CN109055379A - A kind of preparation method of transgenic chicken oviduct bioreactor - Google Patents
A kind of preparation method of transgenic chicken oviduct bioreactor Download PDFInfo
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- CN109055379A CN109055379A CN201811054091.1A CN201811054091A CN109055379A CN 109055379 A CN109055379 A CN 109055379A CN 201811054091 A CN201811054091 A CN 201811054091A CN 109055379 A CN109055379 A CN 109055379A
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Abstract
The invention proposes a kind of preparation methods of transgenic chicken oviduct bioreactor, comprising the following steps: the gRNA of S1. design targeting chicken genomic DNA；S2. the expression vector of gRNA is constructed；S3. building carries the donor vehicle of exogenous gene expression frame: the donor vehicle includes homologous repair vector and non-homogeneous repair vector；S4. by vector introduction chicken individuals.The present invention, which utilizes, combines CRISPR gene editing technology, improve accuracy rate of the exogenous origin gene integrator into genome, realize high efficiency import foreign gene, prepare the transgenic chicken that can express and secrete foreign protein into egg white, and realize foreign gene it is stable be hereditary to offspring.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to a kind of transgenic chicken oviduct bioreactor.
Background technique
Poultry salpingo biological reactor is primarily referred to as the poultry based on chicken, and expression has medicine in oviduct tissue
It is excreted therewith in produced birds, beasts and eggs with the foreign protein of value and being secreted into.Foreign protein type includes having listed
Various recombinant proteins, such as insulin, monoclonal antibody etc..Early in Chinese Scientists Zeng Jie (Zeng Bangzhe) just proposition in 1994
The concept and transgenic poultry eka-gold egg plan of poultry salpingo biological reactor, and it is academic in first national transgenosis in 1996
In seminar with transgenic experiments room and developer's Enterprise linkage such as the U.S., Canada, Japan, Britain, the subsequent U.S.
Avigenics company and Georgia university R.Ivarie professor, which have carried out to expand to communicate with once outstanding person, discusses joint study, until
Avigenics company of the U.S. in 1998 has just carried out project verification and scale investment and exploitation, and Avigenics company exists within 2002
It takes the lead in delivering on Nature Biotechnology and successfully expresses foreign protein in transgenosis egg, but expression quantity only has
1.34mg/L (egg white solution).2003 later Roslyn research institutes of Britain also enter the field, and support project to form by Sang
Li Liao company.There is the company of north America region to start the correlative study and exploitation of salpingo biological reactor successively later, such as
TranXenoGEN, Viragen, GeneWorks etc., wherein the GeneWorks for being found in 1996 is obtaining 18,000,000 at that time
Dollar risk investment, but all research all suffer from expression quantity lowly can not be suitable for reactor, it is very distant from industrialization
Remote problem also embodies the most crucial problem in the field, and the difficulty of prepare transgenosis poultry salpingo biological reactor is very
It is huge.
Poultry Breeding behavior is more special, after sperm enters vagina, poultry infundibulopelvic of faliopian tube in conjunction with ovum and by
Essence, after stop 2 hours or more, the endocrine egg protein of fallopian tubal will wrap up fertile egg during this period, later in isthmus of uterine tube
Egg shell membrane is formed, eggshell is then formed, total time-consuming is up to 18 hours.The fertilized embryo excreted has developed to blastaea evening
Phase is named as the X phase, and having developed has 50,000 to 60,000 cells.Wherein archaeocyte (Primordial germ cells,
PGCs) just originate from and match the central transparent area of disk with the X phase, enter later as chick embryo development enters blood circulation system, finally exist
Into sexual gland and form sperm and ovum.According to the rule of the migration of PGCs, can 11 days or so after embryonic development from embryo
PGCs is collected and separated in tire blood.It is injected into the 15th phase recipient embryo from PGCs isolated in donor, donor PGCs
Be successfully entered receptor sexual gland and be divided into reproduction cell, and by the gene of donor pass to offspring (Vick L, Li Y,
Simkiss K.Transgenic birds from transformed primordial germ
cells.Proc.R.Soc.Lond B Biol Sci,1993,251:179-182.)。
Because the special reproduction feature of poultry, makes it carry out unicellular microinjection like that without image of Buddha mammal, heredity
Property can only rely on the random combine of viral vectors, and this randomness cause efficiently specific expressed difficulty increase.Another party
The research in face concentrates on carrying out avian subject clone by the foundation of stem cell strain and by body cell, but difficulty is still huge.
At least sub- other high level expression of gram-grade in exogenous gene high-efficient, orientation, heritable integration and egg white, at present these two aspects
The problem of foreign countries also solve far away, several method used in present chicken transgenosis be still rely on randomness integration.Recent fowl
The research of egg bioreactor, which is concentrated mainly on, improves the individual problem of transgene efficiency and screening with high level expression.These
Problem cannot solve, and birds, beasts and eggs reactor just cannot achieve.
Prepare transgenosis poultry salpingo biological reactor major technology bottleneck is the uncertainty of random integration.The present invention
On the basis of existing the relevant technologies, in conjunction with newest gene editing (genome editing) technology, to greatly improve whole
The accuracy rate of conjunction really realizes efficiently heritable importing foreign gene.
The emergence and development of gene editing technology open the technical bottleneck for overcoming transgenic poultry bioreactor in recent years
Warded off new approach, including three classes gene editing technology, 1, Zinc finger nuclease (ZFN), 2, transcriptional activation increment effect because
Sub- nuclease (TALEN), 3, the short palindromic sequences of regular intervals repeat cluster (CRISPR).Although three classes gene editing technology is former
Reason and the mode of action are not quite similar, but they finally make target dna generate double-strand break (DSB), so that active cell is repaired
The system of answering a pager's call.It is repaired in the cell by two ways after genomic DNA fracture, i.e. homologous recombination repair (HR) and non-homogeneous end
(NHEJ) is repaired in end connection.When being present in the identical sequence in DNA breakage both ends into the cell, homologous recombination repair could occur,
After the homologous sequence in the exogenous DNA both ends building both ends target DSB, lossless insertion genome can be carried out by homologous recombination repair
DNA；Exist when intracellular without mutually homotactic template, DNA double chain then passes through non-homologous end joining reparation, but result can make
There is different degrees of mutation in double-strand after must connecting.In addition to this, there are also a kind of end engagements that micro- homology arm mediates
(MMEJ), i.e., homology arm is shorter in length than in HR length needed for homology arm.
In three kinds of gene editing technologies, CRISPR system is prevalent in bacterium and archeobacteria and bacterium is suitable
Answering property is immunized related.CRISPR/Cas system by a series of Cas albumen, including Cas1, Cas2, Cas4 and effect protein, than
It further include one section of CRISPR sequence in addition to the encoding gene of these albumen in gene cluster composition if Cas9 and Cpf1 is formed,
That is leader sequence and some repetitive sequences and intervening sequence composition.When CRISPR system functions, effect protein needs
It is combined with gRNA and forms ribonucleoprotein complex, DNA and cutting of the identification with gRNA complementary pairing, to cause double-strand disconnected
It splits.GRNA is generally by two component parts of tracrRNA and crRNA.The 5' end regions of crRNA can with target site complementary pairing,
The end 3' and tracrRNA are bound to each other to form special hairpin structure.TracrRNA and crRNA with specific recognition sequence exist
It in eucaryote and is not present, by artificial synthesized and transcribe, forms gRNA and Cas9 protein binding after transcribing in eukaryocyte
Compound is formed, identifies that former intervening sequence closes on motif (protospacer first during the compound screen dna
Adjacent motif, PAM), when the series of the upstream PAM and the end the 5' complementary pairing of gRNA, Cas9 albumen will be in spy
Anchor point cuts the base-pair of complementary pairing, forms DSB, which is target site,
On target dna sequence；Sequence complementary to it is known as spacer sequence, positioned at the 5' end regions of the crRNA of gRNA.?
In CRISPR system, PAM sequence is particularly significant.Different types of Cas9 albumen has different PAM rules.It is applied at present
CRISPR system includes its mutation.Select different Cas9 albumen, i.e., different PAM rules, the corresponding target site sequence in the upstream PAM
Also not identical, corresponding target site sequence is not also identical, and the end 5' base is mutually paired with target site sequence in gRNA；It is practical
Designed and synthesis two single stranded DNAs of gRNA template, i.e., design according to target site sequence and synthesize in.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of preparation side of transgenic chicken oviduct bioreactor
Method overcomes in the preparation process of transgenic chicken gene random integration not it is intended that the method provided through the invention
Certainty and poor efficiency realize that foreign gene is specific expressed, and compare traditional preparation methods, and the operation is more convenient, and the period is more
It is short, it is more efficient.
The present invention provides a kind of preparation method of transgenic chicken oviduct bioreactor, comprising the following steps:
S1. the chicken genome target DNA sequence dna of the gRNA: selection of design targeting chicken genomic DNA is ovalbumin first
Introne selects the gRNA of 90% or more homology according to the different PAM sequence rules of variety classes Cas9 albumen；
S2. construct the expression vector of gRNA: after determining target site sequence according to PAM rule, synthesis meets expression vector structure
The positive and negative DNA of target site sequence for building requirement is single-stranded, and annealing is connected to CRISPR/Cas system expression carrier after forming DNA double chain
On, gRNA and Cas albumen can be expressed after vectors into cells；
S3. building carries the donor vehicle of exogenous gene expression frame: the donor vehicle include homologous repair vector and
Non-homogeneous repair vector；
S31. the donor vehicle of the homologous reparation is constructed: homologous up and down using single stranded DNA as homologous recombination repair template
Sequence is with 45bp-90bp；Or using double-stranded DNA as homologous recombination repair template, by upstream and downstream at the DSB of target dna site
Each 100bp-1000bp sequence is connected to the end 5' and the end 3' of foreign gene, and as homology arm, donor vehicle can be to close
Ring plasmid or PCR product or the form of linear DNA exist；
S32. construct the donor vehicle of the non-homogeneous reparation: PAM sequence and target site sequence form a cleavage site
The reverse complementary sequence of frame, cleavage site frame is reverse complemental cleavage site frame, and there are one or two in donor vehicle
Reverse complemental cleavage site frame exists in the form of closed circular form plasmid or linear DNA；
S4. by vector introduction chicken individuals: will be made in any gRNA/Cas9 prepared in step S2 combination and step S3
The donor vehicle got ready after mixing, the subgerminal cavity being injected into the fertile egg in the chick embryo development X phase, then to injection
Chicken embryo afterwards carries out electroporation；Or it will be prepared in any gRNA/Cas9 prepared in step S2 combination and step S3
Donor vehicle after mixing, plasmid mixed liquor is mixed in proportion with nucleic acid transfection reagent, is injected into the chicken embryo of X phase later
In fertile egg；Then hatching is until hatching.
Improved as of the invention further, PAM type include 5'-NNN-3', 5'-NNNN-3', 5'-NNNNN-3',
5'-NNNNNN-3', 5'-NNNNNNN-3' and 5'-NNNNNNNN-3'.
As further improvement of the invention, step S2 can also be realized by the following method: be building up to existing gRNA
On expression vector: individually building gRNA expression plasmid, promoter use U6, the Polll polymerase such as T7, and Cas9 expression plasmid,
Or Cas9 albumen cotransfection is to target cell.
As further improvement of the invention, exogenous gene expression frame described in step S3 includes one section of completion ovalbumin
Gene First Intron sequence, one section makes Protein secretion to extracellular signal peptide sequence, one section of arbitrary target foreign protein
Global DNA sequence and one section protection mRNA stability polyA sequence.
As further improvement of the invention, will no longer contain after completion ovalbumin gene First Intron sequence completion
The site of selected gRNA identification and cutting.
As further improvement of the invention, one section of completion ovalbumin gene First Intron sequence, is from selected
GRNA cleavage site starts the sequence terminated to First Intron or constitutes complete egg white together with cleavage site upstream sequence
Protein gene First Intron sequence.
As further improvement of the invention, signal peptide sequence is molten for the original signal peptide sequence of target foreign protein, chicken source
Bacterium enzyme signal peptide, chicken source Ovotransferrin signal peptide, chicken source class ovum mucoglobulin signal peptide or the signal peptide of engineer.
It is improved as of the invention further, arbitrary target foreign protein is that human serum albumins, interleukins, people are solidifying
Blood factor, interferon, tumor necrosis factor, colony stimulating factor, growth factor, chemotactic cytokine, recombinant antibodies or
Other protein moleculars.
It is improved as of the invention further, polyA sequence is using the original polyA sequence of chicken egg white, additionally adds
Add BGH polyA sequence, SV40polyA sequence or other polyA sequences.
As further improvement of the invention, S4 step can also be realized by the following method: will prepare in step S2
Any gRNA/Cas9 combination and step S3 in the donor vehicle for preparing after mixing, pass through electroporation or nucleic acid turn
Transfection reagent is imported into from the PGC cell isolated and purified in chicken embryo, passes through drug screening foreign gene success quiding gene
The PGCs of screening, is then injected into the 15th phase chicken embryo blood vessel by the PGCs cell of group.
The invention has the following beneficial effects:
1. it is whole at random to overcome gene in the preparation process of chicken salpingo biological reactor for the method provided through the invention
The uncertainty and poor efficiency of conjunction realize that foreign gene is specific expressed, and compare traditional preparation methods, and the operation is more convenient,
Period is shorter, more efficient；
2. it is accurate into genome to improve exogenous origin gene integrator using CRISPR gene editing technology is combined by the present invention
Rate realizes that high efficiency imports foreign gene, prepares the transgenic chicken that can express and secrete foreign protein into egg white,
And realize foreign gene it is stable be hereditary to offspring.
Detailed description of the invention
Fig. 1 is the electrophoretogram in the embodiment of the present invention 1 after PCR product T7Endonuclease I digestion；
Fig. 2 cuts donor plasmid pBlue-1/cut-ALB and 2 in the embodiment of the present invention 21 and cuts donor plasmid pBlue-2/
Cut-ALB structural schematic diagram；
Three days embryonic development figures after 3 sealer of Fig. 3 embodiment of the present invention；
Wherein, 1, cleavage site frame in genomic dna sequence；2, reverse complemental cleavage site frame.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, the embodiment described is the embodiment of part of representative of the invention, rather than whole embodiments, this field are general
Other all embodiments obtained belong to protection of the invention to logical technical staff without making creative work
Range.
A kind of preparation method of transgenic chicken oviduct bioreactor of the present invention, comprising the following steps:
S1. the chicken genome target DNA sequence dna of the gRNA: selection of design targeting chicken genomic DNA is ovalbumin first
Introne, sequence is as shown in SEQ.1, according to the different PAM sequence rules of variety classes Cas9 albumen, select homology 90% with
On gRNA；
5'-CTCAAAAGGTAAGCAACTCTCTGGAATTACCTTCTCTCTATATTAGCTCTTACTTGCACCTAAACT
TTAAAAAATTAACAATTATTGTGTTATGTGTTGTATCTTTAAGGGTGAAGTACCTGCGTGATACCCCCTATAAAAAC
TTCTCACCTGTGTATGCATTCTGCACTATTTTATTATGTGTAAAAGCTTTGTGTTTGTTTTCAGGAGGCTTATTCTT
TGTGCTTAAAATATGTTTTTAATTTCAGAACATCTTATCCTGTCGTTCACTATCTGATATGCTTTGCAGTTTGCCTG
ATTAACTTCTAGCCCTACAGAGTGCACAGAGAGCAAAATCATGGTGTTCAGTGAATTCTGGGGAGTTATTTTAATGT
GAAAATTCTCTAGAAGTTTAATTCCTGCAAAGTGCAGCTGCTGATCACTACACAAGATAAAAATGTGGGGGGTGCAT
AAACGTATATTCTTACAATAATAGATACATGTGAACTTGTATACAGAAAAGAAAATGAGAAAAATGTGTGTGCGTAT
ACTCACACACGTGGTCAGTAAAAACTTTTGAGGGGTTTAATACAGAAAATCCAATCCTGAGGCCCCAGCACTCAGTA
CGCATATAAAGGGCTGGGCTCTGAAGGACTTCTGACTTTCACAGATTATATAAATCTCAGGAAAGCAACTAGATTCA
TGCTGGCTCCAAAAGCTGTGCTTTATATAAGCACACTGGCTATACAATAGTTGTACAGTTCAGCTCTTTATAATAGA
AACAGACAGAACAAGTATAAATCTTCTATTGGTCTATGTCATGAACAAGAATTCATTCAGTGGCTCTGTTTTATAGT
AAACATTGCTATTTTATCATGTCTGCATTTCTCTTCTGTCTGAATGTCACCACTAAAATTTAACTCCACAGAAAGTT
TATACTACAGTACACATGCATATCTTTGAGCAAAGCAAACCATACCTGAAAGTGCAATAGAGCAGAATATGAATTAC
ATGCGTGTCTTTCTCCTAGACTACATGACCCCATATAAATTACATTCCTTATCTATTCTGCCATCACCAAAACAAAG
GTAAAAATACTTTTGAAGATCTACTCATAGCAAGTAGTGTGCAACAAACAGATATTTCTCTACATTTATTTTTAGGG
AATAAAAATAAGAAATAAAATAGTCAGCAAGCCTCTGCTTTCTCATATATCTGTCCAAACCTAAAGTTTACTGAAAT
TTGCTCTTTGAATTTCCAGTTTTGCAAGCCTATCAGATTGTGTTTTAATCAGAGGTACTGAAAAGTATCAATGAATT
CTAGCTTTCACTGAACAAAAATATGTAGAGGCAACTGGCTTCTGGGACAGTTTGCTACCCAAAAGACAACTGAATGC
AAATACATAAATAGATTTATGAATATGGTTTTGAACATGCACATGAGAGGTGGATATAGCAACAGACACATTACCAC
AGAATTACTTTAAAACTACTTGTTAACATTTAATTGCCTAAAAACTGCTCGTAATTTACTGTTGTAGCCTACCATAG
AGTACCCTGCATGGTACTATGTACAGCATTCCATCCTTACATTTTCACTGTTCTGCTGTTTGCTCTAG-3'
SEQ.1
The CRISPR system being applied at present includes its mutation, and it is as shown in the table for PAM type.
(N=A, T, C or G；W=A or T；M=A or C；R=A or G；V=G, C or A；Y=C or T, D
=A, G or T；)
S2. construct the expression vector of gRNA: after determining target site sequence according to PAM rule, synthesis meets expression vector structure
The positive and negative DNA of target site sequence for building requirement is single-stranded, and annealing is connected to CRISPR/Cas system expression carrier after forming DNA double chain
On, gRNA and Cas albumen can be expressed after vectors into cells；
S3. building carries the donor vehicle of exogenous gene expression frame: the donor vehicle include homologous repair vector and
Non-homogeneous repair vector；
S31. the donor vehicle of the homologous reparation is constructed: homologous up and down using single stranded DNA as homologous recombination repair template
Sequence is with 45bp-90bp；Or using double-stranded DNA as homologous recombination repair template, by upstream and downstream at the DSB of target dna site
Each 100bp-1000bp sequence is connected to the end 5' and the end 3' of foreign gene, and as homology arm, donor vehicle can be to close
Ring plasmid or PCR product or the form of linear DNA exist；
S32. construct the donor vehicle of the non-homogeneous reparation: PAM sequence and target site sequence form a cleavage site
The reverse complementary sequence of frame, cleavage site frame is reverse complemental cleavage site frame, and there are one or two in donor vehicle
Reverse complemental cleavage site frame exists in the form of closed circular form plasmid or linear DNA；
S4. by vector introduction chicken individuals: will be made in any gRNA/Cas9 prepared in step S2 combination and step S3
The donor vehicle got ready after mixing, the subgerminal cavity being injected into the fertile egg in the chick embryo development X phase, then to injection
Chicken embryo afterwards carries out electroporation；Or it will be prepared in any gRNA/Cas9 prepared in step S2 combination and step S3
Donor vehicle after mixing, plasmid mixed liquor is mixed in proportion with nucleic acid transfection reagent, is injected into the chicken embryo of X phase later
In fertile egg；Then hatching is until hatching.
Embodiment 1
When PAM type is 5'-NNN-3', it is commonly used to the SpCas9 derived from Streptococcus pyogenes, PAM rule
65 target site sequences are generated in target dna sequence for 5'-NGG-3', SpCas9, as shown in table 1.
Target site sequence of 1 SpCas9 of table in target dna sequence
With sequence TGTGCGTATACTCACACACGTGGFor, whereinTGGFor PAM sequence,
TGTGCGTATACTCACACACG is target site sequence.
(1) using pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid as carrier (Addgene plasmid ID:
42230, hereinafter referred to as pX330), gRNA and SpCas9 albumen can be expressed simultaneously, by 1ug pX330 plasmid 1uL BbsI enzyme
It cuts, after 37 DEG C hatch 1 hour, 1% agarose electrophoresis recycles endonuclease bamhi (QIAquick Gel Extraction Kit recycling
Kit), endonuclease reaction system is as follows:
According to selected target site sequence TGTGCGTATACTCACACACG, artificial synthesized two oligonucleotides, according toRule, wherein when the end 5' of target site sequence is not base
When G, need additionally to add bases G.The sequence for needing to synthesize in this example be 5'-CACCGTGTGCGTATACTCACACACG-3' and
Two oligonucleotide chains are annealed, form short double-stranded DNA, reaction system is such as by 5'-AAACCGTGTGTGAGTATACGCACAC-3'
Under:
By above-mentioned reaction system in ep pipe be uniformly mixed, after handle 1 hour in 37 DEG C of environment, 75 DEG C heating 5 minutes, so
After be placed under room temperature environment it is cooling.
By the line style pX330 plasmid by BbsI digestion and purification and recovery and with the double-strand short dna product of cohesive end
Mixing, LigaFastTMRapid DNA Ligation System (Promega, Cat.No.M8221) is attached reaction, room
Temperature is lower to place 1 hour, and reaction system is as follows:
Plasmid conversion: connection product is mixed, ice with bacillus coli DH 5 alpha competent cell (Takara No:D9057A)
Upper to place 10 minutes, then heat shock in 90 seconds in 42 DEG C places at least 5 minutes on ice, is then applied to competent cell and contains
Have on the LB solid medium of the Ampicilin of concentration 100ug/ml, 37 DEG C are incubated overnight.Picking single bacterium falls within 2ml within second day
In 100ug/ml Ampicilin LB culture solution.250rpm, 37 DEG C after shaken cultivation 5 hours, are therefrom drawn the inoculation of 40ul bacterium solution
In 40ml 100ug/ml Ampicilin LB culture solution.250rpm, 37 DEG C of shaken overnight cultures (12h-16h).
Bacterium solution is collected, and is centrifuged 5min under the conditions of 6000g, abandons supernatant.According to QIAGEN EndoFree Plasmid
Operating procedure extracts plasmid in Midi Kit kit specification, obtains endotoxin-free plasmid pX330-gRNA.
T7E1 digestion detects gRNA efficiency: the UMNSAH/DF-1 cell (ATCC that purchase is obtainedRCRL12203TM) recovery,
Add the DMEM culture solution containing 10% fetal calf serum, at 39 DEG C, 5%CO2Secondary culture is carried out in incubator.Secondary culture number
Dai Hou takes cell 1 × 10 with 0.25% trypsin digestion cell5It is plated in 6 orifice plates, the DMEM culture solution of 10%FBS is trained
It supports, remaining cell cryopreservation is in liquid nitrogen.When DF-1 cell grows into the 70%-90% for accounting for 6 orifice plates area, endotoxin-free is taken
PX330-gRNA plasmid 2ug, with opti-MEM (GibcoTM, 31985070) and it is diluted to 100ul, while taking 5ul
lipofectamin2000(InvitrogenTM, 11668027), it is diluted to 100ul with opti-MEM, is stood at room temperature
5min later again mixes the two, after soft pressure-vaccum mixes, stands 20min at room temperature.Mixed DNA is added to DF-1
In cell, after slight oscillatory, 6 orifice plates are put back into 39 DEG C of incubators.Cell culture fluid is replaced after 6h, uses fresh 10%FBS
DMEM culture solution continues to cultivate cell.
After 48h, culture solution is removed, 0.25% trypsin digestion cell is added, after serum stops digestion reaction, is centrifuged digestive juice
Supernatant is removed afterwards and recycles cell, is resuspended with PBS, later according to MagExtractor genome (TOYOBO, NPK-101)
Specification extracts DF-1 cell genomic dna, using the cell genomic dna of extraction as template, carries out PCR amplification, PCR reactant
It is as follows
PCR amplification program: 95 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 68 DEG C of extension 20s, 35 are followed
72 DEG C of extension 2min, last 4 DEG C of heat preservations after ring.
PCR product is recycled with kit NucleoSpin Gel and PCR Clean-up (MACHREY-NAGEL), is taken
200ng product carries out T7Endonuclease I (NEB) digestion, and digestion system is as follows:
Reaction system is mixed and is added in PCR pipe, PCR instrument temperature setting is set, temperature setting is as follows:
95℃ 10min
95℃-85℃ -2℃/second
85℃-25℃ -0.1℃/second
1ul T7Endonuclease I enzyme is added in the reaction system, after 37 DEG C are reacted 1 hour, electrophoresis detection.As a result
As shown in Fig. 1.
It is identified through sequencing, the efficiency of the gRNA is 32.76%,.
(2) using phU6-gRNA plasmid as carrier (Addgene plasmid ID:53188), single expression gRNA.By 1ug
PhU6-gRNA BbsI restriction enzyme digestion, after recycling digestion products, and the double-strand short dna product of (1) middle synthesis and formation of annealing
Mixing, is attached reaction.Connection product is converted later, expands plasmid, it is stand-by after recycling.
Embodiment 2
Using human serum albumin gene as foreign gene, donor plasmid is constructed.
1, homologous reparation donor plasmid is constructed.Homologous donor DNA successively by upstream homology arm, exogenous gene expression frame and under
It includes one section of First Intron sequence that trip homology arm, which is sequentially connected in exogenous gene expression frame, to completion ovalbumin gene
First Intron sequence；Human serum albumin ALB gene removes original signal peptide, adds chicken lysozyme signal peptide.One section of BGH
PolyA sequence protects mRNA structure.
(1) uses single stranded DNA as homologous recombination repair template, and upstream 90bp is as upstream at genome broken site
Homology arm, downstream 90bp take antisense strand to carry out as template artificial synthesized, operation chart is as schemed as downstream homology arm
Show, specific single-stranded DNA templates sequence is as shown in SEQ.2；
SEQ.2
(2) use double-stranded DNA as homologous recombination repair template, circular vectors or linearized vector or PCR product
, upstream and downstream 300bp-1000bp is as upstream homology arm and downstream homology arm at genome broken site, in the present embodiment
1000bp or more homology arm is selected, with pBluescript II KS (+) for carrier framework, design primer is respectively used for amplifying
Lower homology arm；Design primer expands human serum albumin gene, and upstream primer has chicken lysozyme signal peptide sequence, and downstream has 6
× His sequence label；Design primer expands the later First Intron sequence of cleavage site, and downstream primer has 15bp chicken bacteriolyze
Enzyme signal peptide homologous sequence；Design BGH polyA downstream primer；Primer is as shown in the table, and underscore is 15bp homologous sequence；Tiltedly
Body is lysozyme of chicken sequence, and overstriking is 6 × His sequence label.Primer sequence is as follows:
Expand homologous upper arm and homologous lower arm respectively using primer, using the DF-1 cell genomic dna of extraction as template, into
Row PCR amplification, PCR reaction system are as follows
PCR amplification program: 95 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 68 DEG C of extension 20s, 35 are followed
72 DEG C of extension 2min, last 4 DEG C of heat preservations after ring.It is produced with QIAquick Gel Extraction Kit QIAquick Gel Extraction Kit recycling PCR
The homologous upper arm of object and homologous lower arm are sequenced correct rear stand-by.
First Intron sequence after expanding cleavage site using primers F-intron1/R-intron1, with the DF- of extraction
1 cell genomic dna is template, carries out PCR amplification；PCR amplification program are as follows: 95 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s, 68
DEG C extend 30s, 72 DEG C of extensions 2min after 35 circulations, last 4 DEG C keep the temperature.It is returned with QIAquick Gel Extraction Kit
It receives kit and recycles PCR product intron1, be sequenced correct rear stand-by.
Expand human serum albumin using primers F-ssALB/R-ALB, with the ALB plasmid pGEM-ALB of purchase (justice is stuck up, Cat:
HG10968-G) it is template, carries out PCR amplification；PCR amplification program are as follows: 95 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s, 58 DEG C are moved back
Fiery 30s, 68 DEG C of extension 20s, 72 DEG C of extensions 2min after 35 circulations, last 4 DEG C keep the temperature.With QIAquick Gel
Extraction Kit QIAquick Gel Extraction Kit recycles PCR product ssALB, is sequenced correct rear stand-by.
By the carrier pcDNA of purchaseTM4/TO(InvitrogenTM, Catalog number:V102020) and use EcoRV enzyme
Digestion, digestion system are as follows:
37 DEG C of environment are placed 2 hours, recycle digestion products with QIAquick Gel Extraction Kit QIAquick Gel Extraction Kit
For use.
By PCR product ssALB and pcDNATMThe mixing of 4/TO digestion purified product, uses LigaFastTM Rapid DNA
Ligation System is attached reaction, places 1 hour at room temperature, reaction system is as follows:
It then carries out plasmid conversion and extracts plasmid, according to QIAGEN EndoFree Plasmid Midi Kit kit
Operating procedure extracts plasmid in specification, obtains connection product pcDNA4-ssALB, stand-by after digestion identification successful connection.
Human serum albumin expression cassette sequence is expanded using primers F-ssALB/R-BGH, with the plasmid pcDNA4- voluntarily constructed
SsALB is template, carries out PCR amplification；PCR amplification program are as follows: 95 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s, 60 DEG C of annealing 30s,
68 DEG C of extension 20s, 72 DEG C of extensions 2min after 35 circulations, last 4 DEG C keep the temperature.With QIAquick Gel Extraction Kit
QIAquick Gel Extraction Kit recycles PCR product ssALB-BGH, is sequenced correct rear stand-by.
Carrier pBluescript II KS (+) is used into the digestion of EcoRV enzyme, digestion system is as follows:
37 DEG C of environment are placed 2 hours, with kit NucleoSpin Gel and PCR Clean-up (MACHREY-
NAGEL) recycling digestion products are stand-by.
The homologous upper arm that PCR amplification is obtained, homologous lower arm, intron sequences and human serum albumin table after cleavage site
Mixed up to frame PCR product ssALB-BGH with pBluescript II KS (+) digestion products, using seamless Cloning Kit (HD Cloning Kit, Clontech, Code.639633) carry out homologous recombination.
It is as follows that homologous recombination operates reaction system:
After mixing, it reacts 15 minutes for 50 DEG C, places later on ice.According to QIAGEN EndoFree Plasmid
Operating procedure extracts plasmid in Midi Kit kit specification, obtains connection product pBlue-ssALB homologous donor, digestion mirror
Fixed connection is stand-by after striving for.
2, non-homogeneous reparation donor plasmid is constructed
PAM sequence and target site sequence form a cleavage site frame, and the reverse complementary sequence of cleavage site frame is reversed
Complementary cuts site frame, there are one or two reverse complemental cleavage site frames in donor vehicle, with closed circular form plasmid or line
Property DNA form exist.
Reverse complemental cleavage site frame sequence in this example is 5'-CCACGTGTGTGAGTATACGCACA-3', whereinCCA
For the PAM sequence in reverse complemental cleavage site frame.When, there are a reverse complemental cleavage site frame, claiming this confession in donor plasmid
Constitution grain is " 1 cuts donor plasmid "；When, there are two reverse complemental cleavage site frames, this donor plasmid is referred to as " 2 in donor plasmid
Cut donor plasmid ".
(1) when donor plasmid is cut in building 1, design primer expands human serum albumin expression cassette, and the upstream end 5' is anti-with one
To complementary cuts site frame, it is as shown in the table for sequence, and underscore is reverse complemental cleavage site frame.
Human serum albumin expression cassette sequence is expanded using primers F -1/cut-ALB and R-BGH, with the plasmid voluntarily constructed
PBlue-ssALB homologous donor is template, carries out PCR amplification；PCR amplification program are as follows: 95 DEG C of initial denaturation 1min；98 DEG C of denaturation
10s, 60 DEG C of annealing 30s, 68 DEG C of extension 20s, 72 DEG C of extensions 2min after 35 circulations, last 4 DEG C keep the temperature.With QIAquick Gel
Extraction Kit QIAquick Gel Extraction Kit recycles PCR product 1cut/ssALB-BGH, is sequenced correct rear stand-by.
PCR product 1cut/ssALB-BGH is mixed with pBluescript II KS (+) EcoRV digestion products, is used
LigaFastTMRapid DNA Ligation System is attached reaction, places 1 hour at room temperature, reaction system is as follows:
It then carries out plasmid conversion and extracts plasmid, according to QIAGEN EndoFree Plasmid Midi Kit kit
Operating procedure extracts plasmid in specification, obtains 1 and cuts donor plasmid pBlue-1cut/ssALB, after digestion identification connection is correct to
With.
(2) when donor plasmid is cut in building 2, design downstream primer expands human serum albumin expression cassette, and the downstream end 5' has one
A reverse complemental cleavage site frame, it is as shown in the table for sequence, and underscore is reverse complemental cleavage site frame.
Human serum albumin expression cassette sequence is expanded using primers F -1/cut-ALB and R-2/cut-ALB, with what is voluntarily constructed
Plasmid pBlue-ssALB homologous donor is template, carries out PCR amplification；PCR amplification program are as follows: 95 DEG C of initial denaturation 1min；98 DEG C of changes
Property 10s, 68 DEG C of extension 30s, 35 circulation after 72 DEG C of extensions 2min, it is last 4 DEG C keep the temperature.With QIAquick Gel
Extraction Kit QIAquick Gel Extraction Kit recycles PCR product 2cut/ssALB-BGH, is sequenced correct rear stand-by.
PCR product 2cut/ssALB-BGH is mixed with pBluescript II KS (+) EcoRV digestion products, is used
LigaFastTMRapid DNA Ligation System is attached reaction, places 1 hour at room temperature, reaction system is as follows:
It then carries out plasmid conversion and extracts plasmid, according to QIAGEN EndoFree Plasmid Midi Kit kit
Operating procedure extracts plasmid in specification, obtains 2 and cuts donor plasmid pBlue-2cut/ssALB, after digestion identification connection is correct to
With.
It cuts donor plasmid pBlue-1/cut-ALB and 2 and cuts donor plasmid pBlue-2/cut-ALB in 1 obtained.Donor plasmid
Structural schematic diagram is as shown in Fig. 2.
Embodiment 3
Subgerminal cavity microinjection
(1) donor plasmid 1 that will be obtained in the gRNA expression plasmid px330-gRNA obtained in embodiment 1 and embodiment 2:
1 mixing, plasmid concentration are diluted to 1ug/ul with DMEM；The purchase of SPF fertile egg is used in Japanese Chuan Xian Co., Ltd., fresh fertile egg
95% solution actually is wiped and is dried, the circular ring marks for being about 3 centimetres with one diameter of pencil drawing in the small head end of fertile egg, circle
Plane of a loop and fertile egg cross section keeping parallelism；Injection needle is prepared using needle stretch machine (NARISHIGE, PC-100), is prepared
Parameter is 76 DEG C of fusing, stretches 25 DEG C, and grinding needle point makes needle point angle on needle grinding machine (NARISHIGE, EG-401)
60°.The injection needle prepared dries after brewed 24 hours in 95% alcoholic solution, and irradiation-sterilize in the UV lamp
2 hours；(Minimo, H021) cuts eggshell along annulus with a chainsaw, and control makes electric saw in cutting process not enter eggshell
Interior agitation egg white.After removing small head end eggshell, fertile egg and egg white are poured into the experiment evaporating dish after sterilizing, then in entity
It is operated under microscope (Olympus, SZH10), is infused using microinjection instrument (eppendorf, transjector 5246)
Enter mixed solution, is injected successfully when blastodisc central, clear area reddens.Using electroporation apparatus (NEAP, type II) to chicken embryo into
Row point perforation procedure, it is as shown in the table for Electroporation parameters, and after electroporation experiment operates, fertilization egg yolk and egg liquid are refunded egg
It in shell, is sealed and is sealed using ventilated membrane, be subsequently placed into incubator hatching.
After hatching 3 days, fertile egg is carried out to change shell operation, take the common egg not less than former eggshell, 95% alcohol is molten
Liquid is wiped and is dried, the circular ring marks for being about 4 centimetres with one diameter of pencil drawing in eggshell stub end, circular planes and fertile egg
Cross section keeping parallelism removes stub end eggshell along annulus cutting eggshell with a chainsaw, the fertile egg hatched 3 days is slowly shifted
Into the new eggshell of removal stub end, appropriate preservative film sealing sealing after supplementing egg white solution is put into incubator until hatching and nestling.
Incubation condition be 1-3 days 38.5 DEG C, humidity 55%, egg-turning is primary within 90 minutes；4-21 days 37.2 DEG C, humidity 60%.
(2) donor plasmid 1 that will be obtained in the gRNA expression plasmid px330-gRNA obtained in embodiment 1 and embodiment 2:
1 mixing, plasmid concentration are diluted to 2ug/ul with DMEM；50ul plasmid mixed solution is taken, 100ul is diluted to opti-MEM, takes
50ul 2000 and it is diluted to 100ul with opti-MEM, is placed at room temperature for after five minutes, the two is mixed equal
It is placed at room temperature for after even stand-by after twenty minutes；Fresh fertile egg is wiped and is dried with 95% alcoholic solution, with a chainsaw along annulus
Eggshell is cut, fertile egg and egg white are poured into the experiment evaporating dish after sterilizing, injects mixed solution using microinjection instrument, when
It is injected successfully when reddening in blastodisc central, clear area.After injecting successfully, fertilization egg yolk and egg liquid are refunded in eggshell, using saturating
Air film sealing sealing, is subsequently placed into incubator hatching.
Three days embryonic development figures are as shown in Fig. 3 after sealer.
After hatching 3 days, fertile egg is carried out to change shell operation, changes preservative film sealing sealing after shell, be put into incubator until incubating
Dissolve young bird.Incubation condition be 1-3 days 38.5 DEG C, humidity 55%, egg-turning is primary within 90 minutes；4-21 days 38 DEG C, humidity 60%.
Embodiment 4
PGCs mediated method
(1) building has the donor plasmid of resistant gene: design downstream primer is drawn to expand Zeocin gene expression frame
It is as shown in the table for object sequence, uses primers F -1/cut-ALB and R-Zeocin amplification human serum albumin expression cassette and Zeocin gene
Expression cassette sequence carries out PCR amplification using the plasmid pcDNA4-IRES/ssALB voluntarily constructed as template；PCR amplification program are as follows:
95 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s, 68 DEG C of extension 30s, 72 DEG C of extensions 2min after 35 circulations, last 4 DEG C keep the temperature.With
QIAquick Gel Extraction Kit QIAquick Gel Extraction Kit recycles PCR product 1cut/IRES-ssALB-Zeocin, and sequencing is just
It is stand-by after really.
PCR product 1cut/IRES-ssALB-Zeocin and pBluescript II KS (+) EcoRV digestion products is mixed
It closes, uses LigaFastTMRapid DNA Ligation System is attached reaction, places 1 hour at room temperature, reactant
It is as follows:
It then carries out plasmid conversion and extracts plasmid, according to QIAGEN EndoFree Plasmid Midi Kit kit
Operating procedure extracts plasmid in specification, obtains 1 and cuts donor plasmid pBlue-1cut/IRES-ssALB-Zeocin, digestion identification
It is stand-by after connection is correct.
(2) PGCs cell obtains
SPF fertile egg is bought in Japanese Chuan Xian Co., Ltd., and fresh fertile egg is wiped and dried with 95% alcoholic solution,
Small head end is put into incubator upward and is hatched, and 38.5 DEG C, 55% condition of humidity is hatched 48 hours to 50 hours, and embryonic development is arrived
After the 13-14 phase, takes out the small head end of fertile egg and place upward, gently chisel a hole in small head end with tip tweezers, then use
Tweezers slowly remove eggshell, so that being open substantially in 3cm or so, and it is observed that entire chicken embryo.It takes ground and disinfects
Glass needle with removal needle point syringe be connected, glass needle along chicken embryo vessel directions piercing, be pierced into successfully moment have it is micro-
Amount blood, which enters glass needle, makes needle point take on a red color, the subsequent slow suction blood of syringe；It can be operated in chicken embryo blood vessel many places,
More blood are extracted as far as possible.
(3) PGCs cell culture
Culture medium is configured to cultivate PGCs cell, under the culture medium, haemocyte can not be grown, thus in incubation
Heteroproteose cell is removed, PGCs is individually grown；Medium component such as following table, basal medium are birds Knockout DMEM (Life
Technology)。
It takes 3ul chicken embryo blood to be added in the configured culture medium of 1000ul, is added in 24 orifice plates, be not necessarily to feeder cells.
The culture medium of every two days replacement one thirds, culture cell quantity to 2 × 105When, replace whole culture mediums.
(4) PGCs cell transfecting
By the donor plasmid pBlue- in the gRNA expression plasmid px330-gRNA and the present embodiment that are obtained in embodiment 1
1cut/IRES-ssALB-Zeocin is mixed in 1:1 ratio, is taken 50ul plasmid mixed solution, is diluted to 100ul with opti-MEM,
Take 50ul2000 and it is diluted to 100ul with opti-MEM, is placed at room temperature for after five minutes, then the two is mixed
It is placed at room temperature for after closing uniformly stand-by after twenty minutes；Liposome and nucleic acid mixed liquor are added to containing 1 × 105The training of PGCs cell
It supports in ware, after 37 DEG C hatch 6 hours, culture medium is replaced after centrifugation, 6 orifice plates is transferred to and continues to cultivate.It is added in culture medium after 2 days
Zeocin makes its concentration 50ug/ml.Continue culture in the environment containing Zeocin more than 2 week.
(5) PGCs cell is injected again
SPF fertile egg is bought in Japanese Chuan Xian Co., Ltd., and fresh fertile egg is wiped and dried with 95% alcoholic solution,
Small head end is put into incubator upward and is hatched, and 38.5 DEG C, 55% condition of humidity is hatched 48 hours to 50 hours, and embryonic development is arrived
After the 13-14 phase, takes out the small head end of fertile egg and place upward, gently chisel a hole in small head end with tip tweezers, then use
Tweezers slowly remove eggshell, so that being open substantially in 3cm or so, and it is observed that entire chicken embryo.It takes ground and disinfects
Glass needle with removal needle point syringe be connected, the PGCs cell that glass needle absorption is isolated and purified with fresh culture, glass
Needle is pierced into along chicken embryo vessel directions, and syringe is slowly advanced after being pierced into successfully；Culture solution containing PGCs is pushed into chicken embryo blood
Guan Zhong.After the completion of operation, chicken embryo is put into incubator by sealer hatches, and 38.5 DEG C of incubation condition temperature, 50%-60%'s is wet
Degree, until hatching and nestling.
Those skilled in the art is not under conditions of departing from the spirit and scope of the present invention that claims determine, also
Various modifications can be carried out to the above content.Therefore the scope of the present invention is not limited in above explanation, but by
The range of claims determines.
Claims (10)
1. a kind of preparation method of transgenic chicken oviduct bioreactor, which comprises the following steps:
S1. the chicken genome target DNA sequence dna of the gRNA: selection of design targeting chicken genomic DNA is that ovalbumin first includes
Son selects the gRNA of 90% or more homology according to the different PAM sequence rules of variety classes Cas9 albumen；
S2. construct the expression vector of gRNA: after determining target site sequence according to PAM rule, synthesis meets expression vector establishment and wants
The positive and negative DNA of the target site sequence asked is single-stranded, and annealing is connected on CRISPR/Cas system expression carrier after forming DNA double chain,
GRNA and Cas albumen can be expressed after vectors into cells；
S3. building carries the donor vehicle of exogenous gene expression frame: the donor vehicle includes homologous repair vector and non-same
Source repair vector；
S31. the donor vehicle of the homologous reparation is constructed: using single stranded DNA as homologous recombination repair template, upper and lower homologous sequence
With 45bp-90bp；It is or using double-stranded DNA as homologous recombination repair template, upstream and downstream at the DSB of target dna site is each
100bp-1000bp sequence is connected to the end 5' and the end 3' of foreign gene, and as homology arm, donor vehicle can be with closed loop
Plasmid or PCR product or the form of linear DNA exist；
S32. construct the donor vehicle of the non-homogeneous reparation: PAM sequence and target site sequence form a cleavage site frame, cut
The reverse complementary sequence for cutting site frame is reverse complemental cleavage site frame, and there are one or two reverse mutuals in donor vehicle
Cleavage site frame is mended, is existed in the form of closed circular form plasmid or linear DNA；
S4. by vector introduction chicken individuals: will be prepared in any gRNA/Cas9 prepared in step S2 combination and step S3
Donor vehicle after mixing, the subgerminal cavity being injected into the fertile egg in the chick embryo development X phase, then to injection after
Chicken embryo carries out electroporation；Or the confession that will be prepared in any gRNA/Cas9 prepared in step S2 combination and step S3
Body carrier is mixed with nucleic acid transfection reagent in proportion after mixing, by plasmid mixed liquor, is injected into the chicken embryo fertilization of X phase later
In egg；Then hatching is until hatching.
2. a kind of preparation method of transgenic chicken oviduct bioreactor according to claim 1, which is characterized in that described
PAM type includes 5'-NNN-3', 5'-NNNN-3', 5'-NNNNN-3', 5'-NNNNNN-3', 5'-NNNNNNN-3' and 5'-
NNNNNNNN-3'。
3. a kind of preparation method of transgenic chicken oviduct bioreactor according to claim 1, which is characterized in that described
Step S2 can also be realized by the following method: it is building up on existing gRNA expression vector: individually building gRNA expression plasmid,
Promoter uses U6, the Polll polymerase such as T7, with Cas9 expression plasmid or Cas9 albumen cotransfection to target cell.
4. a kind of preparation method of transgenic chicken oviduct bioreactor according to claim 1, which is characterized in that step
Exogenous gene expression frame described in S3 includes one section of completion ovalbumin gene First Intron sequence, and one section makes Protein secretion
To extracellular signal peptide sequence, the global DNA sequence of one section of arbitrary target foreign protein and one section of protection mRNA stability
PolyA sequence.
5. a kind of preparation method of transgenic chicken oviduct bioreactor according to claim 4, which is characterized in that described
The site that no longer will be identified and cut containing selected gRNA after completion ovalbumin gene First Intron sequence completion.
6. a kind of preparation method of transgenic chicken oviduct bioreactor according to claim 4, which is characterized in that described
One section of completion ovalbumin gene First Intron sequence is to terminate since selected gRNA cleavage site to First Intron
Sequence constitutes complete ovalbumin gene First Intron sequence together with cleavage site upstream sequence.
7. a kind of preparation method of transgenic chicken oviduct bioreactor according to claim 4, which is characterized in that described
Signal peptide sequence is original signal peptide sequence, chicken source lysozyme signal peptide, the chicken source Ovotransferrin signal of target foreign protein
The signal peptide of peptide, chicken source class ovum mucoglobulin signal peptide or engineer.
8. a kind of preparation method of transgenic chicken oviduct bioreactor according to claim 4, which is characterized in that described
Arbitrary target foreign protein is human serum albumins, interleukins, human blood coagulation, interferon, tumor necrosis factor, colony
Stimulating factor, growth factor, chemotactic cytokine, recombinant antibodies or other protein moleculars.
9. a kind of preparation method of transgenic chicken oviduct bioreactor according to claim 4, which is characterized in that described
PolyA sequence be using the original polyA sequence of chicken egg white, additional addition BGH polyA sequence, SV40polyA sequence or
Other polyA sequences of person.
10. a kind of preparation method of transgenic chicken oviduct bioreactor according to claim 1, which is characterized in that institute
Stating S4 step can also be realized by the following method: will be in any gRNA/Cas9 that prepared in step S2 combination and step S3
The donor vehicle prepared after mixing, is imported by electroporation or nucleic acid transfection reagent and is isolated and purified from chicken embryo
To PGC cell in, by the PGCs cell of drug screening foreign gene success quiding gene group, then the PGCs of screening is infused
It is mapped in the 15th phase chicken embryo blood vessel.
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