CN108642054A - Reduce immunogenicity sgRNA, low immunogenicity dressing and preparation method thereof - Google Patents
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- CN108642054A CN108642054A CN201810470742.9A CN201810470742A CN108642054A CN 108642054 A CN108642054 A CN 108642054A CN 201810470742 A CN201810470742 A CN 201810470742A CN 108642054 A CN108642054 A CN 108642054A
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- A01K67/0276—Knock-out vertebrates
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Abstract
The invention discloses a kind of reduction immunogenicity sgRNA, low immunogenicity dressing and preparation method thereof.Its length of the reduction immunogenicity sgRNA is in 18nt between 22nt；3 ' ends of its target sequence on GGTA1 genes contain GG, and G/C content is 40% 60%；Its with targeting binding site genome sequence in be not present SNPs；It carries out full genome undershooting-effect analysis, maximum allowable 5 base mispairings in site of missing the target with pig GGTA1 genes.The low immunogenicity dressing is pigskin, and the pigskin derives from GGTA1 gene knock-out pigs.Reduction immunogenicity sgRNA provided by the invention and low immunogenicity dressing immunogenicity is extremely low or even non-immunogenicity, can be used as human skin equivalent, reduces the risk of complication.
Description
Technical field
The invention belongs to technical field of biological materials, more particularly, to a kind of low immunogenicity biological dressing and its system
Preparation Method.
Background technology
In the treatment to serious deep burn, defect of skin, Wound dressing is needed to promote the surface of a wound quickly to heal.Wound
For face biological dressing as a kind of emerging Wound dressing, the market impetus is full, and it is (such as self skin, same to be broadly divided into Animal Skin dressing
Kind allograft skin, xenogenesis skin etc.) and non-animal skin dressing (such as collagen, chitosan, honey biological dressing).Self skin is in curative effect
It is best, but skin source is extremely limited.The permeability and adhesiveness of alloskin are similar to the skin of burn patient, can be effective
Antigenicity is reduced, rejection is reduced.But it is mainly derived from cadaver skin, cadaver skin source is extremely limited, at high price, preserves item
Part requirement is high, using the problem of there is also religion and ethics aspects.The dressing of xenogenesis skin is relative to self skin of the same race and of the same race
For allograft skin, source is relatively extensive, cheap.
Currently, pigskin plays a role as interim skin substitutes in the treatment of clinical burn or skin injury.
But immunogenicity existing for pigskin itself so that (about one week) is ostracised in a short time for it, and patient needs over the course for the treatment of
Multiple dressing, cutting scab, skin-grafting increase patient suffering, and easily leave scar or pigmentation in skin donor site again.
Thus, the immunogenicity of pigskin how is reduced, immunological rejection is then reduced, realizes that dressing is lacked in treatment on the way,
The hardship removed changing medicine for patient from, cut scab reduces complication, has a very important significance.
Invention content
For the disadvantages described above or Improvement requirement of the prior art, the present invention provides a kind of reduction immunogenicity sgRNA, low
Immunogenicity dressing and preparation method thereof, it is immune its object is to reduce the expression of immunogenicity gene or silence by acquisition
Thus source property gene pigskin solves that existing pigskin dressing immunogenicity is high as dressing, need multiple dressing cutting scab, skin-grafting skill
Art problem.
To achieve the above object, according to one aspect of the present invention, a kind of reduction immunogenicity sgRNA is provided, it is special
Sign is, is combined with pig GGTA1 gene orders, and have following characteristics：
(1) its length in 18nt between 22nt；
(2) 3 ' ends of its target sequence on GGTA1 genes contain GG, and G/C content is in 40%-60%；
(3) its with targeting binding site genome sequence in be not present SNPs；
(4) it carries out full genome undershooting-effect analysis, maximum allowable 5 base mispairings in site of missing the target with pig GGTA1 genes.
Preferably, the reduction immunogenicity sgRNA is any one in following sequence：
GCTGCTTGTCTCAACTGTAATGG；
CCCAGAAGGTTCTTTGTTCTGG；
AAGGCTCCAGTGGTATGGGAAGG。
Preferably, the reduction immunogenicity sgRNA is following sequence：AAGGCTCCAGTGGTATGGGAAGG.
Other side according to the invention, provides a kind of low immunogenicity dressing, and the dressing is pigskin, the pig
Skin derives from GGTA1 gene knock-out pigs.
Preferably, the low immunogenicity dressing, GGTA1 gene knock-out pigs, with sgRNA provided by the invention.
Other side according to the invention provides the preparation method of the low immunogenicity dressing, which is characterized in that
Include the following steps：
(1) preparation of transfection carrier：By the positive-sense strand mould of the sgRNA target sequences as described in claims 1 to 3 any one
5 ' end addition RNA sequence TAGG of plate, 5 ' end addition CAAA of antisense strand template, synthesize the single-stranded annealed shape of oligonucleotide chain
At double-strand, the transfection carrier CRISPR/Cas skeleton plasmids of linearisation and in-vitro transcription CRISPR/Cas skeleton plasmids are distinguished
It is connect with the double-strand, obtains transfection carrier plasmid and in-vitro transcription carrier；
(2) adult fibroblast is transfected：By the transfection carrier electroporation transfection pig adult obtained in step (1) at fiber
Cell, the pig adult fibroblast of the knockout of the identified determining GGTA1 genes of culture of single cell clone；
(3) preparation of clone embryos：By the pig adult fibroblast of the knockout of the GGTA1 genes obtained in step (2)
Nucleus inject enucleation oocyte, and endochylema injecting step (1) obtain in-vitro transcription carrier and Cas9 mRNA, obtain
Clone embryos；
(4) prepared by GGTA1 gene knock-out pigs：The clone embryos obtained in step (3) are implanted into Gilt Uterus, cultivates and obtains
GGTA1 gene knock-out pigs；
(5) take leather for dressing：The GGTA1 gene knock-out pigs obtained in step (4) are taken into pachydermia in head layer, after flushing
Hydrophobic treatment, proteolysis processing, blocking antigen processing, frozen dried are carried out successively, obtain the low immunogenicity dressing.
Preferably, the preparation method of the low immunogenicity dressing, step (1) the transfection carrier CRISPR/Cas bones
Frame plasmid is U6-CRISPR/Cas skeleton plasmids；The in-vitro transcription CRISPR/Cas skeleton plasmids are T7-CRISPR/Cas bones
Frame plasmid.
Preferably, the preparation method of the low immunogenicity dressing, the outer transcription vector of step (3) the endochylema injecting body
With the mRNA of Cas9, the mass ratio of the mRNA of in-vitro transcription carrier and Cas9 are 1 in injection:9~1:Between 3.
Preferably, the preparation method of the low immunogenicity dressing, the middle pachydermia thickness 0.4mm~0.5mm it
Between.
Preferably, the preparation method of the low immunogenicity dressing, the hydrophobic treatment are to use a concentration of 5~10%
(m/v) polyglycol solution impregnates 50~60min；
The proteolysis processing is using the immersion of a concentration of 0.3~0.4% trypsin solution normal saline solution
70~100min；
The blocking antigen processing is using a concentration of 1~2% 12~20h of sodium dodecyl sulfate solution sustained oscillation
Afterwards, glutaraldehyde cross-linking is finally used again, closes residue antigens.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain down and show
Beneficial effect：
Using CRISPIR/Cas9 gene knockouts and acellular dermal matrix, can dual reduction pigskin immunogenicity, system
Pigskin immunogenicity is extremely low or even non-immunogenicity, can be used as human skin equivalent, reduce the wind of complication
Danger.
Description of the drawings
Fig. 1 is flow diagram of the embodiment of the present invention；
Fig. 2 is the structure figures of digestion DNA during external digestion Activity determination in the embodiment of the present invention；
Fig. 3 is the gray-scale map of digestion band during external digestion Activity determination in the embodiment of the present invention；
Fig. 4 is the CRISPR/Cas9 systemic vectors structure schematic diagram used in the embodiment of the present invention；
Fig. 5 is cell transfecting efficiency design sketch in the embodiment of the present invention；
Fig. 6 is clone's embryo Validation in vitro CRISRP/Cas9 cutting effect figures in the embodiment of the present invention；
Fig. 7 is the GGTA1 gene knock-out pigs that microinjection obtains in the embodiment of the present invention；
Fig. 8 is the identification of GGTA1 gene knockout effects in the respectively tissue of GGTA1 gene knock-out pigs in the embodiment of the present invention；
Fig. 9 is the α -1,3Gal content detections that pigskin in the embodiment of the present invention, primary type pigskin and market take off cell pigskin
Result figure.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below
It does not constitute a conflict with each other and can be combined with each other.
Pigskin is sufficiently close to mankind's affinity, and soft texture, also similar with human skin structure.Pigskin is as dressing
Using, there is good elasticity, surface of a wound viscosity and water penetration, be easy to survive after transplanting, the growth of self microparticle skin will not be hindered,
It can the quick closure surface of a wound.
The α 1,3- of alpha-galactoside invertase 1 (alpha-galactosyltransferase 1, GGTA1) gene code
The galactose residue that galactosyltransferase (α -1,3GT) can be catalyzed UDP- galactose molecules (UDP-Gal) is transferred to gala
On the receptor N- acetyl galactoses amine molecule (GalNAc) of sugar, α -1,3 galactosides (α -1,3Gal) are formed.The cell surface of pig
Express this saccharide compound α -1,3Gal.Human cell cannot synthesize α -1,3Gal due to lacking functional GGTA1 genes
Compound, it is experimentally confirmed that and human body will produce under the stimulation of bacterium be largely directed to α -1 in vivo, the antibody of 3Gal.When pigskin
After being contacted with the body fluid in patient skin damage face, human antigen is combined with the heterogenetic antigen on chief cell surface, activating complement, is generated
Immunological rejection.Therefore, GGTA1 genes strike subtract can silence or reduce even be eliminated porcine arterial endothelial cell α -1,3Gal
Expression, to reduce the immunogenicity of pig.
Regularity repeats short palindromic sequence cluster (clustered regularly interspaced short
Palindromic repeats, CRISPRs) it is that one kind is widely present in prokaryotic gene group, it is one kind of prokaryotes
Acquired immune system.CRISPR/Cas9 is a kind of complex with endonuclease activity, identifies specific DNA sequence dna,
The cutting for carrying out specific site causes double-strand DNA cleavage, and under conditions of no template, non-homogeneous recombination end link occurs,
Frameshift mutation is caused, gene knockout is caused.Include Cas9 nucleases, tracrRNA and CrRNA in the system.TracrRNA and
CrRNA is combined and is formed a RNA chain, abbreviation sgRNA (single guide RNA).SgRNA identifies specific DNA sequence, Cas9
The RuvC and HNH of albumen are responsible for the cutting of target location.This technology can not only quickly, simply, efficiently target gene group
Gene, and it is easy to operate, it high-throughput can prepare, it is at low cost, thus be concerned.
However CRISPRs systems is used to carry out gene silencing, high miss rate may be allowed in site similar with target sequence
Non-specific cleavage occurs, destroys genome structure, causes gene knockout or silencing efficiency bad, it is also possible to cause individual can not
Survival.Since the targeting accuracy of gene is highly dependent on sgRNA target sequences, design and prepare the sgRNA that precisely targets at
Therefore key technology and difficult point to knock out target gene need silencing efficiency and the safety of design and experimental demonstration sgRNA,
Otherwise GGTA1 genes can not be prepared by effective reticence simultaneously, ensures individual survival and development.
Reduction immunogenicity sgRNA provided by the invention, which is characterized in that it is combined with pig GGTA1 gene orders, and
Have following characteristics：
(1) its length in 18nt between 22nt；
(2) its 3 ' end of target sequence on GGTA1 genes contains GG, and G/C content is in 40%-60%；
(3) its with targeting binding site genome sequence in be not present SNPs；
(4) it carries out full genome undershooting-effect analysis, maximum allowable 5 base mispairings in site of missing the target with pig GGTA1 genes.
Any one in preferably following sequence：
GCTGCTTGTCTCAACTGTAATGG；
CCCAGAAGGTTCTTTGTTCTGG；
AAGGCTCCAGTGGTATGGGAAGG。
More preferably it is following sequence：AAGGCTCCAGTGGTATGGGAAGG.
Low immunogenicity dressing provided by the invention, which is characterized in that the dressing is pigskin, and the pigskin derives from
GGTA1 gene knock-out pigs.
The GGTA1 gene knock-out pigs preferably have sgRNA provided by the invention.
The preparation method of low immunogenicity dressing provided by the invention, which is characterized in that include the following steps：
(1) preparation of transfection carrier：By 5 ' end addition RNA of the positive-sense strand template of sgRNA target sequences provided by the invention
Sequence TAGG, 5 ' end addition CAAA of antisense strand template, complementary with vector plasmid cohesive terminus,cohesive termini, synthesis oligonucleotide chain is single-stranded
The double-strand of annealed formation；.It is with I digestion CRISPR/Cas skeleton plasmids of restriction enzyme Bbs, the plasmid after digestion is pure
Change；The plasmid of linearisation is connect with sgRNA oligonucleotides double-strands, conversion, sequencing, extracting endotoxin-free plasmid turn for cell
Dye.By the transfection carrier CRISPR/Cas skeleton plasmids of linearisation and in-vitro transcription CRISPR/Cas skeleton plasmids respectively with it is described
Double-strand connects, and obtains transfection carrier plasmid and in-vitro transcription carrier；The transfection carrier CRISPR/Cas skeleton plasmids are U6-
CRISPR/Cas skeleton plasmids；The in-vitro transcription CRISPR/Cas skeleton plasmids are T7-CRISPR/Cas skeleton plasmids.
(2) adult fibroblast is transfected：By the transfection carrier electroporation transfection pig adult obtained in step (1) at fiber
Cell, the pig adult fibroblast of the knockout of the identified determining GGTA1 genes of culture of single cell clone；It is at fibre by described
Cell extraction genomic DNA, PCR amplification GGTA1 include the genetic fragment of target sequence, by denaturation, renaturation and digestion, determine
The knockout situation of GGTA1 genes, enrichment GGTA1 knock out cell, carry out the culture and identification of single cell clone：PCR amplification goes out
The genetic fragment for including 1 target sequence ensures that target sequence is not at the centre position of amplified fragments, after recovery purifying product, uses
T1NE1 endonucleases carry out endonuclease reaction.If there is mutation at target site, two bands can be cut by T1NE1, it is on the contrary then
It cuts not open.
(3) preparation of clone embryos：By the pig adult fibroblast of the knockout of the GGTA1 genes obtained in step (2)
Nucleus inject enucleation oocyte, and endochylema injecting step (1) obtain in-vitro transcription carrier and Cas9 mRNA, obtain
Clone embryos；The mRNA of the endochylema injecting body outer transcription vector and Cas9, in-vitro transcription carrier and Cas9 in injection
The mass ratio of mRNA is 1:9~1:Between 3.Specifically：
It is collected from slaughterhouse in qualified pig ovary and picks out the mature oocyte with first polar body, be enucleated, will close
Suitable body cell injects enucleation oocyte.After the mRNA of the in-vitro transcription carrier and Cas9 are mixed, clone embryos are injected.
The mRNA mixed liquors of in-vitro transcription carrier, T7-Cas9, final concentration mass ratio is 1:9~1:Between 3.
(4) prepared by GGTA1 gene knock-out pigs：The clone embryos obtained in step (3) are implanted into the portable Gilt Uterus of heat,
It cultivates and obtains GGTA1 gene knock-out pigs；
(5) take leather for dressing：It is proceeded as follows in 37 DEG C, 110~150 revs/min of constant-temperature table：By step
(4) the middle GGTA1 gene knock-out pigs obtained take pachydermia in head layer, flushing carries out hydrophobic treatment successively later, proteolysis is handled,
Blocking antigen processing, frozen dried, obtain the low immunogenicity dressing.The middle pachydermia thickness 0.4mm~0.5mm it
Between.
Specifically：
From pachydermia in head layer is taken on GGTA1 gene knock-out pigs, it is placed in antibiotic phosphate buffer and rinses for several times,
Be placed in polyethylene glycol in, after enhancing intermolecular hydrophobic effect, it is fully impregnated with antibiotic phosphate buffer with
Washing, is put into the physiological saline containing trypsase and impregnates, finally used again after lauryl sodium sulfate (SDS) sustained oscillation
Glutaraldehyde cross-linking closes residue antigens.It is fully rinsed with aqua sterilisa, is preserved at 4 DEG C after freeze-drying.
The hydrophobic treatment is to impregnate 50~60min using a concentration of 5~10% (m/v) polyglycol solutions；It is described poly-
Ethylene glycol is any one in Mw=400,600,800,2000,4000.
The proteolysis processing is using the immersion of a concentration of 0.3~0.4% trypsin solution normal saline solution
70~100min；
The blocking antigen processing is using a concentration of 1~2% 12~20h of sodium dodecyl sulfate solution sustained oscillation
Afterwards, glutaraldehyde cross-linking is finally used again, closes residue antigens.
It is embodiment below：
Embodiment one, the design of GGTA1 genes sgRNA, screening and CRISPR/Cas9 systemic vectors preparation
1, the design of sgRNA
For GGTA1 genes, the principle of sgRNA designs is as follows：
(1) length 20nt or so；
(2) 3 ' sgRNAs of the end containing GG, G/C content is in 40%-60%；
(3) sgRNA, which is targeted, is not present SNPs in binding site genome sequence；
(4) full genome undershooting-effect is analyzed, maximum allowable 5 base mispairings in site of missing the target.
Based on the above design principle, target sequence set shown in table 1 is designed.
The sequence for the sgRNA that table 1 designs (grey is labeled as PAM sequences)
2, the screening of sgRNA and the preparation of in-vitro transcription carrier
6 suitable sgRNA are filtered out according to the above principle, are further imitated by external digestion Activity determination gRNA target spots
Rate is screened sgRNA, is as follows:
(1) in-vitro transcription carrier sgRNA
Synthetic primer T7-gRNA-FPg, refers to table 2, and wherein NNNNNNNNNNNNNN is gRNA target spots (without PAM sequences
Row).Use T7-gRNA-FPg and gRNA-RP primer pairs, standard sgRNA1 (g1) primer g1-FP or standard sgRNA2 (g2)
Primer g2-FP and gRNA-RP primer pair is PCR, PCR product 120bp or so by template of standard sgRNA segments, excessively full formula gold
Column EP101-01) purifying, use DEPC H2Concentration is surveyed after O eluted dnas, the DNA profiling as subsequent in vitro transcription.
The synthetic primer pair of 2 in-vitro transcription sgRNA of table
Reaction system (50 μ l) is as follows：
SgRNA plasmids：10ng
T7-gRNA-FPg(10μM)：1.5μl
gRNA-RP(10μM)：1.5μl
2×Pfu Mix：25μl
DEPC H2O：Add to 50 μ l
Above-mentioned reaction system is put into PCR instrument, and is reacted by following procedure：
(2) preparation of digestion DNA
SgRNA target spots (including PAM sequences) can be building up to by way of bridging PCR in PCR product, as shown in Figure 2.
Sequence wherein used is as follows：
NNNNNNNNN (270bp) MMMMMMMMMMM (gRNA target site positions contain PAM sequences) NNNNNNNNNNN
(450bp)
Clip size is 740bp before digestion, and purpose band size should be respectively after digestion：280bp+460bp or so.
(3) the external endonuclease reactions of Cas9/sgRNA
The reaction system of the external endonuclease reactions of 3 Cas9/sgRNA of table
It is loaded according to 3 reaction system order of table, after being sufficiently mixed, 37 DEG C of reaction 0.5h, 65 DEG C are boiled 5min.It is solidifying with agarose
Gel electrophoresis tests and analyzes digestion result.Each endonuclease reaction all does the sgRNA endonuclease reactions of standard target spot simultaneously, as
Positive reference carries out active comparison.The gray-scale map of digestion band such as Fig. 3 is scaled the Activity Results such as table 4 of sgRNA.Usually,
The digesting efficiency of sample sgRNA<Standard sgRNA1 (g1) shows that sample sgRNA target actives are poor, it is not recommended that uses；If
The digesting efficiency of 40%-50% >=sample sgRNA >=standard sgRNA1 (g1) shows that sample sgRNA target actives are qualified；Sample
Digesting efficiency >=40%-50% of sgRNA shows that sample sgRNA target actives are good, it is proposed that uses；If the enzyme of sample sgRNA
Efficiency >=standard sgRNA2 (g2) is cut, shows that sample sgRNA target actives are very high, it is proposed that is used.It follows that digestion activity
Good and workable sgRNA has sgRNA-1, sgRNA-2, sgRNA-5.
The target spot efficiency of 4 external digestion Activity determination gRNA of table
3, the preparation of CRISPR/Cas9 system transfections carrier
As shown in figure 4, Cas9gRNA expression vectors are the px330-U6-Chimeric_BB-Cbh-hSpCas9 with purchase
Carrier is skeleton, and required boot sequence is inserted into according to fixed oligo design patterns.It is as follows：
(1) oligonucleotide chain double-strand is synthesized：Respectively in 5 ' end addition CACC antisense strand moulds of sgRNA-5 positive-sense strand templates
5 ' end addition CAAA of plate, with vector plasmid cohesive terminus,cohesive termini complementation.The single-stranded annealed formation double-strand of oligonucleotide chain of synthesis.
(2) I digestion px330-U6-Chimeric_BB-Cbh-hSpCas9 skeleton plasmids of restriction enzyme Bbs are used.It presses
It is configured according to following digestion system：
10×NEB Buffer4：5uL
100×BSA：0.5uL
BsaⅠ：2uL
PDR274 cyclic plasmids：25uL
ddH2O water：Add to 50uL
Digestion system is placed at 37 DEG C and reacts 2-3h, 20min is inactivated at 65 DEG C.
(3) Plasmid DNA digestion purifies：Plasmid after digestion is utilized into High pure PCR product
Purification Kit purifying.
(4) plasmid of linearisation is connect with sgRNA-5 oligonucleotides double-strands, linked system is as follows：10×Buffer：
2uL
T4 ligases：0.5uL
SgRNA-5 oligonucleotides double-strands：1uL
Linearization plasmid：20ng
ddH2O：Add to 20uL
It is connected in 22 DEG C of constant incubators overnight, 65 DEG C of inactivation 20min.
(5) plasmid conversion, sequencing, extracting endotoxin-free plasmid carry out cell transfecting.
Embodiment two, transfection adult fibrocyte
1, cell transfecting and screening
U6-gRNA-3 plasmid electroporations are transfected into pig adult fibroblast.It is carried out by step in detail below：
1. microscopically observation cell growth condition, select grow fine, cell of the degree of converging up to 80% or so, use
0.25% trypsin digestion 2-3min, 1500g centrifugation 5min, outwells upper liquid；
2. 500uL DPBS buffer solution suspension cells precipitate, 1500g centrifuges 5min, thoroughly washes pancreas remaining in cell
Protease；
3. certain volume electricity, which is added, turns the above-mentioned cell of buffer solution suspension, electricity, which is turned liquid, for 50uL by every group of electricity swivel system divides equally
Into each group centrifuge tube, and the corresponding electric Pignus pignoris grain of certain mass, liquid-transfering gun mixing cell suspension are added thereto；
4. opening ECM2001Elector Cell ManipuLator electroporations, setting electricity turns required voltage (130V-
170V), electric shock time (3ms), number of shocks (1 time)；
Turn in suspension to the electric shock tank of electric revolving cup 5. 10uL liquid-transfering guns draw 50uL electricity in batches, electric revolving cup is put into electroporation
Tank, screwing screw make electric revolving cup be come into full contact with electrode metal face in slot, start to shock by electricity by Automatic start；
6. electric shock finishes when indicator light stops flicker, rotary screw takes out electric revolving cup, and 10uL liquid-transfering guns absorption electricity turns liquid and arrives
In corresponding culture dish, certain volume culture medium is added into culture dish.It is sequentially completed each group electroporation transfection；
7. electricity turns to complete, each group cell is put into 39 DEG C, culture in 5%CO2 saturated humidities incubator, for 24 hours after, wait for that cell pastes
Wall washes away the cell that electricity turns dead with DPBS, adds new culture medium；After 36h, cell growth shape is observed under fluorescence inverted microscope
Condition, rough estimate green fluorescence account for the ratio of total cell number, calculate transfection efficiency and take pictures.
After sgRNA expression plasmids are stably transfected into cell genomic dna, the EGFP gene in plasmid also can be with genome
Expression and express, the cell percentages for sending out fluorescence can be counted under 400 times of (or 200 times) fluorescopes.As shown in figure 5, with
The EGFP-N1 plasmid encoding luciferase amounts of control group transfection are compared, and experimental group sgRNA plasmid transfection efficiency is up to 80% or so.
After transfecting 36h, genomic DNA is extracted, the genetic fragment of target sequence is included using it as template amplification GGTA1, passed through
Denaturation, renaturation and digestion, determine the knockout situation of GGTA1 genes.It is enriched with GGTA1 and knocks out cell, carry out the training of single cell clone
It supports and identifies.
2, the culture and identification of single cell clone
It transfects successful cell and its monoclonal cell is selected using limiting dilution assay.Concrete operation step：In fluorescence microscopy
Cell is observed under mirror blue-light source, selects the cell with green fluorescent protein, takes 0.25% trypsase 500uL digestion
5min；Trypsase is sopped up, culture medium is added into the cell digested, is blown and beaten repeatedly with pipettor, cell is made all to suspend
At unicellular；It is drawn in the cell to new culture hole that 1uL has suspended, is observed under fluorescence inverted microscope glimmering with 10uL liquid-transfering guns
Photo-cell number；The cell crossed through limiting dilution is continued to cultivate, when fluorecyte grows up to clone's spot, spot method and point are cloned with choosing
Digestion method is selected.When fluorecyte purity is up to 90%, digestion is collected, and is divided to two parts, and portion is subsequently identified, a
It freezes.
Embodiment three, the identification of GGTA1 gene knockout porcine clone embryos
Genomic DNA is extracted using TIANamp Genomic DNA Kit, for each target position point design pair of primers
PCR amplification is carried out, primer sequence is shown in Table 5.PCR reaction system 50uL, reaction condition are as follows：94℃7min；(94 DEG C of 30s, 58 DEG C
1min, 72 DEG C of 30s) × 35；72℃7min；4 DEG C of preservations.Amplified fragments size is respectively 603bp and 504bp, PCR purified reagents
Box purified pcr product.It takes 15uL PCR products in 1.5mL centrifuge tubes, boils 5min denaturation after sealing in boiling water, then room temperature
Renaturation 30min.T7NE1 digestion systems 15uL：PCR product, 12.5uL；
10 × NEB2,1.5uL；T7NE1 enzymes, 1uL.After 37 DEG C of water-bath digestion 30min, 1uL is taken to carry out 2% gel electrophoresis.
Sample to be tested takes double in experiment, carries out denaturation annealing, and experimental group sample carries out digestion, negative control group sample not digestion.
The two is placed in 37 DEG C of water-bath 30min.
5 primer sequence of table
With gene order near PCR amplification target spot, then by TA clones, be sequenced come further verify mutation exist and
Catastrophe.The mRNA mixtures of T7-gRNA-M and T7-Cas9 are injected into clone embryos.Clone embryo Validation in vitro
CRISRP/Cas9 cutting effects are as shown in Figure 6.
(final concentration of 30ng/ μ L, the Cas9mRNA final concentrations of sgRNA after the mRNA of T7-gRNA-M and T7-Cas9 is mixed
For 180ng/ μ L) 1500 clone embryos are injected into, after washing 5 times with embryo medium, it is transferred to the Embryo Culture pre-equilibrated
Liquid, be placed in 39 DEG C, saturated humidity, hypoxemia (5%O2+ 5%CO2+ 90%N2) under conditions of overnight incubation.
Example IV GGTA1 gene knock-out pigs prepare and identification
Morning next day is entered the embryo transfer developed to two generation cells using common oviduct transplantation method female to four-head heat receptor
Pig uterus.Routine operation sutures, postoperative continuous 4h injection of antibiotics anti-inflammatory, records the physiological conditions of receptor sow daily.Transplanting
30 and 60d or so carry out 2 B ultrasound detections afterwards, determine receptor pig pregnancy status.Finally obtain 5 clone pigs (as shown in Figure 7).
It acquires liver, heart and ear skin tissue sample respectively to it, extracts the RNA in each tissue, GGTA1 genes are analyzed using RT-PCR
Expression variation, the results are shown in Figure 8 for Preliminary Determination.As a result it shows：The expression quantity of GGTA1 in liver, heart and ear skin tissue
There is significant decrease.
Example IV, acellular dermal matrix GGTA1 knock-out pig pigskins preparation
Fresh porcine skin is derived from 6 months big GGTA1 gene knock-out pigs, removes subcutaneous tissue, carry out degreasing, secondary degreasing,
Liming, deliming soak nitre, cut open the preprocessing process such as layer.Pachydermia in the head layer of 0.45mm thickness is taken with splitting machine, merging contains antibiotic
Phosphate buffer in rinse for several times, place into 8% polyethylene glycol (Mw=800) solution, at 37 DEG C, 150 revs/min
60min is impregnated in constant-temperature table, then it is fully impregnated and washed with antibiotic phosphate buffer；It is subsequently put on
In 0.35% trypsin solution (being configured using physiological saline volume ratio by weight), in 37 DEG C, 150 revs/min of constant temperature
Immersion treatment 1.5h in shaking table is fully impregnated and is washed to it with antibiotic phosphate buffer, then is placed in containing 1.2%
In the aqueous solution of SDS, 30h is impregnated in 37 DEG C, 150 revs/min of constant-temperature table, with antibiotic phosphate buffer pair
It is fully washed；Then it is soaked in the glutaraldehyde solution of 0.32% (v/v) of Fresh and is crosslinked 12min, with sterilizing
Water fully rinses, and is freeze-dried to obtain the pigskin of low immunogenicity, can save 1~2 year at 4 DEG C.
Embodiment five, Immunity identification and compare
By GGTA1 knock-out pigs (GGTA1 in example IV-/+Type) obtained by pigskin, respectively with it is (primary with batch normal pig
Type), market takes off the α -1,3Gal protein contents of cell pigskin and is compared.It is detected using immunoblotting, specific side
Method is as follows：
The mortar disinfected is taken out, 100mg pigskins is weighed, is put into mortar, liquid nitrogen is added, grinding is added in a moment
[50mmol/L Tris-Cl (pH=8.0), 150mmol/L NaCl, 1%NP-40,1% are gone 150 microlitres of RIPA protein lysates
Oxycholic acid sodium, 1mmol/L EDTA, 0.1%SDS, 0.2mmol/L PMSF, 1 μ g/ml protease inhibitors], continue to grind, make
Pigskin fully cracks；Lysate is moved into the EP pipes of 1.5ml again, ice stands 5min on ice；4 DEG C, 12000rpm centrifugations
15min, in careful Aspirate supernatant to another new EP pipes；Protein quantification is carried out using BCA methods, protein sample is slow with 5X loadings
Fliud flushing presses 4：1 ratio mixes, and boiling water heats 10min, high speed centrifugation 5min, with 10%SDS-PAGE gel electrophoresis protein isolates,
After electrophoresis, gel is removed, cuts off extra separation gel and concentration glue；Consider paper, gel, PVDF by cathode, sponge, three layers
Film, three layers consider paper, sponge, anode sequence place, bubble side by side；Constant pressure 100V electricity turns 1h, and gel is transferred to by deenergization
It fills and is dyed in the container of suitable Coomassie brilliant blue；PVDF after transferring film makes marks, and is put into the degreasing that 1 × TBST prepares 5%
Milk powder closes 1h；Confining liquid is abandoned, film is washed three times with 1 × TBST, 1：1000 dilution primary antibodies, are incubated at room temperature 2h, and 1 × TBST is washed 4 times,
Each 10min；IgG (1 × TBST dilution ratios 1 that incubation at room temperature goat antirabbit is marked with HRP：10000) film, is washed 5 times, every time
10min；Enhance luminescent solution in the luminous colour developing of chemiluminescence imaging system according to specification.
Testing result such as Fig. 9, GGTA1-/+α -1,3Gal the protein contents of type pigskin are less than the α -1,3Gal of primary type pigskin
Protein content (about 68%) and market take off cell pigskin (about 41%).This explanation compared with primary type and market take off cell pigskin,
GGTA1 in the present embodiment-/+Type pigskin has lower α -1, and 3Gal protein contents, immunogenicity is relatively low, is answered in human skin
In, the generation of immunological rejection can be effectively reduced.
Through zoopery and clinical proof, prepared acellular dermal matrix GGTA1 knock-out pig pigskins, antigenic pole
Low, the holding time is long, and solves the expensive of existing artificial skin product, and antigenicity is strong, infection is higher, elastic
The shortcomings of poor.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, all within the spirits and principles of the present invention made by all any modification, equivalent and improvement etc., should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of reduction immunogenicity sgRNA, which is characterized in that it is combined with pig GGTA1 gene orders, and has following spy
Sign：
(1) its length in 18nt between 22nt；
(2) 3 ' ends of its target sequence on GGTA1 genes contain GG, and G/C content is in 40%-60%；
(3) its with targeting binding site genome sequence in be not present SNPs；
(4) it carries out full genome undershooting-effect analysis, maximum allowable 5 base mispairings in site of missing the target with pig GGTA1 genes.
2. reducing immunogenicity sgRNA as described in claim 1, which is characterized in that it is any one in following sequence：
GCTGCTTGTCTCAACTGTAATGG；
CCCAGAAGGTTCTTTGTTCTGG；
AAGGCTCCAGTGGTATGGGAAGG。
3. reducing immunogenicity sgRNA as claimed in claim 2, which is characterized in that it is following sequence：
AAGGCTCCAGTGGTATGGGAAGG。
4. a kind of low immunogenicity dressing, which is characterized in that the dressing is pigskin, and the pigskin derives from GGTA1 clpp genes
Except pig.
5. low immunogenicity dressing as claimed in claim 4, which is characterized in that the GGTA1 gene knock-out pigs have such as
SgRNA described in claims 1 to 3 any one.
6. the preparation method of low immunogenicity dressing as described in claim 4 or 5, which is characterized in that include the following steps：
(1) preparation of transfection carrier：It will be the positive-sense strand template of the sgRNA target sequences as described in claims 1 to 3 any one
5 ' end addition RNA sequence TAGG, 5 ' end addition CAAA of antisense strand template, the synthesis single-stranded annealed formation of oligonucleotide chain
Double-strand, by the transfection carrier CRISPR/Cas skeleton plasmids of linearisation and in-vitro transcription CRISPR/Cas skeleton plasmids respectively with institute
Double-strand connection is stated, transfection carrier plasmid and in-vitro transcription carrier are obtained；
(2) adult fibroblast is transfected：By the transfection carrier electroporation transfection pig adult obtained in step (1) at fiber finer
Born of the same parents, the pig adult fibroblast of the knockout of the identified determining GGTA1 genes of culture of single cell clone；
(3) preparation of clone embryos：By the thin of the pig adult fibroblast of the knockout of the GGTA1 genes obtained in step (2)
Karyon injects enucleation oocyte, and the mRNA of the in-vitro transcription carrier and Cas9 of endochylema injecting step (1) acquisition, is cloned
Embryo；
(4) prepared by GGTA1 gene knock-out pigs：The clone embryos obtained in step (3) are implanted into Gilt Uterus, cultivates and obtains
GGTA1 gene knock-out pigs；
(5) take leather for dressing：The GGTA1 gene knock-out pigs obtained in step (4) are taken into pachydermia in head layer, after rinsing successively
Hydrophobic treatment, proteolysis processing, blocking antigen processing, frozen dried are carried out, the low immunogenicity dressing is obtained.
7. the preparation method of low immunogenicity dressing as claimed in claim 6, which is characterized in that step (1) transfection carries
Body CRISPR/Cas skeleton plasmids are U6-CRISPR/Cas skeleton plasmids；The in-vitro transcription CRISPR/Cas skeleton plasmids are
T7-CRISPR/Cas skeleton plasmids.
8. the preparation method of low immunogenicity dressing as claimed in claim 6, which is characterized in that step (3) the endochylema note
The mRNA of beam outer transcription vector and Cas9, the mass ratio of the mRNA of in-vitro transcription carrier and Cas9 are 1 in injection:9
~1:Between 3.
9. the preparation method of low immunogenicity dressing as claimed in claim 6, which is characterized in that the middle pachydermia thickness exists
Between 0.4mm~0.5mm.
10. the preparation method of low immunogenicity dressing as claimed in claim 6, which is characterized in that the hydrophobic treatment is to adopt
50~60min is impregnated with a concentration of 5~10% (m/v) polyglycol solutions；
Proteolysis processing for impregnate 70 using a concentration of 0.3~0.4% trypsin solution normal saline solution~
100min；
After the blocking antigen processing is 12~20h of sodium dodecyl sulfate solution sustained oscillation using a concentration of 1~2%,
Glutaraldehyde cross-linking is finally used again, closes residue antigens.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113368306A (en) * | 2020-03-23 | 2021-09-10 | 成都中科奥格生物科技有限公司 | Low-immunogenicity biological material and preparation method and application thereof |
| CN113425905A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Blood vessel material and preparation method and application thereof |
| CN113425907A (en) * | 2020-03-23 | 2021-09-24 | 四川大学 | Pericardium material and preparation method and application thereof |
| CN113425912A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Acellular dermal material and preparation method and application thereof |
| CN113429475A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Glue raw material and preparation method and application thereof |
| CN113425906A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Cartilage material and preparation method and application thereof |
| CN113425900A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Glue water gel material and preparation method and application thereof |
| CN113425904A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Oral cavity patch material and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103990179A (en) * | 2014-05-24 | 2014-08-20 | 河北爱能生物科技有限公司 | Method for preparing xenogeneic acellular matrix and product thereof |
| CN105492609A (en) * | 2015-06-11 | 2016-04-13 | 深圳市第二人民医院 | Method for CRISPR-Cas9 specific knockout of pig GGTA1 gene and sgRNA for specific targeted GGTA1 gene |
-
2018
- 2018-05-17 CN CN201810470742.9A patent/CN108642054A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103990179A (en) * | 2014-05-24 | 2014-08-20 | 河北爱能生物科技有限公司 | Method for preparing xenogeneic acellular matrix and product thereof |
| CN105492609A (en) * | 2015-06-11 | 2016-04-13 | 深圳市第二人民医院 | Method for CRISPR-Cas9 specific knockout of pig GGTA1 gene and sgRNA for specific targeted GGTA1 gene |
Non-Patent Citations (2)
| Title |
|---|
| RAMM R.,等: "GGTA1-KO pig heart valves do not bind preformed human xenoantibodies", 《BASIC RESEARCH IN CARDIOLOGY》 * |
| 李国玲等: "利用CRISPR/Cas9系统建立Xist基因敲除猪模型", 《遗传》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113368306A (en) * | 2020-03-23 | 2021-09-10 | 成都中科奥格生物科技有限公司 | Low-immunogenicity biological material and preparation method and application thereof |
| CN113425905A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Blood vessel material and preparation method and application thereof |
| CN113425907A (en) * | 2020-03-23 | 2021-09-24 | 四川大学 | Pericardium material and preparation method and application thereof |
| CN113425912A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Acellular dermal material and preparation method and application thereof |
| CN113429475A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Glue raw material and preparation method and application thereof |
| CN113425906A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Cartilage material and preparation method and application thereof |
| CN113425900A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Glue water gel material and preparation method and application thereof |
| CN113425904A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Oral cavity patch material and preparation method and application thereof |
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