CN108424929A - The Cas9/sgRNA coexpression vectors and its construction method of pig MC1R genes and application - Google Patents
The Cas9/sgRNA coexpression vectors and its construction method of pig MC1R genes and application Download PDFInfo
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- CN108424929A CN108424929A CN201810063376.5A CN201810063376A CN108424929A CN 108424929 A CN108424929 A CN 108424929A CN 201810063376 A CN201810063376 A CN 201810063376A CN 108424929 A CN108424929 A CN 108424929A
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Abstract
The present invention provides the Cas9/sgRNA coexpression vectors of a boar MC1R genes, it includes the sgRNA of pX458 carriers and the targeting pig MC1R genes being connected on pX458 carriers, targets the nucleotide sequence of the sgRNA of pig MC1R genes as shown in SEQ ID NO.1 and SEQ ID NO.2.The present invention can realize accurate genetic modification in the genome of pig, while not introduce any exogenous DNA array, retain that its meat is excellent, the merits such as in good taste, efficiently avoid the issuable bio-safety risk of traditional transgenic technology.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to the structure of the Cas9/sgRNA coexpression vectors of pig MC1R genes
Method and its application.
Background technology
In the hair color field for changing pig kind, generally use is different cultivars intermolecular hybrid method, the implementation steps of the technology
To be that female parent carries out breeding acquisition hybridization first filial generation with another pig as male parent using a boar, then repeatedly returned with female parent
Hand over fixed hair color phenotype.However, can also change its meat and reproductive capacity etc. while changing a certain pig kind hair color, other are economical
Character, character mutation result are difficult to control.This is because changing a certain pig kind hair color by traditional crossbreeding technology
Meanwhile the genome because introducing another boar kind, the expression of most genes can be changed, therefore its except hair color can be changed
His important economical trait.
Invention content
The purpose of the present invention is being directed to the above technical problems to be solved, change bristles color can be effectively orienting by providing one kind
Technical solution.
In order to achieve the goal above, the present invention provides the Cas9/sgRNA coexpression vectors of a boar MC1R genes,
SgRNA, the targeting pig MC1R including pX458 carriers and the targeting pig MC1R genes being connected on the pX458 carriers
The nucleotide sequence of the sgRNA of gene is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Preferably, the pig is Guangdong and Guangxi Provinces little Hua pigs.
The present invention also provides a kind of host cells comprising the Cas9/sgRNA coexpressions of above-mentioned pig MC1R genes carry
Body.
The present invention also provides the construction methods of the Cas9/sgRNA coexpression vectors of pig MC1R genes comprising following step
Suddenly：
(1) sgRNA of targeting pig MC1R genes, the nucleotide of the sgRNA of the targeting pig MC1R genes are designed and synthesized
Sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；
(2) phosphorylation and procedural is carried out to positive and negative two oligonucleotide chains of the sgRNA of the targeting pig MC1R genes
Annealing, is allowed to form the double-stranded DNA short-movie section with I corresponding cohesive terminus,cohesive terminis of Bbs；
(3) I digestion pX458 carriers of Bbs are used, the plasmid of linearisation returns kit after electrophoresis, by glue and recycled；
(4) pX458 for using T4 ligases to be linearized the double-stranded DNA short-movie section that step (2) obtains with step (3)
Carrier is attached, and then converts connection product into DH5 α bacterium competent cells, and the LB for being applied to ammonia benzyl resistance is flat
On plate, bacterial colony is finally selected, identifies whether the sgRNA sequences of targeting pig MC1R genes are properly inserted by being sequenced
In pX458 carriers, selects correctly clone and shake bacterium culture, the Cas9/sgRNA coexpression vectors of extraction targeting pig MC1R genes.
In the construction method, it is preferable that the pig is Guangdong and Guangxi Provinces little Hua pigs.
The present invention also provides Cas9/sgRNA coexpression vectors the answering for changing bristles color of above-mentioned pig MC1R genes
With.
The present invention also provides the Cas9/sgRNA coexpression vectors of above-mentioned pig MC1R genes to prepare for changing pig hair
Application in terms of the reagent of color.
The present invention targets the CRISPR/Cas9 expression vectors of pig MC1R genes by structure, and transfection Guangdong and Guangxi Provinces little Hua pigs are newborn
The nephrocyte of piglet extracts genome T7E1 restriction analysis and the analysis of PCR product TA cloning and sequencings, has detected CRISPR/Cas9
To editor's activity of MC1R.Its editorial efficiency is being verified up to after 45% or more, the EGFP positives are enriched to by flow sorting techniques
Cell builds gene editing clone embryos as donorcells, by somatic cell nuclear transfer technique, and is further moved by embryo
Operation is implanted into the fallopian tubal of replace-conceive large white sow, and the MC1R that white hair color is obtained after normal pregnancy edits Guangdong and Guangxi Provinces little Hua
Pig.
The MC1R genes of present invention application CRISPR/Cas9 technical editors Guangdong and Guangxi Provinces little Hua pigs, can be in Guangdong and Guangxi Provinces little Hua pigs
Accurate genetic modification is realized in genome, while not introducing any exogenous DNA array, maintains Guangdong and Guangxi Provinces little Hua pig genomes
" purity ", retains that its meat is excellent, the merits such as in good taste, efficiently avoids the issuable life of traditional transgenic technology
Object security risk.In addition, the present invention changes the Coat Color of Chinese native pig breed using gene editing oriented approach, to avoid
In conventional hybridization breeding process while changing Coat Color, the blood relationship of external pig kind is introduced, place of china pig is affected
The outstanding characters such as the original excellent pork quality of kind and resistance are strong.This meets the new hair color of the different market demands for cultivating
Guangdong and Guangxi Provinces little Hua pigs have important breeding meaning.
Description of the drawings
Fig. 1 is the collection of illustrative plates schematic diagram of Cas9/sgRNA coexpression vectors pX458.
Fig. 2 shows gene editing efficiency of the T7E1 and TA cloning and sequencings analysis MC1R-sgRNA-1 in PK cells.
Fig. 3 shows gene editing efficiency of the TA cloning and sequencings detection MC1R-sgRNA-1 in clone embryos.
Fig. 4 is the hair color comparing result of wild type and MC1R gene editing types Guangdong and Guangxi Provinces little Hua pigs.
Fig. 5 is the colour of skin comparing result of wild type and MC1R gene editing types Guangdong and Guangxi Provinces little Hua pigs.
Specific implementation mode
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention
Rather than the application range of the limitation present invention.It is such as unspecified, details parameter is not described in detail involved in embodiment but
What operation was well known to those skilled in the art.
Embodiment
1.MC1R gene target site selects
From NCBI download place of china pig MC1R full length gene sequences, using sgRNA Photographing On-lines software (http:// crispr.mit.edu/) analyzed, the sgRNA of 2 targeting pig MC1R genes are designed, the particular sequence of the sgRNA is shown in following
Table 1 and sequence table SEQ ID NO.1 and SEQ ID NO.2.
Table 1 targets pig MC1R gene sgRNA sequence informations
Note：PAM (protospacer-adjacent motif) refers to sgRNA binding motifs.
2. targeting the Cas9/sgRNA coexpression vectors structure of pig MC1R genes
The Cas9 albumen and reporter gene that pX458 carriers can express sgRNA simultaneously, codon optimizes through humanized
EGFP, collection of illustrative plates is as shown in the figure (Fig. 1).According to the 2 sgRNA sequences designed above, it is positive and negative single-stranded to synthesize corresponding oligonucleotides,
Every single-stranded upper added with I corresponding cohesive terminus,cohesive terminis of Bbs.I digestions of Bbs, 1 μ g pX458 carriers, the plasmid of linearisation are used first
After electrophoresis, kit is returned by glue and is recycled.Meanwhile to positive and negative two oligonucleotide chains of sgRNA carry out phosphorylation and
Procedural annealing is allowed to be formed the double-stranded DNA short-movie section with cohesive terminus,cohesive termini, using T4 ligases by itself and linearisation
PX458 carriers are attached, and are then converted connection product into DH5 α bacterium competent cells, and be applied to ammonia benzyl resistance
On LB tablets, bacterial colony is finally selected, identifies whether sgRNA sequences are properly inserted into pX458 carriers by sequencing,
It selects correctly clone and shakes bacterium culture, the Cas9/sgRNA coexpression vectors of extraction targeting pig MC1R genes.
3. Guangdong and Guangxi Provinces little Hua porcine kidney cells culture and transfection
Guangdong and Guangxi Provinces little Hua porcine kidney cells (porcine kidney cell, PK cell) are detached from the kidney of newborn piglet and are built
It is vertical.PK cells are in the DMEM culture mediums containing 20% fetal calf serum and dual anti-(100U/mL penicillin and 100mg/mL streptomysins)
Middle growth.A subculture is replaced in cell culture after 1-2 days.When the degree of converging of PK cell growths to 80% or so, PBS is first used
Washing twice, then with pancreatin digest 2min, then be added serum terminate digestion, by postdigestive cell collect to 15mL from
Heart pipe is centrifuged, and is abandoned after supernatant and is washed second of centrifugation of a progress with PBS again.Supernatant is abandoned after centrifugation, is then used appropriate
Cell is resuspended in Buffer R solution, and cell density is made to reach 1.0 × 107Cell/mL, uses NeonTMTransfection system is to PK cells
Carry out electric shock transfection.Transfection conditions are：Voltage：1600V, width：40ms, pulse number：3.24 h replace training after cell transfecting
Base is supported, addition is dual anti-, in 5% CO2, cultivated under the conditions of 37 DEG C.Flow cytometer showed is carried out after 3d, sorts or collect cell
It extracts DNA and carries out subsequent experimental.
4.T7E1 is tested
Digestion is carried out using T7E1, designs suitable primer such as following table 2 and SEQ ID NO.3 and SEQ ID NO.4
It is shown.
Primer is tested in 2 T7E1 digestions of table
T7E1 enzymes (T7 endonuclease I) can be used for detecting the gene mutation of CRISPR/Cas9 mediations, positive gram of screening
It is grand.It is further cultured for after one week, is extracted using tissue/cell extracts kit thin after PK cells 3d after transfection, or sorting
Born of the same parents' genome goes out the MC1R genetic fragments comprising sgRNA recognition sites, using Axygen PCR cleaning agents by PCR amplification
Box purified pcr product.PCR product after purification is first denaturalized at high temperature, then anneals to form heteroduplex through Programmed cryopreservation
DNA, carry out digestion to above-mentioned product using T7E I makes abrupt climatic change site be located at PCR segments by the primer of above-mentioned design
Asymmetric point on, can be distinguished in polyacrylamide gel according to stripe size difference after digestion, finally by
Image J softwares calculate the cutting efficiency of CRISPR/Cas9, estimate its activity of practicing shooting.
5. sequencing analysis
The MC1R genetic fragments for including sgRNA institutes recognition site are amplified using high-fidelity Taq enzyme, utilize Ago-Gel
The segment is separated by electrophoresis, then returns kit with glue and recycles segment, the segment of recycling is cloned into pMD 18-T carriers, is converted
After select several single bacterium colonies, carry out the efficiency of sequencing analysis CRISRP/Cas9 mutagenesis at cleavage site.
6. flow cytometry and sorting
First with the adherent PK cells of 2% trypsin digestion, then it is resuspended into individual cells with PBS, via hole diameter is 50 μ
The nylon membrane of m is filled into streaming pipe, and the Fluorescence Ratio and intensity of cell are analyzed on stream type cell analyzer.Using streaming
Cell sort instrument sub-elects EGFP positive cells, is seeded in 12 orifice plates and continues to cultivate.
7. the In-vitro maturation of egg mother cell
Sow ovary is acquired from slaughterhouse, the content in the ovarian follicle of a diameter of 3-5mm of Ovarian surface is extracted with syringe,
Content is diluted and is resuspended to form suspension in TL-PVA.Suspension is stood under 37 DEG C of condition of culture to particle
The egg mother cell of cell is settled down to culture dish bottom completely, and precipitation is sucked out to be placed under stereoscope and is chosen with pipettor or mouth suction pipe
Select the complete egg mother cell of ovum pericyte.The healthy egg mother cell selected is put into containing 10% liquor folliculi, FSH, LH, EGF's
22h is cultivated in TCM- 199.Egg mother cell is moved on to containing 10% liquor folliculi, the TCM-199 of EGF with pipettor or mouth suction pipe again
In continue cultivate 22h.Selected after 44h cultures are ripe have been drained off second polar body healthy mature egg mother cell it is spare.
8. body-cell neucleus transplanting operates
First with the adherent PK cells of 2% trypsin digestion, then cell is uniformly dispelled and is added in DMEM culture solutions
It is spare.The healthy mature porcine oocytes for cultivating 44h are moved on into the operation of the nuclear transfer containing hyaluronidase and cytochalasin
In liquid, removal granular cell is gently blown and beaten with pipettor, selects the egg mother cell progress nuclear transfer for being developed to the M II phases.It answers
With micromanipulation instrument, determine needle by holding egg mother cell is sucked, and using injection needle by the polar body and nucleus in egg mother cell
It is sucked out, then draws the single body cell digested with injection needle, pierce through oolemma, be injected into non-nucleus egg mother cell
In perivitelline, so that body cell and egg mother cell is merged by electro photoluminescence, is placed in PZM-5 culture solutions in 37 DEG C of incubators
Observation fusion situation after middle culture 2h.
9. embryo transfer
First receptor sow inject containing 0.025% yellow Jackets by ear vein behind Baoding with restrainer
Physiological saline, anaesthesia dosage are that per kilogram of body weight injects 1ml.It is first molten with 0.5% potassium permanganate after the anesthesia completely of receptor sow
Liquid cleans body surface, and in body side shaving, reuses medical Iodophor and medicinal alcohol disinfection body surface.From receptor sow side into
Knife and blunt separation tissue and peritonaeum extract ovary and fallopian tubal out from abdominal cavity.Suction clone embryos are taken with embryo needle is moved, from fallopian tubal
Umbrella portion is put it into fallopian tubal, is resetted ovary and fallopian tubal, is finally sewed up a wound and sterilize.
10. the nursing and midwifery of receptor sow
After operation three days continuous to receptor sow injection cefalexin injection prevent wound infection, after two weeks to wound into
Row is taken out stitches.Observe whether receptor sow returns feelings, 30 days pregnancy situations that receptor sow is monitored by B ultrasound after 24 days.113 days right
Receptor sow oxytocin injection is hastened parturition, and 16-24h starts to produce piglet after injection, and every piglet production interval is more than 40min
It needs to carry out artificial aiding-birth, can be taken by injecting prostaglandin or artificial draw.
Editorial efficiency qualification result
CRISPR/Cas9 editorial efficiencies are identified in 1.PK cells and in clone embryos
After the sgRNA/Cas9 expression vectors for being inserted into targeting MC1R genes are transferred to PK cells by electrotransfection, pass through streaming
The method of sorting out cultivates the cell sorting for expressing EGFP, and part cell is used for body-cell neucleus transplanting, remaining cell extraction
The efficiency of T7E1 and TA clone's assessment gene editings is carried out after genomic DNA.The clone embryos obtained by body-cell neucleus transplanting,
The overwhelming majority is used for embryo transfer, and remaining clone embryos are placed in PZM-5 culture solutions, and 7 days are cultivated in 37 DEG C of incubators extremely
Each blastaea is collected into different EP pipes and carries out PCR amplification and TA cloning and sequencings by blastula stage respectively, identification gene editing effect
Rate.
T7E1 abrupt climatic changes are the result shows that the activity of 2 sgRNA is chosen after screening and knocks out that efficient (editorial efficiency is
45.7%) the gene editing efficiency of MC1R-sgRNA-1 is up to 45.7%, TA cloning and sequencings the result shows that at 10 of acquisition
In PCR clones, what MC1R-sgRNA-1 target sites mutated has 6, and gene editing efficiency is 60% (Fig. 2)；In summary
As a result, gene editing efficiency of the MC1R-sgRNA-1 in PK cells is higher, it can be used for subsequent experimental.TA cloning and sequencings are analyzed
The clone embryos of blastula stage the result shows that in 10 cloned blastocysts of acquisition, what MC1R-sgRNA-1 target sites mutated
There are 4, gene editing efficiency is 40% (see Fig. 3).2. CRISPR/Cas9 editorial efficiencies in piglet of being born are identified
A small amount of tail portion sample extraction genomic DNA when the Guangdong and Guangxi Provinces little Hua pigs docking of birth is taken to carry out genotype identification, it is right
Each individual PCR amplification goes out MC1R genetic fragments, then carries out TA cloning and sequencings and determine gene editing efficiency, embryo transfer and birth
The statistical result of piglet editorial efficiency is as shown in the following Table 3.
3 embryo transfer of table and birth piglet editorial efficiency
Edit the hair color and the colour of skin of MC1R gene alterations Guangdong and Guangxi Provinces little Hua pigs：
The MC1R genes of newborn piglet are edited, affect the expression of pigment related gene, make " both ends crow " (head originally
Portion and tail portion, and major part portion hair color are black) Guangdong and Guangxi Provinces microtias spend pig to become the individual of entire body white hair (see Fig. 4).In addition, logical
It crosses and examines and can find, the skin at Guangdong and Guangxi Provinces little Hua pig black wools position is aobvious black originally, edited in MC1R genes, should
The skin at position also becomes white skin, while chloasma (see Fig. 5) is manifested on skin, similar MC1R genic mutation types
The red white dermal phenotype of hair.
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Claims (7)
1. the Cas9/sgRNA coexpression vectors of a boar MC1R genes, it is characterised in that including pX458 carriers and be connected to
The sgRNA of targeting pig MC1R genes on the pX458 carriers, the nucleotide sequence of the sgRNA of the targeting pig MC1R genes
As shown in SEQ ID NO.1 and SEQ ID NO.2.
2. Cas9/sgRNA coexpression vectors according to claim 1, which is characterized in that the pig is Guangdong and Guangxi Provinces little Hua pigs.
3. a kind of host cell comprising Cas9/sgRNA coexpression vectors according to claim 1 or 2.
4. the construction method of the Cas9/sgRNA coexpression vectors of pig MC1R genes comprising following steps：
(1) sgRNA of targeting pig MC1R genes, the nucleotide sequence of the sgRNA of the targeting pig MC1R genes are designed and synthesized
As shown in SEQ ID NO.1 and SEQ ID NO.2；
(2) phosphorylation and procedural annealing are carried out to positive and negative two oligonucleotide chains of the sgRNA of the targeting pig MC1R genes
Processing, is allowed to form the double-stranded DNA short-movie section with I corresponding cohesive terminus,cohesive terminis of Bbs；
(3) I digestion pX458 carriers of Bbs are used, the plasmid of linearisation returns kit after electrophoresis, by glue and recycled；
(4) the pX458 carriers for using T4 ligases to be linearized the double-stranded DNA short-movie section that step (2) obtains with step (3)
It is attached, then converts connection product into DH5 α bacterium competent cells, and be applied on the LB tablets of ammonia benzyl resistance,
Bacterial colony is finally selected, whether the sgRNA sequences by the way that the identification targeting pig MC1R genes are sequenced are properly inserted into institute
It states in pX458 carriers, selects correctly clone and shake bacterium culture, the Cas9/sgRNA coexpressions of extraction targeting pig MC1R genes carry
Body.
5. according to the method described in claim 4, it is characterized in that, the pig is Guangdong and Guangxi Provinces little Hua pigs.
6. Cas9/sgRNA coexpression vectors as claimed in claim 1 or 2 are for changing the application of bristles color.
7. Cas9/sgRNA coexpression vectors as claimed in claim 1 or 2 are in terms of preparing for changing the reagent of bristles color
Using.
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Application publication date: 20180821 |