CN104342457A - Method for targetedly integrating exogenous gene into target gene - Google Patents
Method for targetedly integrating exogenous gene into target gene Download PDFInfo
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- CN104342457A CN104342457A CN201410553171.7A CN201410553171A CN104342457A CN 104342457 A CN104342457 A CN 104342457A CN 201410553171 A CN201410553171 A CN 201410553171A CN 104342457 A CN104342457 A CN 104342457A
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Abstract
The invention discloses a method for targetedly integrating an exogenous gene into a target gene of a mammal, which is implemented by targetedly integrating an exogenous gene into a target gene by utilizing a CRISPR/Cas9 system, and especially targetedly integrating a Cre gene to a first exon of a mouse Enpp1 gene. The technique has the advantages of high integration efficiency (20%) and simple operation step, only needs to construct one exogenous gene vector, and can not be influenced by the position effect.
Description
(1) technical field
The invention belongs to genetically engineered field, specifically, relate generally to the construction process of foreign gene knock-in and over-express vector and rely on CRISPR/Cas9 technology the goal gene of the foreign gene on this carrier and process LAN can be incorporated into genome specific site.
(2) background technology
In recent years, China's pharmaceutical industries development is very rapid, but also there is a lot of problem simultaneously, such as, research and develop weak foundation, imitated foreign patent is spread unchecked, lacked independent intellectual property right etc., and these all seriously govern the pharmaceutical industries development of China.Therefore, the drug research and development ability strengthening China is very urgent.Drug development process is exactly the process of disease pathogenesis and drug targets screening, the process of screening is mostly by means of the animal disease model of all kinds of human diseases, and particularly nearer with people's sibship all kinds of animal models come the pathogenesis of analysis of disease and the research and development of newtype drug.And transgenic technology is a good instrument in structure animal model, can be inserted in genome by goal gene, works orderly in vivo, this is that the molecule mechanism of study of disease provides exceedingly useful instrument.
In traditional transgenic research, by exogenous genetic fragment microinjection to zygote, by radom insertion mode by exogenous origin gene integrator on body genome; But radom insertion mode can be subject to impact and the restriction of carrier self size of position effect, can not comprise complete regulation and control original paper and often make the expression level of foreign gene lower, even not express or ectopic expression.Meanwhile, because the multiple copied radom insertion of foreign gene is in host genome, there is certain risk in this mode concerning host.These drawbacks limit the development of transgenic technology.Therefore, need one to make exogenous gene high-efficient expressed, there is the transgenic technology of higher safety and reliability simultaneously.
In order to efficient integrate foreign genes and in host normal expression, overcome the impact that transgene is subject to position effect, investigators add the elements such as nuclear matrix attached component, site controlling elements, insulator to weaken the impact of position effect on carrier, but due to the complicacy of gene expression regulation, still there is impact in position effect; Meanwhile, foreign gene is still and integrates in a random basis in host genome, reduces genetically modified security and reliability, and these drawbacks are still traditional transgenic research urgent problem.The appearance of gene targeting overcomes traditional genetically modified drawback, substantially increase genetically modified controllability, this technology utilizes the homology between foreign gene and genome sequence to carry out homologous recombination to realize exogenous origin gene integrator to a special kind of skill in host genome, has high specificity, Absorbable organic halogens heredity and foreign gene not by advantages such as position effect affect.But it is low that gene recombination Knockout technology faces recombination efficiency, construction step is complicated, simultaneously with shortcomings such as abnormal restructuring.Therefore, be badly in need of a kind of fast, the method for efficient, simple syndication foreign gene.
At present, CRISPR/Cas9 system has very large advantage in genome editor, and not only can fix a point to delete goal gene can also site-directed integration goal gene.
(3) summary of the invention
The object of this invention is to provide a kind of method of efficient targeted exogenous gene integration, to improve foreign gene normal expression in host, there is safe reliability simultaneously, the present invention utilizes CRISPR/Cas9 system by fixed point integration of foreign gene to target gene, particularly by Cre Gene targeting in mouse Enpp1 gene first exon, this technology is integration efficiency high (20%) not only; Operation steps is simple, only needs structure foreign gene carrier; Simultaneously not location effects.Utilize this technology to carry out transgenosis to there is unique advantage there is high controllability and security, reduce the risk of genetically modified organism.
The technical solution used in the present invention is:
By the method for fixed point integration of foreign gene to mammal target gene, it is characterized in that described method is: (1) is connected on foreign gene recombinant expression vector after being increased by foreign gene, build restructuring foreign gene carrier; (2) target gene is connected with carrier, build restructuring target gene carrier; (3) restructuring foreign gene carrier and restructuring target gene carrier are cut respectively through AatII and SphI enzyme, sepharose reclaims, acquisition foreign gene-target gene fragment; (4) with px330 plasmid (Addgene:42230) for template, PCR reaction is carried out under the primer Cas9-F containing T7 promoter sequence and primer Cas9-R effect, PCR reaction product is connected with pGME-T carrier, obtain plasmid pGEM-Cas9, again linearized for plasmid pGEM-Cas9, in-vitro transcription and RNA purification kit are reclaimed, obtain Cas9mRNA; (5) according to target gene sequence, fragment T7-sgRNA containing the sgRNA of T7 promoter sequence, i.e. fragment T7-sgRNA, then is cloned into pGME-T carrier by synthetic, linearized, in-vitro transcription and RNA purification kit reclaim, and obtain T7-sgRNA-target gene mRNA; (6) Cas9mRNA, T7-sgRNA-target gene mRNA and foreign gene-target gene fragment are mixed into mixed solution, injection mammal zygote (preferred mouse fertilized egg), realizes the object of fixed point integration of foreign gene to target gene;
Cas9-F：
5’-TTAATACGACTCACTATAGGATGGACTATAAGGACCACGAC-3’
Cas9-R：5’-GCGAGCTCTAGGAATTCTTAC-3’。
Further, in described every 20 μ l mixed solutions, the concentration of Cas9mRNA, T7-sgRNA-target gene mRNA and foreign gene-target gene fragment is respectively 50ng/ μ l, 20ng/ μ l and 10ng/ μ l.
Further, described foreign gene is Cre gene.
Further, described target gene is mouse Enpp1 gene.
The present invention also provides a kind of by the method for foreign gene Cre site-directed integration to mouse target gene Enpp1, described method is: the structure of (1) restructuring foreign gene carrier: with GFP-Cre carrier for template, PCR reaction is carried out under the effect of primer pCre-F and primer pCre-R, reclaim pcr amplification product, be cloned into pGME-T carrier, obtain restructuring foreign gene carrier pCre-Knockin, nucleotides sequence is classified as shown in SEQ ID NO.1;
pCre-F:
5’-GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGTCCAATTTACTGACCGT-3’
pCre-R:5’-ATAAGAGAAGAGGGACAGCT-3’；
(2) structure of restructuring target gene carrier: mouse Enpp1 gene is connected with carrier pGEM-T, build restructuring target gene carrier pGEM-Enpp1, nucleotides sequence is classified as shown in SEQ ID NO.2;
(3) foreign gene-target gene fragment: the restructuring target gene carrier pGEM-Enpp1 that the restructuring foreign gene carrier pCre-Knockin and the step (2) that step (1) are obtained obtain is connected after enzyme is cut, carrier construction pCre-Enpp1, nucleotides sequence is classified as shown in SEQ ID NO.3;
(4) T7-sgRNA-Enpp1 mRNA: according to the nucleotide sequence of Enpp1, synthetic T7-sgRNA sequence, sequence is for shown in SEQ ID NO.6, by the T7-sgRNA sequence clone shown in SEQ ID NO.6 to pGME-T carrier, obtain plasmid T7-sgRNA-Enpp1, plasmid is carried out linearizing, in-vitro transcription obtains T7-sgRNA-Enpp1 mRNA;
(5) with px330 plasmid for template, PCR reaction is carried out under the primer Cas9-F containing T7 promoter sequence and primer Cas9-R effect, PCR primer is connected with pGME-T carrier, obtain plasmid pGEM-Cas9, again linearized for plasmid pGEM-Cas9, in-vitro transcription and RNA purification kit are reclaimed, obtain Cas9mRNA;
Cas9-F：
5’-TTAATACGACTCACTATAGGATGGACTATAAGGACCACGAC-3’
Cas9-R：5’-GCGAGCTCTAGGAATTCTTAC-3’；
(6) T7-sgRNA-Enpp1 mRNA, Cas9 mRNA are mixed with carrier pCre-Enpp1, then inject mouse fertilized egg, then zygote is hatched, realize foreign gene Cre site-directed integration to the object in mouse Enpp1.
The method of targeted exogenous gene integration provided by the invention, fixed point integration of foreign gene on mouse loci, makes foreign gene specific expressed by main employing CRISPR/Cas9 system.This be utilize first CRISPR/Cas9 systemic characteristic by foreign gene Cre site-directed integration in first exon of mouse Enpp1, Cre success at mouse expression in vivo.Utilize this new method can overcome traditional genetically modified random integration problem, improve integration efficiency; Construction step is simple simultaneously, makes the structure of genetically modified biology be reduced to 1 week, decreases expensive molecular agents.
According to the present invention's employing of the present invention CRISPR/cas9 technology by fixed point integration of foreign gene to the method on the locus of Enpp1 gene, obtain a kind of targeting vector built according to aforesaid method, it has Cre-Knock-in carrier structure as shown in Figure 1.Can in Mice Body high expression cre albumen (Figure 10).
The method of targeted exogenous gene integration provided by the invention, fixed point integration of foreign gene on the target gene seat of the biologies such as mouse, zebra fish or pig, makes foreign gene specific expressed by main employing CRISPR/Cas9 system.This be utilize first CRISPR/Cas9 systemic characteristic by fixed point integration of foreign gene in host genome, foreign gene success at host's expression in vivo.Utilize this new method can overcome traditional genetically modified random integration problem, improve integration efficiency; Construction step is simple simultaneously, makes the structure of genetically modified biology be reduced to 1 week, decreases expensive molecular agents.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
(1) characteristic of the fixed point cutting double-stranded DNA of CRISPR/Cas9 system is utilized, goal gene can be made to integrate at specific site, overcome the random integration problem of goal gene and multiple copied and insert expression impact on contiguous important gene (activation of the chromosome instability caused as inserted fixed, contiguous gene functionally inactive, proto-oncogene and inactivation cancer suppressor gene etc.), improve genetically modified security, controllability.
(2) utilize Enpp1 internal promoter to instruct the expression of foreign gene, the high expression of foreign gene can be realized, decrease due to the imperfect ectopic expression caused of controlling element in traditional gene studies simultaneously, improve genetically modified success ratio.
(3) two ends of the present invention homologous sequence only needs 60bp just can realize by fixed point integration of foreign gene in host genome, therefore the method homology arm without the need to multistep structure ~ 5Kbp simple to operate, obtains the Transgenics time greatly to shorten.
(4) this invention utilizes CRISPR/Cas9 technology successfully to mediate first exon of Cre gene site-directed insertion Enpp1 gene first, thereby is achieved the transgenic mice of expressing Cre gene.With routine obtain compared with Cloning of mouse by homologous recombination technique, CRISPR/cas9 technology mediates homologous is utilized to recombinate, in Mice Body, recombination efficiency is 20%, and the gene targeting efficiency of routine is only 1%, its target practice efficiency improves 20 times, for fixed point transgenic animal provide great convenience.
(4) accompanying drawing explanation
Fig. 1 is the vector construction mode chart of targeted exogenous gene integration.
Fig. 2 is restructuring foreign gene carrier pCre-Knockin forming types figure.
Fig. 3 is the gel electrophoresis figure that foreign gene Cre increases, and swimming lane pCre is the pcr amplification product of Cre, and swimming lane M is Marke.
Fig. 4 is the gel electrophoresis figure that target gene Enpp1 increases, and swimming lane Enpp1 is the pcr amplification product of target gene Enpp1, and swimming lane M is Marke.
Fig. 5 is the gel electrophoresis figure of Cas9 expression vector, and swimming lane Cas9 is the pcr amplification product of px330 carrier, and swimming lane M is Marke.
Fig. 6 is the gel electrophoresis figure of Cas9mRNA in-vitro transcription product, and swimming lane Cas9 IVT mrna is Cas9mRNA in-vitro transcription product, and swimming lane M is Marke.
Fig. 7 is the gel electrophoresis figure of T7-sgRNA-Enpp1 mRNA in-vitro transcription product, and swimming lane Cas9 IVT mrna is Cas9mRNA in-vitro transcription product, and swimming lane M is Marke.
Fig. 8 is the gel electrophoresis figure that foreign gene Cre is incorporated into the carrier pCre-Enpp1 that mouse Enpp1 genome builds, and swimming lane 1 ~ swimming lane 8 is that the pCre of 8 mouse detects electrophorogram, and swimming lane M is Marke.
Fig. 9 is the gel electrophoresis figure of T7-sgRNA, and swimming lane T7-sgRNA is the pcr amplification product of T7-sgRNA, and swimming lane M is Marke.
Figure 10 is for foreign gene Cre is in the expression (shown in arrow color portion) of mouse Head And Face.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Material therefor, reagent etc. in following embodiment, if no special instructions, all can obtain from commercial channels.
PGEM-T purchased from Promega company, catalog number A3600.LA Taq purchased from TAKARA, catalog number RR02MA.
LB liquid nutrient medium consists of: peptone 10g/L, yeast extract 5g/L, sodium-chlor 10 g/L, and solvent is water, pH value 7.4.
LB flat board consists of: peptone 10g/L, yeast extract 5g/L, sodium-chlor 10 g/L, agar 15 g/L, and solvent is water, pH value 7.4.
Embodiment 1
1, recombinate the structure of foreign gene Cre carrier and preparation, called after: pCre-Knockin, sequence is as shown in SEQ ID NO.1.
(1) pCre-Knockin recombinant expression vector forming types figure, as Fig. 2, ApaI, AatII, SphI, NcoI, SacII are the multiple clone site connected designed by goal gene.
(2) primer is designed
According to GFP-Cre carrier sequence design pair of primers, before forward primer, add T2A sequence (underscore part).
PCre-F (forward primer):
GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCC TATGTCCAATTTACTGACCGT
PCre-R (reverse primer): ATAAGAGAAGAGGGACAGCT
(3) to recombinate the preparation of foreign gene Cre carrier and checking
1) acquisition of restructuring foreign gene Cre carrier: make template with GFP-Cre plasmid, PCR reaction is carried out under the effect of primer pCre-F and primer pCre-R, after reaction terminates, agarose gel electrophoresis is carried out to pcr amplification product and cuts glue recovery, synthesis object clip size is 1612bp (as shown in Figure 3), is restructuring foreign gene carrier pCre-Knockin.
PCR reaction system is:
PCR reaction conditions is:
2) pCre gene recombination carrier T builds: get 1 μ l step 1) PCR primer that obtains places 1h with pGEM-T carrier in room temperature (25 DEG C) and is connected, acquisition connecting fluid.
Linked system is:
3) acquisition of recombinant plasmid: get connecting fluid 2 μ l and transform 50 μ l bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of thermal shock 60s, 500 μ l LB liquid nutrient mediums are added after ice bath 2min, be placed in 37 DEG C, 200rpm shaking table 1h, get 200 μ l nutrient solutions and coat X-gal+ final concentration 24mg/ml IPTG+ final concentration 100mg/ml Amp containing final concentration 50mg/ml
+lB dull and stereotyped, 37 DEG C of overnight incubation.Picking hickie, is inoculated in 5ml containing Amp
+(100mg/ml), in LB liquid nutrient medium, 37 DEG C, 200rpm incubator overnight, extract plasmid, sequencing result shows step 1) the restructuring foreign gene carrier pCre-Knockin nucleotides sequence that obtains is classified as shown in SEQ ID NO.1.
2, the structure of restructuring target gene carrier, called after pGEM-Enpp1, sequence is as SEQ ID NO.2.
(1) primer is designed
According to GeneBank:NC_000076.6 primers, and before primer Enpp1-F, add restriction enzyme site AatII (underscore part), before primer Enpp1-R, add restriction enzyme site SphI (underscore part).
Enpp1-F：5’-
GACGTCCACGGGTCAGCGGGAAACGGTC-3’
Enpp1-R：5’-
GCATGCCAGCGACAGCACTTTGTAGGTGT-3’
(2) pcr amplification
With mouse embryo stem cell DNA (phenol chloroform) for template, carry out PCR reaction, carry out gel electrophoresis analysis to pcr amplification product under the effect of primer Enpp1-F and primer Enpp1-R, obtaining object clip size is 160bp, as shown in Figure 4.
PCR reaction system is:
PCR reaction conditions is:
(3) checking of pcr amplification product
Get 1 μ l step (2) pcr amplification product to place 1h with pGEM-T carrier in room temperature (25 DEG C) and be connected.
Linked system:
Get connecting fluid 2 μ l and transform 50 μ l bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of thermal shock 60s, 500 μ l LB liquid nutrient mediums are added after ice bath 2min, be placed in 37 DEG C, 200rpm shaking table 1h, get 200 μ l nutrient solutions and coat X-gal+ final concentration 24mg/ml IPTG+ final concentration 100mg/ml Amp containing final concentration 50mg/ml
+lB dull and stereotyped, 37 DEG C of overnight incubation.Picking hickie, is inoculated in 5ml containing Amp
+(100mg/ml) in LB liquid nutrient medium, 37 DEG C, 200rpm incubator overnight, extract plasmid, and sequencing result shows that restructuring target gene carrier pGEM-Enpp1 successfully constructs.
3, the structure of Enpp1/Cre recombinant expression vector and preparation, called after pCre-Enpp1, sequence is as SEQ ID NO.3.
(1) linearizing pCre-Knockin carrier
Get the pCre-Knockin carrier of 2 μ g steps 1 preparations, cut with AatII and SphI enzyme, 37 DEG C of water-baths are spent the night, and it is as follows that concrete enzyme cuts system:
Reclaim test kit with digestion products and reclaim linearizing pCre-Knockin.
(2) Enpp1 fragment obtains
Get the restructuring target gene carrier pGEM-Enpp1 of 20 μ g steps 2 preparations, cut with AatII and SphI enzyme, 37 DEG C of water-baths are spent the night, and it is as follows that concrete enzyme cuts system:
The agarose of configuration 2% carries out gel electrophoresis, cuts glue and reclaims 153bp target stripe, be fragment Enpp1.
(3) be connected on linearizing pCre-Knockin carrier by step (2) fragment Enpp1,16 DEG C of connections of spending the night, concrete linked system is as follows:
Get connecting fluid 2 μ l and transform 50 μ l bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of thermal shock 60s, 500 μ l LB liquid nutrient mediums are added after ice bath 2min, be placed in 37 DEG C, 200rpm shaking table 1h, get 200 μ l nutrient solutions and coat X-gal+ final concentration 24mg/ml IPTG+ final concentration 100mg/ml Amp containing final concentration 50mg/ml
+lB dull and stereotyped, 37 DEG C of overnight incubation.Picking hickie, is inoculated in 5ml containing Amp
+(100mg/ml) in LB liquid nutrient medium, 37 DEG C, 200rpm incubator overnight, extract plasmid, and sequencing result shows that restructuring target gene carrier pCre-Enpp1 successfully constructs.
4, the structure of external Cas9 expression vector and preparation, called after pGEM-Cas9, sequence is as SEQ ID NO.4.
(1) design of primers
With px330 (Addgene:42330) sequence for template, design primer Cas9-F (underscore part is T7 promoter sequence) and primer Cas9-R.
Cas9-F：
5’-
TTAATACGACTCACTATAGGATGGACTATAAGGACCACGAC-3’
Cas9-R：5’-GCGAGCTCTAGGAATTCTTAC-3’。
(2) pcr amplification
Take px330 as template, under the effect of primer Cas9-F and primer Cas9-R, carry out PCR reaction, amplified production carries out gel electrophoresis analysis, and obtaining object clip size is shown in 5309bp, Fig. 5.
PCR reaction system is:
PCR reaction conditions is:
(3)
Get 1 μ l step (2) PCR primer to be connected with pGEM-T, room temperature places 1h, and linked system is as follows:
Get connecting fluid 2 μ l and transform 50 μ l bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of thermal shock 60s, 500 μ l LB liquid nutrient mediums are added after ice bath 2min, be placed in 37 DEG C, 200rpm shaking table 1h, get 200 μ l nutrient solutions and coat X-gal+ final concentration 24mg/ml IPTG+ final concentration 100mg/ml Amp containing final concentration 50mg/ml
+lB dull and stereotyped, 37 DEG C of overnight incubation.Picking hickie, is inoculated in 5ml containing Amp
+(100mg/ml) in LB liquid nutrient medium, 37 DEG C, 200rpm incubator overnight, extract plasmid, and sequencing result shows the success of pGEM-Cas9 expression vector establishment.
(4) linearizing pGEM-Cas9 carrier
Reaction system is as follows, and 37 DEG C are spent the night:
(5) in-vitro transcription Cas9
Getting the linearizing pGEM-Cas9 carrier of 1 μ g utilizes in-vitro transcription test kit (ambion AM1340) to carry out in-vitro transcription, 37 DEG C of reaction 3h, and obtain pGEM-Cas9 vector in vitro transcript, system of specifically transcribing is as follows:
(6) utilize the Cas9 mRNA of RNA purification kit (ambion AM1908) purifying in-vitro transcription, concrete purification system is as follows:
Refined solution crosses RNA purification kit (ambion AM1908) adsorption column, 12000rpm, 30s, and 70% alcohol washes twice, finally uses 80 μ l without RNase water elution, collects liquid and carries out gel electrophoresis analysis, as Fig. 6, obtains Cas9 mRNA.
5, the vector construction of in-vitro transcription sgRNA skeletal coding sequence and preparation, name pGEM-sgRNA, sequence is as SEQ ID NO.5.
The T7-sgRNA nucleic acid fragment of synthetic, with the sgRNA frame sequence (i.e. T7-sgRNA nucleic acid fragment) of T7 promotor, is cloned on pGEM-T carrier by synthetic.Specific as follows:
(1) primer is designed
SgRNA-F (underscore is T7 promotor):
5’-
TTAATACGACTCACTATAGGGTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCT-3’
sgRNA-R：5’-AAAAGCACCGACTCGGTGCC-3’
(2) pcr amplification
Take px330 as template, under the effect of primer sgRNA-F and primer sgRNA-R, carry out PCR reaction, pcr amplification product carries out gel electrophoresis analysis, and obtaining object clip size is 120bp, as Fig. 9.
PCR reaction system is:
PCR reaction conditions is:
(3) get 1 μ l step (2) pcr amplification product to be connected with pGEM-T, room temperature places 1h.
Get connecting fluid 2 μ l and transform 50 μ l bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of thermal shock 60s, 500 μ l LB liquid nutrient mediums are added after ice bath 2min, be placed in 37 DEG C, 200rpm shaking table 1h, get 200 μ l nutrient solutions and coat X-gal+ final concentration 24mg/ml IPTG+ final concentration 100mg/ml Amp containing final concentration 50mg/ml
+lB dull and stereotyped, 37 DEG C of overnight incubation.Picking hickie, is inoculated in 5ml containing Amp
+(100mg/ml), in LB liquid nutrient medium, 37 DEG C, 200rpm incubator overnight, extract plasmid, sequencing result shows that pGEM-sgRNA successfully constructs, for subsequent use.Containing two BbsI restriction enzyme sites between T7 and sgRNA sequence, the recognition sequence for certain gene can be inserted between two restriction enzyme sites.
Embodiment 2
1) for the sgRNA of Enpp1 gene design Enpp1, sequence is as SEQ ID NO.6; BbsI restriction enzyme site is added, following (underscore restriction enzyme site) at sgRNA5 ' end.
sgRNA-Enpp1-F:
CACCGGGCTTCGCTGCTCGCGCCCA
sgRNA-Enpp1-R:
AAACTGGGCGCGAGCAGCGAAGCCC
Sex change, annealing reaction system is as follows:
In PCR instrument, reaction system is as follows: 37 DEG C of 30min; 95 DEG C of 5min; 95-25 DEG C, 5 DEG C/min.
After sex change is annealed, the product sgRNA-Enpp1 of acquisition is connected to pGEM-sgRNA carrier (sequence is as shown in SEQ ID NO.5), 25 DEG C of ligation 1h, after reaction terminates, get connecting fluid 2 μ l and transform 50 μ l bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of thermal shock 60s, 500 μ l LB liquid nutrient mediums are added after ice bath 2min, be placed in 37 DEG C, 200rpm shaking table 1h, get 200 μ l nutrient solutions and coat X-gal+ final concentration 24mg/ml IPTG+ final concentration 100mg/ml Amp containing final concentration 50mg/ml
+lB dull and stereotyped, 37 DEG C of overnight incubation.Picking hickie, is inoculated in 5ml containing Amp
+(100mg/ml), in LB liquid nutrient medium, 37 DEG C, 200rpm incubator overnight, extract plasmid, obtains plasmid T7-sgRNA-Enpp1.
Ligation system is as follows:
2) linearization plasmid T7-sgRNA-Enpp1 reaction system is as follows:
Use phenol chloroform purify DNA again.
3) in-vitro transcription T7-sgRNA-Enpp1
Get the linearizing T7-sgRNA-Enpp1 plasmid of 1 μ g, utilize in-vitro transcription test kit (ambion AM1340) to carry out in-vitro transcription, 37 DEG C of reaction 3h, concrete system is as follows:
4) utilize RNA purification kit (ambion AM1908) to extract the mRNA of in-vitro transcription T7-sgRNA-Enpp1 plasmid, concrete system is as follows:
Cross RNA purification kit (ambion AM1908) wash-out post, 12000rpm, 30s, 70% alcohol washes twice, finally uses without RNase water elution, agarose gel electrophoresis analysis, as Fig. 9, for subsequent use.
5) transgenic mice preparation
CRISPR/Cas9 injection mouse system is as follows:
sgRNA-Enpp1 mRNA 20ng/μl
Cas9 mRNA 50ng/μl
pCre-Enpp1 10ng/μl
Mixed population amasss: 20 μ l
Injection:
Utilize Eppendorf 2xTransferMan NK2 microinjection instrument to draw the above-mentioned mixed solution of 2 μ l, inject 60 ~ 80 zygotes.
After mouse is born 5 days, get 8 respectively, clip mouse nail extracts genomic dna.PCR identifies Cre gene, as Fig. 8.13.5 days embryonic stages, Cre gene in the expression of mouse Head And Face, as shown in Figure 10 (shown in arrow color portion).
Claims (6)
1. by the method for fixed point integration of foreign gene to target gene, it is characterized in that described method is: (1) is connected on foreign gene recombinant expression vector after being increased by foreign gene, build restructuring foreign gene carrier; (2) target gene is connected with carrier, build restructuring target gene carrier; (3) restructuring foreign gene carrier and restructuring target gene carrier are cut respectively through AatII and SphI enzyme, sepharose reclaims, acquisition foreign gene-target gene fragment; (4) with px330 plasmid for template, PCR reaction is carried out under the primer Cas9-F containing T7 promoter sequence and primer Cas9-R effect, PCR reaction product is connected with pGME-T carrier, obtain plasmid pGEM-Cas9, again linearized for plasmid pGEM-Cas9, in-vitro transcription and RNA purification kit are reclaimed, obtain Cas9mRNA; (5) according to target gene sequence, fragment T7-sgRNA containing the sgRNA of T7 promoter sequence, i.e. fragment T7-sgRNA, then is cloned into pGME-T carrier by synthetic, linearized, in-vitro transcription and RNA purification kit reclaim, and obtain T7-sgRNA-target gene mRNA; (6) Cas9mRNA, T7-sgRNA-target gene mRNA and foreign gene-target gene fragment are mixed into mixed solution, injection mammal zygote, realizes the object of fixed point integration of foreign gene to target gene;
Cas9-F：
5’-
TTAATACGACTCACTATAGGATGGACTATAAGGACCACGAC-3’
Cas9-R：5’-GCGAGCTCTAGGAATTCTTAC-3’。
2. the method for claim 1, is characterized in that the concentration of Cas9mRNA, T7-sgRNA-target gene mRNA and foreign gene-target gene fragment in described every 20 μ l mixed solutions is respectively 50ng/ μ l, 20ng/ μ l and 10ng/ μ l.
3. the method for claim 1, is characterized in that described foreign gene is Cre gene.
4. the method for claim 1, is characterized in that described target gene is mouse Enpp1 gene.
5. the method for claim 1, is characterized in that described mammal zygote is mouse fertilized egg.
6. the method as described in one of claim 3 ~ 5, it is characterized in that described method is: the structure of (1) restructuring foreign gene carrier: with GFP-Cre carrier for template, PCR reaction is carried out under the effect of primer pCre-F and primer pCre-R, reclaim pcr amplification product, be cloned into pGME-T carrier, obtain restructuring foreign gene carrier pCre-Knockin, nucleotides sequence is classified as shown in SEQ ID NO.1;
pCre-F:
5’-GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGTCCAATTTACTGACCGT-3’
pCre-R:5’-ATAAGAGAAGAGGGACAGCT-3’；
(2) structure of restructuring target gene carrier: mouse Enpp1 gene is connected with carrier pGEM-T, build restructuring target gene carrier pGEM-Enpp1, nucleotides sequence is classified as shown in SEQ ID NO.2;
(3) foreign gene-target gene fragment: the restructuring target gene carrier pGEM-Enpp1 that the restructuring foreign gene carrier pCre-Knockin and the step (2) that step (1) are obtained obtain is connected after enzyme is cut, carrier construction pCre-Enpp1, nucleotides sequence is classified as shown in SEQ ID NO.3;
(4) T7-sgRNA-Enpp1mRNA: according to the nucleotide sequence of Enpp1, synthetic T7-sgRNA sequence, sequence is for shown in SEQ ID NO.6, by the T7-sgRNA sequence clone shown in SEQ ID NO.6 to pGME-T carrier, obtain plasmid T7-sgRNA-Enpp1, plasmid T7-sgRNA-Enpp1 is carried out linearizing, in-vitro transcription obtains T7-sgRNA-Enpp1mRNA;
(5) with px330 plasmid for template, PCR reaction is carried out under the primer Cas9-F containing T7 promoter sequence and primer Cas9-R effect, PCR primer is connected with pGME-T carrier, obtain plasmid pGEM-Cas9, again linearized for plasmid pGEM-Cas9, in-vitro transcription and RNA purification kit are reclaimed, obtain Cas9mRNA;
Cas9-F：
5’-
TTAATACGACTCACTATAGGATGGACTATAAGGACCACGAC-3’
Cas9-R：5’-GCGAGCTCTAGGAATTCTTAC-3’；
(6) T7-sgRNA-Enpp1mRNA, Cas9mRNA are mixed with carrier pCre-Enpp1, then inject mouse fertilized egg, then zygote is hatched, realize foreign gene Cre site-directed integration to the object in mouse Enpp1.
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