CN105821049B - A kind of preparation method of Fbxo40 gene knock-out pig - Google Patents
A kind of preparation method of Fbxo40 gene knock-out pig Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods of Fbxo40 gene knock-out pig.CRISPR/Cas9 targeting vector and PGK-Neo resistant gene are transfected porcine fetus fibroblasts by the present invention jointly, obtain the positive monoclonal cell of G418 resistance, insertion/deletion mutation occurs for the Fbxo40 gene in positive monoclonal cell, and reading frame generates frameshit and terminates in advance, using such cell clone as the donorcells of nuclear transfer, using egg mother cell as the receptor oocytes of nuclear transfer, clone embryos are obtained by somatic cell nuclear transfer technique, it will be in good clone embryo transplantation to heat sow fallopian tubal, the clone pig of Fbxo40 gene knockout is developed and obtained by full period.The present invention utilizes the gene editing technology of CRISPR/Cas9, and low cost expeditiously obtains Fbxo40 knock-out pig, provides animal model for research muscle development and muscle related disease.
Description
Technical field
The invention belongs to animal genetic engineering and gene genetics to modify field, specifically, being related to a kind of utilizing CRISPR/
Cas9 system edits pig Fbxo40 gene, and obtains Fbxo40 gene knock-out pig by somatic cell nuclear transfer technique.
Background technique
IGF1 can lead to the hypertrophy of muscle, but in the muscle of differentiation by activating IGF1R/IRS1/PI3K/AKT access
In cell, the substrate IRS1 of IGF1 can degrade by ubiquitination, and by proteasome, so as to cause the interruption of IGF1 signal path.
The study found that mediate IRS1 rapid conversion is E3 ubiquitin ligase Fbxo40.Fbxo40-SCF-E3 protein complexes can be straight
It connects IRS1 ubiquitination, and when the tyrosine phosphorylation of IRS1, this ubiquitination effect can enhance.It is knocked out in mouse
After Fbxo40, the significant thickening of muscle fibre.In the fast growing period of mouse, IGF1 is in highly expressed state, and Fbxo40 is knocked out
Later, IRS1 expression quantity can raise, and the weight and muscle mass of mouse dramatically increase.
Fbxo40 can be used as a candidate gene of big animal breeding.The expression of Fbxo40 is muscle specific.Small
Report in mouse shows that Fbxo40 is expressed the 4th day after birth, reaches top in puberty.Firstly, in mouse,
Fbxo40 blocks the signal path of IGF1 by degradation IRS1.Document report, pig it is each tissue sxemiquantitative the result shows that
Fbxo40 is logical in longissimus dorsi muscle and Expression in Myocardium amount highest, the low expression in its hetero-organization, but signal of the Fbxo40 in pig
It studies not yet on road.Are the related pathways of Fbxo40 consistent with mouse so in pig? existing documents and materials are not
Provide the report of this respect.
Secondly, the Fbxo40 reported in mouse only in skeletal muscle, Expression in Myocardium, and be confined to differentiation muscle cell
Middle expression.Fbxo40 knock-out mice shows significant muscle and increases, and occurs without other abnormal phenotypes.Thus
Fbxo40 gene for agriculturally big animal breeding improvement, and medically it is amyotrophic treatment have great importance.
Thus by preparation Fbxo40 gene knock-out pig, Fbxo40 is illustrated on the basis of this model in pig muscle development
The effect of signal path, has great importance.
CRISPR/Cas9 is a kind of adaptive immune system being present in bacterium and Archimycetes.Using artificial synthesized
The fixed point cutting of genome may be implemented in the base pair complementarity of sgRNA sequence and genomic DNA, Cas9 endonuclease, from
And generate the double-strand break of DNA.DNA double chain fracture can be repaired by two ways: one is taking nonhomologous end
It connects repair mode (NHEJ), this mode can generate the insertion/deletion reparation of casual cnalogy at double-strand break, may make
At the frameshift mutation of gene, gene lacks functionality is caused.Another repair mode is with single-stranded oligonucleotide or double-strand
Donor plasmid vector is that expected precisely reparation is realized by way of homologous recombination (HR) under the guidance of template.CRISPR/
Cas9 system can be used as a kind of gene editing system with locus specificity, and maximum feature is easy to operate, cost
Low, effect is efficiently.Its huge advantage of CRISPR/Cas9 system addresses rapidly becomes the outstanding person in gene editing tool, in base
Because the fields such as functional study, disease model, gene therapy are widely used.
Summary of the invention
It is to utilize CRISPR-Cas9 system the object of the present invention is to provide a kind of preparation method of Fbxo40 gene knock-out pig
System edits Fbxo40 gene, and obtains Fbxo40 gene knock-out pig by somatic cell nuclear transfer technique.
Present invention firstly provides pig Fbxo40 gene third exons to prepare the purposes in Fbxo40 gene knock-out pig.
The nucleotide sequence of the pig Fbxo40 gene third exon is as shown in SEQ ID NO.1.
Present invention firstly provides the 4th exons of pig Fbxo40 gene to prepare the purposes in Fbxo40 gene knock-out pig.
The nucleotide sequence of the 4th exon of pig Fbxo40 gene is as shown in SEQ ID NO.4.
The present invention provides the sgRNA of selectively targeted pig Fbxo40 gene third exon, sequences 5 '-
Tgtctgtgctcccctggcggagg-3 ' (as shown in SEQ ID NO.2).The oligonucleotide sequence for being complementary pairing is 5 '-
Cctccgccaggggagcacagaca-3 ' (as shown in SEQ ID NO.3).
The present invention provides above-mentioned sgRNA to prepare the application in Fbxo40 gene knock-out pig.
The present invention provides application of the above-mentioned sgRNA in the improvement of animal germ plasm resource.
The present invention provides above-mentioned sgRNA to construct the animal model with muscle development and muscle disease relevant medical research
In application.
The present invention also provides the CRISPR/Cas9 targeting vectors of the DNA sequence dna containing above-mentioned sgRNA.
CRISPR/Cas9 targeting vector of the present invention, is prepared by the following method to obtain, by SEQ ID NO.2,
Oligonucleotide shown in 3 at 94 DEG C, 5min, 37 DEG C, put anneal on ice to oligonucleotide immediately after by 10min
5min；Px330 skeleton carrier carries out digestion with restriction enzyme Bbs I, after recycling, connect with the oligonucleotide of annealing.
Naming the CRISPR/Cas9 targeting vector in one embodiment of the invention is pX330 1-8, and the present invention provides
The application of the CRISPR/Cas9 targeting vector in pig breeding.
The present invention provides CRISPR/Cas9 targeting vectors in building and muscle development and muscle disease relevant medical research
Animal model in application.The muscle disease includes muscular atrophy.
The present invention provides application of the CRISPR/Cas9 targeting vector in the transgene pig for preparing muscle mass raising.
The present invention also provides a kind of methods for preparing Fbxo40 gene knock-out pig, and CRISPR/Cas9 of the invention is beaten
Targeting vector pX330 1-8 and PGK-Neo gene linear carrier corotation enter in porcine fetus fibroblasts, and G418 screening is had
The positive cell clone of resistance；Using positive cell as nuclear transfer donor cell, egg mother cell is nuclear transfer recipient cell, passes through body
Nuclear transfer technology obtains clone embryos；Clone embryos are moved into gram that pig Tubal pregnancy obtains Fbxo40 gene knockout
Grand pig.
Wherein, CRISPR/Cas9 targeting vector pX330 1-8 and PGK-Neo gene linear carrier corotation enter pig into fiber
The method of cell are as follows: CRISPR/Cas9 targeting vector pX330 1-8 be added 2 μ g, and with PGK-Neo gene linear carrier according to
Molar ratio 3:1 mixing is transferred to pig fibroblast with the method for electric shock transfection or liposome transfection.
The present invention has studied Fbxo40 gene third, the 4th exon, has screened in the sgRNA of prepare more of comforming choosing optimal
SgRNA, selectively targeted Fbxo40 gene third exon.Using the gene editing technology of CRISPR/Cas9, inexpensive,
Expeditiously obtain Fbxo40 gene knock-out pig.One aspect of the present invention for illustrate Fbxo40 signal path in pig provide it is dynamic
Object model facilitates medical domain further to study muscle development and muscle related disease with this model, on the other hand of the invention
Method provides technical basis to cultivate the new lines of the pig of high lean meat percentage.
Detailed description of the invention
Fig. 1 is the sgRNA design position and sequence diagram of CRSIPR/Cas9 in the embodiment of the present invention 1.
Fig. 2 is in the embodiment of the present invention 4 using sequence electrophoresis result figure around PCR method amplification target spot.By with it is wild
Type compares stripe size, detects the target practice situation of monoclonal cell.Swimming lane 1-56 is respectively the number of cell monoclonal in figure, figure
Middle Marker is 100bp DNA ladder, and the 5th swimming lane is 5# cell monoclonal, and PCR product size is about 643bp, the 31st swimming
Road is 31# cell monoclonal, and PCR product size is about 1331bp.
Fig. 3 A- Fig. 3 D is in the embodiment of the present invention 4, after monoclonal cell 5# and 31# are sequenced, the knot of sequence alignment
Fruit figure.Fig. 3 A, Fig. 3 B shows that 7 monoclonal colonies of 31# cell monoclonal compared with wild-type sequence, have occurred 3bp's
Replacement, while the insertion of 323bp has occurred, produce the termination in advance of frameshift mutation and translation.Fig. 3 C, Fig. 3 D show that 5# is thin
6 monoclonal colonies of born of the same parents' monoclonal have lacked 365bp, have deleted the important feature of Fbxo40 compared with wild-type sequence
Domain, and produce the termination in advance of frameshift mutation and translation.
Fig. 4 A and Fig. 4 B are to use the mutation type of PCR method identification neonatal pig in the embodiment of the present invention 5.It is each in figure
Swimming lane corresponds to the PCR result for the different number Fbxo40 gene knock-out pigs that the present invention is prepared.
Fig. 5 is to use Fbxo40 in the method detection neonatal pig longissimus dorsi muscle of Western blot in the embodiment of the present invention 5
The expression of albumen.
Fig. 6 is to detect Fbxo40 albumen in newborn pig myocardium using the method for Western blot in the embodiment of the present invention 5
Expression.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
PX330 carrier, PGK-Neo carrier are purchased from Addgene company；The super fidelity enzyme of T4DNA ligase, Q5, Bbs I and
T7EN1 is purchased from NEB company；Primer synthesis is completed by the raw work in Shanghai；Sequencing is synthesized by Mei Ji biotech firm.Plasmid goes endotoxin to mention
Kit and genome extraction kit is taken to be purchased from QIAGEN company.Plastic recovery kit is purchased from GENSTAR company.Digestion, company
It connects, gel extraction, conversion, the routine experiments operating procedure such as PCR amplification are detailed in " molecular cloning (third edition) ".
The building of 1 CRSIPR/Cas9 targeting vector pX330 1-8 of embodiment
1, the design and screening of pig Fbxo40 gene specific sgRNA
The important structural domain of third exon, the 4th exon sequence coding Fbxo40 albumen of pig Fbxo40 gene ---
Zinc-finger structural domain and F-box structural domain.
It is devised altogether according to the action principle of CRISPR/Cas9 in third exon and the different location of the 4th exon
17 sgRNA sequences.Its sequence and the sequence of complementation are as shown in table 1.
Table 1
Bolded section is the PAM sequence of CRISPR/Cas9 identification in table 1.
PX330 carrier framework needs to carry out digestion using Bbs I, so needing to mend out the I digestion position Bbs in sgRNA sequence
The cohesive end of point, so that it is connected into pX330 carrier framework.
By the designed sgRNA that I restriction enzyme site cohesive end of Bbs is added and its complementary series in a manner of synthetic primer
It is synthesized.The oligonucleotides of synthesis is subjected to annealing operation, forms it into the DNA double chain with cohesive end.Cycle of annealing
It is as follows: 94 DEG C, 5min；37 DEG C, 10min；On ice, 5min.PX330 carrier framework uses I digestion of Bbs, 37 DEG C of water-bath 4h.Then
Carry out agarose gel electrophoresis, and gel extraction purpose band.Carrier framework is connect with sgRNA sequence.By the carrier bone of recycling
Frame and sgRNA sequence anneals product are attached overnight in 16 DEG C of connection instrument.By connection product convert DH5 α competent cell, 37
DEG C incubator culture, after it grows monoclonal colonies, the scribing line of picking monoclonal colonies, and carry out sequencing identification positive monoclonal
Bacterium colony.Sequencing primer is PX-F:5 '-GAGGGCCTATTTCCCATGAT-3 ', is located on pX330 carrier framework.
Correct positive monoclonal bacterium colony is sequenced in picking, is added into the LB culture medium of 2-5ml ammonia benzyl resistance, in shaking table
In 37 DEG C, 300rpm is aggressively shaken 8h.The bacterium solution of initial incubation is added to 100ml ammonia with the dilution ratio of 1/500-1/1000
In the LB culture medium of benzyl resistance, 37 DEG C in shaking table, 300rpm is aggressively shaken 12h-16h.
CRISPR/Cas9 targeting vector plasmid goes endogenous toxic material to extract: referring to the EndoFree Plasmid of QIAGEN company
Maxi Kit specification.The plasmid extracted is surveyed after concentration and dispenses, freeze, for turning for subsequent porcine fetus fibroblasts
Dye.
By in six orifice bores, the porcine fetus fibroblasts that 80-90% converges are had reached, digested, be centrifuged, obtained
Obtain quantity about 2 × 105-2×106Porcine fetus fibroblasts.By 2 μ g of CRISPR/Cas9 targeting vector, it is added to Lonza and turns
It in transfection reagent, mixes, cell is resuspended using the transfection reagent that plasmid is added, and cell suspension is added in electric shock cup, T-016
Program electric shock cell.After the completion of electric shock, cell is sucked out immediately, adds a hole of DMEM to six orifice plate of the 3ml containing 10% serum
In.37 DEG C, 5%CO2After incubator culture 48h, cell reaches 80%-90% and converges, and cell dissociation is got off, and extracts cell
Genome, the template as PCR amplification.In the upstream design PCR upstream amplification primer F of sgRNA target spot, in sgRNA target spot
Design PCR downstream amplification primer R in downstream.Include the upstream and downstream sequence of sgRNA target spot using PCR amplification, PCR product is connected
Connection product is converted DH5 α competent cell by peasy-blunt-simple carrier, and 37 DEG C of incubator cultures grow list to it
After colonies, the random a certain number of monoclonal colonies of picking are sequenced.By each monoclonal colonies sequencing result and open country
The sequence of raw type Fbxo40 is compared, and assert that the monoclonal that base insertion/deletion has occurred is positive monoclonal bacterium colony.It will be positive
Property monoclonal colonies number divided by sequencing monoclonal colonies sum, be exactly difference CRISPR/Cas9 targeting vector cutting imitate
Rate.As shown in table 2.
Table 2
As shown in Table 2, the 17 articles of sgRNA target spots designed in pig Fbxo40 gene third exon and the 4th exon are cut
Efficiency is cut between 2%-68%.In order to avoid excessively high cutting efficiency causes undershooting-effect, it is moderate that the present invention has chosen efficiency
(34.6%), and position it is earlier (convenient for generate frameshift mutation, so that the subsequent protein sequence of cleavage site is changed and is mentioned
Preceding termination) 1-8 target spot as the sgRNA for being used to prepare target practice clone pig.
2, the building of CRSIPR/Cas9 targeting vector pX330 1-8
Have chosen cas9 target spot 1-8:5 '-tgtctgtgctcccctggcggagg-3 '.According to the original of base pair complementarity
Then, reverse complementary sequence 5 '-cctccgccaggggagcacagaca-3 '.
PX330 carrier framework needs to carry out digestion using Bbs I, so needing to mend out the I digestion position Bbs in sgRNA sequence
The cohesive end of point, so that it is connected into pX330 carrier framework.The sgRNA sequence and its complementary sequence of I cohesive end of Bbs is added
Column are respectively 5 '-CACCGtgtctgtgctcccctggcgg-3 ', 5 '-AAACccgccaggggagcacagaca C-3 '.
By the designed sgRNA that I restriction enzyme site cohesive end of Bbs is added and its complementary series in a manner of synthetic primer
It is synthesized.The oligonucleotides of synthesis is subjected to annealing operation, forms it into the DNA double chain with cohesive end.Cycle of annealing
It is as follows: 94 DEG C, 5min；37 DEG C, 10min；On ice, 5min.PX330 carrier framework uses I digestion of Bbs, 37 DEG C of water-bath 4h.Then
Carry out agarose gel electrophoresis, and gel extraction purpose band.Carrier framework is connect with sgRNA sequence.By the carrier bone of recycling
Frame and sgRNA sequence anneals product are attached overnight in 16 DEG C of connection instrument.By connection product convert DH5 α competent cell, 37
DEG C incubator culture, after it grows monoclonal, the scribing line of picking monoclonal, and carry out sequencing identification positive monoclonal.Sequencing is drawn
Object is PX-F:5 '-GAGGGCCTATTTCCCATGAT-3 ', is located on pX330 carrier framework.The plasmid built is named as
pX330 1-8。
Correct positive monoclonal bacterium colony is sequenced in picking, is added into the LB culture medium of 2-5ml ammonia benzyl resistance, in shaking table
In 37 DEG C, 300rpm is aggressively shaken 8h.The bacterium solution of initial incubation is added to 100ml ammonia with the dilution ratio of 1/500-1/1000
In the LB culture medium of benzyl resistance, 37 DEG C in shaking table, 300rpm is aggressively shaken 12h-16h.PX330 1-8 plasmid goes endogenous toxic material to mention
It takes referring to step 1.
Building for 2 porcine fetus fibroblasts of embodiment be
By gestation 30 days Mini-musk swines anesthesia, from the sterile taking-up fetus of its intrauterine, fetus is cleaned with containing dual anti-PBS
Afterwards, it is placed in superclean bench, with head, four limbs, internal organ and the cartilaginous tissue of eye scissors removal fetus, is rinsed well with PBS；
Residue tissue is shredded into about 1mm with eye scissors in Tissue Culture Dish3Fritter；Suitable FBS is added, tissue is kept to be unlikely to
Overdrying.The tissue block shredded is transferred in 1 T75 Tissue Culture Flask, tissue block is uniformly spread out；5mL cell is added
Culture medium will be covered with the one of tissue block and face upward, and not be cultured base submergence, in 37 DEG C, 5%CO23~5h is cultivated in incubator
Afterwards, T75 is overturn, tissue block is made to be cultured base submergence；Culture 3 days or so, observing around tissue block has a large amount of cells to climb out of,
When cell grows to about 90% convergence degree, cell is digested and is frozen spare.
The transfection of 3 porcine fetus fibroblasts of embodiment and the screening of middle target cell monoclonal
1, the digestion of PGK-Neo plasmid
Using the restriction enzyme Not I and Nhe I of NEB company to PGK-Neo plasmid enzyme restriction, 37 DEG C of water-bath 4h.It uses
The segment that the plastic recovery kit recycling PGK-Neo size of Genstar is 1.8kb.It dispenses, freezes in -20 DEG C of refrigerators after surveying concentration
It is spare.
2, by six orifice bores, the porcine fetus fibroblasts that 80-90% converges is had reached, digested, be centrifuged,
Obtain quantity about 2 × 105-2×106Porcine fetus fibroblasts.
3, CRISPR/Cas9 targeting vector pX330 1-8 be added 2 μ g, and with PGK-Neo gene linear carrier according to mole
It is added in Lonza transfection reagent, mixes than 3:1.Cell is resuspended using the transfection reagent that plasmid is added, and cell suspension is added
Into electric shock cup, T-016 program electric shock cell.After the completion of electric shock, cell is sucked out immediately, 3ml is added to contain the DMEM of 10% serum
Into a hole of six orifice plates.37 DEG C, 5%CO2After incubator culture 48h, cell reaches 80%-90% and converges, and cell is disappeared
Change is got off, and is diluted in 20-30 10cm Tissue Culture Dish.After 24-48h, and state adherent to the cell in 10cm ware is good
It is good, the G418 of 400-600 μ g/ml is added, every other day adds a G418, dosage is flexible according to cell state and convergence degree
It controls, but maximum concentration is no more than 1000 μ g/ml.After G418 is screened 8-12 days, it is seen that high-quality cell monoclonal.
4, the picking of cell monoclonal and expansion culture.Under the microscope, using marking pen by monoclonal in good condition
It is irised out with circle.The culture medium in 10cm culture dish is discarded, PBS cleaning is primary, and clone's ring is dipped gelatin, will be thin with clone's ring
Born of the same parents' monoclonal circle is lived, and the trypsase of 10-30 μ l 0.1%, 37 DEG C of digestion 1min are added.It observes under the microscope, cell becomes
It is round, free, the DMEM containing 20%FBS is added and terminates digestion, cell is sucked out and is added in 24 orifice plates.After 48-72h, in 24 orifice plates
When cell confluency is to 80-90%, cell is reached in 12 orifice plates.When cell in 12 orifice plates reaches 80%-90% and converges, to thin
Born of the same parents freeze.
The identification of target positive cell monoclonal in embodiment 4
Since the cutting of Cas9 causes double-strand break, insertion/deletion mutation can be randomly generated in the repair mode of NHEJ, therefore
It needing using the genome of target practice cell monoclonal as template, its region is sequenced in PCR amplification target practice site region,
The case where detecting the insertion/deletion mutation of its base.
The genomic DNA for extracting 48 cell monoclonals carries out PCR amplification using it as template, and PCR amplification primer is xin-
1- (0,1,2) -2-F 5 '-gcaacaggtcaaggatccag-3 ' and xin-1- (0,1,2) -2-R 5 ' -
Gcgcactgatgagcttgtta-3 ', using the genome of wild-type cell as negative control, PCR product size is about
1008bp.PCR program is as follows: 98 DEG C, 30s；98 DEG C, 10s；67 DEG C, 30s；72 DEG C, 1min；72 DEG C, 2min.35 circulations.It lacks
The cell monoclonal for losing or being inserted into large fragment can be used agarose gel electrophoresis and carry out preliminary judgement.Know 5# cell list
Large fragment deletion occurs for clone, and the insertion of large fragment has occurred in 31# cell monoclonal.As shown in Fig. 2, 5# cell monoclonal occurs
The missing of large fragment, compared with 6# cell monoclonal (its stripe size is identical with wild type size), it is seen then that 5# cell Dan Ke
Grand band is smaller.Similarly, the insertion of large fragment has occurred in 31# cell monoclonal, with 29# cell monoclonal (its band
Size is identical with wild type size) compared with, it is seen then that 31# cell monoclonal band wants larger.38# cell monoclonal and 25# are thin
Born of the same parents' monoclonal is similar with 5# cell monoclonal, and large fragment deletion also has occurred in they.Comprehensively consider the shape of above-mentioned cell monoclonal
State, the present invention have selected the optimal 5# cell monoclonal of state and 31# cell monoclonal, have been more suitable for body-cell neucleus transplanting
Donorcells.
In order to further determine 5# cell monoclonal, the mutation type of 31# cell monoclonal, PCR product is connected
Connection product is converted DH5 α competent cell by peasy-simple blunt carrier, and 37 DEG C of incubator cultures grow list to it
After colonies, the random a certain number of monoclonal colonies of picking are sequenced.By each monoclonal colonies sequencing result and open country
The sequence of raw type Fbxo40 is compared.
For 31# cell monoclonal, 7 monoclonal colonies of random picking are compared with wild type Fbxo40 sequence, by scheming
Thus 3A, Fig. 3 B prove 31# cell monoclonal it is found that the replacement of 3bp has occurred in they, while the insertion of 323bp has occurred
The replacement of 3bp has occurred, while the insertion of 323bp has occurred, produces the termination in advance of frameshift mutation and translation.Fig. 3 A is 5 '
Terminal sequence comparison result, Fig. 3 B are 3 ' terminal sequence comparison results.
For 5# cell monoclonal, 6 monoclonal colonies of random picking are compared with wild type Fbxo40 sequence, by scheming
3C, Fig. 3 D it can thus be appreciated that 5# cell monoclonal has lacked 365bp, delete Fbxo40's it is found that they have lacked 365bp
Important feature domain, and produce the termination in advance of frameshift mutation and translation.Fig. 3 C is 5 ' terminal sequence comparison results, and Fig. 3 D is 3 ' ends
Sequence alignment result.
The preparation of 5 Fbxo40 gene knock-out pig of embodiment
1, the positive porcine fetus fibroblasts obtained using embodiment 4 is nuclear transfer donor cells.Fetus is cultivated into fiber
Cell converges 1-2 days to 100%, removes culture medium in culture dish, and PBS is added and washs 1 time, is then disappeared with 0.1% trypsase
Change about 2min, is terminated immediately with the cell culture fluid containing serum afterwards after cell rounding and is digested, 1000rpm centrifugation 5min, in abandoning
Clearly, the cell of centrifugation is resuspended with operation liquid T2, ice bath is placed spare.
Using the egg mother cell of maturation in vitro as nuclear transfer receptor archiblast.It is compound that cumulus oocyte is acquired from sow ovary
Body sloughs cumulus cell by maturation in vitro and with hyaluronidase, then selected under Stereo microscope discharge first polar body,
Form is normal, the uniform mature oocyte of cytoplasm is spare.
Under micromanipulation instrument, nuclear transfer donor cell is moved into the mature oocyte of stoning.By electro' asion and
Chemokinesis, inducing cell and egg fusion simultaneously activate egg mother cell simultaneously.It is built into recombination embryo, fusion embryo is put into hypoxemia training
It supports under environment (hypoxemia incubator or being filled with the closed culture of hypoxemia mixing distribution envelope) and cultivates.Using droplet or four orifice plate cultures,
Gas phase condition is the mixed gas containing 7%O2,88%N2 and 5%CO2, and cultivation temperature is 39 DEG C, humidity 100%.External hair
Spilting of an egg situation and developmental condition are observed after educating to 1-4 cell stage, and are used for embryo transfer.
The clone embryos that form is normal, development is excellent are selected to be implanted into modus operandi in the sow of embryo's same period.Transplanting step
Suddenly blood vessel is avoided as far as possible by sow Baoding on operation bracket for free from worries general anaesthesia, the notch at ventrimeson exposes ovary,
Fallopian tubal and uterus draw embryo using embryo transplantation tube, then enter along fimbriae tubae portion clone embryos being discharged into defeated ovum
Pipe ampulla, isthmus junction.Anti-inflammatory needle is injected to replace-conceive sow after embryo transfer, B ultrasound is carried out after 30 days and detects Pregnancy.
2, the DNA level detection of Fbxo40 gene knock-out pig:
Genomic DNA is extracted using the ear tissue of neonatal pig, carries out PCR amplification using it as template.Newborn boar 7801#,
The PCR amplification primer of 7802#, 7803#, 7804#, 7901#, 9402# are xin-1- (0,1,2) -2-F 5 ' -
5 '-gcgcactgatgagcttgtta-3 ' of gcaacaggtcaaggatccag-3 ' and xin-1- (0,1,2) -2-R.Wild type
The PCR product size of pig is about 1008bp.If neonatal pig clones point from 5#, PCR product size is about 643bp.If newborn
Pig clones point from 31#, then PCR product size is about 1331bp.
As shown in Figure 4 A and 4 B shown in FIG., it is known that number is the newborn boar of (7802#, 7803#, 7804#, 7901#, 9402#)
Point is cloned from 5#, number is that the newborn boar of (7801#) clones point from 31#.
3, the protein level detection of Fbxo40 gene knock-out pig:
7801#, 7802#, 7803#, 7804# four-head Fbxo40 Gene Double is taken to strike and 304#, 6#, 254#, 364#, 11#
Etc. wild types with age in days control pig cardiac muscle and longissimus dorsi muscle tissue 100mg, extract total protein, carry out Western blot detection
The expression of Fbxo40.
In Western blot experiment, albumen applied sample amount is 50 μ g.SDS-PAGE resolving gel concentration is 10%.60V electrophoresis
30min, 90V electrophoresis 1h.After electrophoresis, transferring film, 300mA constant current, 1h are carried out using Bio-Rad transferring film instrument.5% defatted milk
Overnight, Fbxo40 primary antibody (1:1000 dilution) is incubated at room temperature 1h for powder closing.TBST washes 6 × 5min of film.Goat antirabbit secondary antibody (1:
10000 dilutions) incubation at room temperature 1h.TBST washes 6 × 5min of film.Develop.As can be seen from the results, in double longissimus dorsi muscles for striking pig and
Non- method detects the expression of Fbxo40 albumen in cardiac muscular tissue, and can be detected with the wild type control individual of age in days
The expression of Fbxo40 albumen.Such as Fig. 5, shown in Fig. 6.As a result illustrate, the CRSIPR/Cas9 targeting vector pX330 that the present invention constructs
1-8 can efficiently and accurately knock out Fbxo40 gene, to successfully obtain Fbxo40 gene knock-out pig.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (7)
1. pig Fbxo40 gene third exon is preparing the purposes in Fbxo40 gene knock-out pig, the pig Fbxo40 gene
The nucleotide sequence of three exons is as shown in SEQ ID NO.1.
2. the sgRNA of selectively targeted pig Fbxo40 gene third exon, which is characterized in that its DNA sequence dna such as SEQ ID
Shown in NO.2 or as shown in SEQ ID NO.3.
3. sgRNA described in claim 2 is preparing the application in Fbxo40 gene knock-out pig.
4. application of the sgRNA described in claim 2 in the improvement of animal germ plasm resource.
5. the CRISPR/Cas9 targeting vector of the DNA sequence dna containing sgRNA described in claim 2.
6. application of the CRISPR/Cas9 targeting vector in pig breeding described in claim 5.
7. a kind of method for preparing Fbxo40 gene knock-out pig, which is characterized in that by CRISPR/Cas9 described in claim 5
Targeting vector and PGK-Neo gene linear carrier corotation enter in porcine fetus fibroblasts, and G418 screening obtains resistant
Positive cell clone；Using positive cell clone as nuclear transfer donor cell, egg mother cell is nuclear transfer recipient cell, thin by body
Karyon implantation technique obtains clone embryos；Clone embryos are moved into the clone that pig Tubal pregnancy obtains Fbxo40 gene knockout
Pig.
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| CA2853829C (en) | 2011-07-22 | 2023-09-26 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US20150044192A1 (en) | 2013-08-09 | 2015-02-12 | President And Fellows Of Harvard College | Methods for identifying a target site of a cas9 nuclease |
| US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US9322037B2 (en) | 2013-09-06 | 2016-04-26 | President And Fellows Of Harvard College | Cas9-FokI fusion proteins and uses thereof |
| US9228207B2 (en) | 2013-09-06 | 2016-01-05 | President And Fellows Of Harvard College | Switchable gRNAs comprising aptamers |
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| WO2016022363A2 (en) | 2014-07-30 | 2016-02-11 | President And Fellows Of Harvard College | Cas9 proteins including ligand-dependent inteins |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
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| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
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