA recognition sequence and the 2nd gRNA recognition sequence design complementary sequence, to build the first double-stranded DNA containing a described gRNA recognition sequence and the second double-stranded DNA containing the 2nd gRNA recognition sequence;
Step 3: respectively according to described first double-stranded DNA and described second double-stranded DNA design gRNA expression vector, be designated as a gRNA carrier and the 2nd gRNA carrier, wherein, containing described first double-stranded DNA in a described gRNA carrier, containing described second double-stranded DNA in described 2nd gRNA carrier;
Step 4: a described gRNA carrier, described 2nd gRNA carrier and the expression vector transfection containing Cas9 nickase gene are entered in miniature porcine fetus fibroblasts, filters out the positive colony cell that tyrosinase cdna knocks out;
Step 5: the enucleation oocyte described positive colony cell being injected sow, forms reconstructed eggs.
2. the construction process of the reconstructed eggs of albinism swine model as claimed in claim 1, is characterized in that, in described step one, the sequence of the tyrosinase cdna of described pig species is numbered the sequence shown in 407745 for gene in ncbi database; The normal chain of described First Exon meets G (N)
19the sequence of NGG sequence pattern, as shown in SEQ ID No.1, the complementary strand of described First Exon meets G (N)
19the sequence of NGG sequence pattern is as shown in SEQ ID No.2; A described gRNA recognition sequence is as shown in SEQ ID No.3, and described 2nd gRNA recognition sequence is as shown in SEQ ID No.4.
3. the construction process of the reconstructed eggs of albinism swine model as claimed in claim 2, it is characterized in that, described step 2 specifically comprises the steps:
Respectively to a described gRNA recognition sequence and the 2nd gRNA recognition sequence design complementary sequence;
CACC sequence fragment is added at 5 ' end of a described gRNA recognition sequence, form the sequence fragment as shown in SEQ ID No.7, and add AAAC sequence fragment at 5 ' end of the complementary sequence of a described gRNA recognition sequence, form the sequence fragment as shown in SEQID No.8, the gRNA recognition sequence being added with sticky end and the complementary sequence that is added with sticky end are carried out anneal, obtains described first double-stranded DNA with sticky end;
CACC sequence fragment is added at 5 ' end of described 2nd gRNA recognition sequence, form the sequence fragment as shown in SEQ ID No.9, and add AAAC sequence fragment at 5 ' end of the complementary sequence of described 2nd gRNA recognition sequence, form the sequence fragment as shown in SEQID No.10, the 2nd gRNA recognition sequence being added with sticky end and the complementary sequence that is added with sticky end are carried out anneal, obtains described second double-stranded DNA with sticky end.
4. the construction process of the reconstructed eggs of albinism swine model as claimed in claim 3, it is characterized in that, described step 3 specifically comprises the steps:
In gRNA-GFP-T1 plasmid vector, introduce two BbsI restriction enzyme sites, obtain intermediate plasmid;
Restriction enzyme BbsI enzyme is used to cut to described intermediate plasmid, and by with sticky end described first double-stranded DNA and described second double-stranded DNA correspondence be connected to enzyme cut after intermediate plasmid on, obtain a described gRNA carrier and described 2nd gRNA carrier respectively.
5. the construction process of the reconstructed eggs of albinism swine model as claimed in claim 4, it is characterized in that, in described step 4, by a described gRNA carrier, before miniature porcine fetus fibroblasts is entered in described 2nd gRNA carrier and the expression vector transfection containing Cas9 nickase gene, also comprise respectively to a described gRNA carrier, described 2nd gRNA carrier and the expression vector enlarged culturing after conversion processing containing Cas9 nickase gene, extract a described gRNA carrier again, the step of described 2nd gRNA carrier and the expression vector containing Cas9 nickase gene.
6. the construction process of the reconstructed eggs of the albinism swine model according to any one of claim 1-5, is characterized in that, also comprises the step merging the reconstructed eggs obtained and activate, specific as follows:
Described reconstructed eggs is gone in embryo medium to be fused with activation from stoning operation liquid;
Described reconstructed eggs is transferred to fusion liquid activation solution to balance, the reconstructed eggs balanced is moved in converged container, stir reconstructed eggs gently, the contact surface of the cell of ovocyte and injection is made to be parallel to two strip electrodes, 1mm is spaced apart between two strip electrodes, then carry out electric pulse stimulation, electrofusion parameter is: 120 volts/mm, 30 μ s, 2 times;
After electric pulse stimulation, reconstructed eggs is moved in embryo operation liquid, filter out and merge successful reconstructed eggs.
7. the construction process of the reconstructed eggs of the albinism swine model according to any one of claim 1-6 builds the reconstructed eggs obtained.
8. a construction process for albinism swine model, is characterized in that, comprises the steps:
Reconstructed eggs is built according to the construction process of the reconstructed eggs of the albinism swine model according to any one of claim 1-5;
Cytogamy and activation are carried out to described reconstructed eggs, obtains the reconstructed eggs activated;
By in the uterine tube of the reconstructed eggs of described activation as replace-conceive sow, or the reconstructed eggs of described activation is cultivated in vitro or in body, form reconstructed embryo, and then described reconstructed embryo is transplanted to the intrauterine of replace-conceive sow;
Raise described replace-conceive sow, produce albinism swine model.
9. the construction process of albinism swine model as claimed in claim 8, is characterized in that, describedly carries out cytogamy and activation to described reconstructed eggs, obtains the reconstructed eggs activated, specifically comprises the steps:
Described reconstructed eggs is gone in embryo medium to be fused with activation from stoning operation liquid;
Described reconstructed eggs is transferred to fusion liquid activation solution to balance, the reconstructed eggs balanced is moved in converged container, stir reconstructed eggs gently, the contact surface of the cell of ovocyte and injection is made to be parallel to two strip electrodes, 1mm is spaced apart between two strip electrodes, then carry out electric pulse stimulation, electrofusion parameter is: 120volts/mm, 30 μ s, 2 times;
After electric pulse stimulation, reconstructed eggs is moved in embryo operation liquid, filter out and merge successful reconstructed eggs.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101898959A (en) * | 2010-07-20 | 2010-12-01 | 暨南大学 | Biphenyl compound and preparation method and use thereof |
-
2014
- 2014-08-29 CN CN201410438927.3A patent/CN104263754B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101898959A (en) * | 2010-07-20 | 2010-12-01 | 暨南大学 | Biphenyl compound and preparation method and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| HIROKO KOIKE-YUSA1,ET AL: "Genome-wide recessive genetic screening inmammalian cells with a lentiviral CRISPR-guide RNA library", 《NATURE BIOTECHNOLOGY》 * |
| 黄威: "腺相关病毒介导的西藏小型猪酪氨酸酶基因打靶的研究", 《中国优秀硕士学位论文全文数据库》 * |
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