CN110468133A - Utilize the method for CRISPR/Cas9 system knock-out pig GOT1 gene - Google Patents
Utilize the method for CRISPR/Cas9 system knock-out pig GOT1 gene Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, and in particular to utilizes the method for CRISPR/Cas9 system knock-out pig GOT1 gene.The present invention provides the detection method of the sgRNA of selectively targeted pig GOT1 gene, the CRISPR/Cas9 gene knockout carrier comprising the sgRNA, the method that pig GOT1 gene knockout is carried out using the carrier and pig GOT1 gene.CRISPR/Cas9 knockout carrier provided by the invention can be realized higher pig GOT1 gene knockout efficiency, the building efficiency of GOT1 Knockout cells and GOT1 gene knock-out pig is significantly improved, provides effective ways and basis for the functional study and its application of pig GOT1 gene.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to the sgRNA of selectively targeted pig GOT1 gene, contains and is somebody's turn to do
The CRISPR/Cas9 gene knockout carrier of sgRNA, and method and pig GOT1 using carrier progress pig GOT1 gene knockout
The detection method of gene knockout.
Background technique
GOT1 is a kind of transaminase that phosphopyridoxal pyridoxal phosphate (Pyridoxal phosphate, PLP) relies on, in cytoplasm base
Aspartic acid (Aspartate, Asp) is participated in matter and α-ketoglutaric acid (α-Ketoglutarate, α-KG) generates glutamic acid
The reversible reaction of (Glutamate, Glu) and oxaloacetic acid (Oxaloacetate, OAA), maintain cell Redox balance,
Promote to play a significant role in terms of tumor cell proliferation, adjusting amino acid metabolism.
Currently, the research of GOT1 gene is concentrated mainly in mouse, it is fewer in the research of the mammals such as pig.Pass through benefit
With CRISPR/Cas9 system knock-out pig GOT1 gene, the function of pig GOT1 gene is studied in vitro and in vivo, for studying GOT1
Function of the gene in pig growth course and its mechanism to play a role are of great significance.
CRISPR/Cas9 is a kind of adaptive immunity defence that bacterium and archeobacteria are formed during long-term evolution, can
For fighting the virus and exogenous DNA of invasion.And CRISPR/Cas9 gene editing technology, then it is specific to target gene progress
The technology of DNA modification, this technology are also the method in the comparison forward position currently used for gene editing.Using CRISPR/Cas9 as base
Application neck of the gene editing technology of plinth in a series of gene therapy of diseases (such as blood disease, tumour and other genetic diseases)
Domain all shows great application prospect.The CRISPR/Cas9 system knockout technique of efficient pig GOT1 gene is developed for mentioning
The building efficiency of high pig GOT1 gene knockout efficiency and GOT1 Knockout cells or animal is of great significance.
Summary of the invention
In order to solve the problems in the existing technology, the object of the present invention is to provide high using CRISPR/Cas9 system
Imitate the method for knock-out pig GOT1 gene and the efficient detection method of pig GOT1 gene knockout.
To achieve the above object, technical scheme is as follows:
In a first aspect, the present invention provides the sgRNA of selectively targeted pig GOT1 gene, the of targeting pig GOT1 gene
517 to the 536th sequences.
The present invention is by largely screening discovery using the 517th to the 536th sequence on pig GOT1 gene as target
Mark sequence, knockout efficiency with higher.
For the target sequence on above-mentioned pig GOT1 gene, the present invention devises special sgRNA, and is further discovered that adopt
It can be realized higher target practice efficiency and GOT1 clpp gene with the corresponding sgRNA of nucleotide sequence as shown in SEQ ID NO.3
Except efficiency.
Preferably, the nucleotide sequence such as SEQ of the encoding gene of the sgRNA of the selectively targeted pig GOT1 gene
Shown in ID NO.3.
SEQ ID NO.3:sgRNA-3:5 '-ACATTCGGTCCTATCGCTAT-3 '.
Second aspect, the present invention provide the CRISPR/Cas9 base of the sgRNA containing the selectively targeted pig GOT1 gene
Because of knockout carrier.
As the preferred embodiment of the present invention, the CRISPR/Cas9 gene knockout carrier is described special to be connected with
Property targeting pig GOT1 gene sgRNA pHS-CR054 carrier.
The third aspect, the present invention also provides the construction method of the CRISPR/Cas9 gene knockout carrier, are as follows: by institute
SgRNA and the pHS-CR054 carrier for stating selectively targeted pig GOT1 gene are connect, and obtain the CRISPR/Cas9 gene knockout
Carrier.
Preferably, the construction method the following steps are included:
(1) BsmBI digestion carrier pHS-CR054 is used, linearized vector is obtained；
(2) the segment annealing of the sgRNA sequence with BsmBI restriction enzyme site, forms and contains BsmBI restriction enzyme site cohesive end
Segment；
(3) annealed product that step (2) obtains is connect with the linearized vector that step (1) obtains, building connection is
State the pHS-CR054 carrier of the sgRNA of selectively targeted pig GOT1 gene.
It is further preferred that the reaction system of digestion is as follows in above-mentioned steps (1): carrier pHS-CR054 500ng, limit
1 μ L, NEB 3.1Buffer of property restriction endonuclease BsmBI 1 μ L, ddH processed2O polishing is to 10 μ L.The response procedures of digestion are as follows: 55
DEG C, 60min；80 DEG C, 20min.
It is further preferred that the reaction system of annealing is as follows in above-mentioned steps (2): 2 μ L of PNK buffer, upstream sequence
100 μM, 100 μM of downstream sequence, ddH2O polishing is to 20 μ L.The response procedures of annealing are as follows: 95 DEG C, 5min；95 DEG C to 25 DEG C slow
Slow cool down drops 2 DEG C per minute.
It is further preferred that the reaction system of connection is as follows in above-mentioned steps (3): annealed product 1 μ L, 1 μ of digestion products
L, T4DNA Ligase 1 μ L, 10 × buffer 1 μ L, ddH2O polishing is to 10 μ L.The response procedures of connection are as follows: 16 DEG C of mistakes
Night.
Fourth aspect the present invention also provides the sgRNA of the selectively targeted pig GOT1 gene or includes the sgRNA's
Application of the CRISPR/Cas9 gene knockout carrier in pig GOT1 gene knockout.
5th aspect, the present invention provide a kind of method for preparing GOT1 Knockout cells comprising: the spy will be contained
The CRISPR/Cas9 gene knockout carrier of the sgRNA of opposite sex targeting pig GOT1 gene imports cell, knocks out GOT1 gene.
It is above-mentioned that CRISPR/Cas9 gene knockout carrier importing cell can be passed through into the conventional biology methods such as transfection reality
It is existing.
The above-mentioned method for preparing GOT1 Knockout cells will contain the sgRNA of the selectively targeted pig GOT1 gene
CRISPR/Cas9 gene knockout carrier import cell after, can be used the conventional methods such as PCR screening positive cell.
The present invention obtains the pig cell of GOT1 gene knockout using the above-mentioned method for preparing GOT1 Knockout cells.
6th aspect, the present invention provide a kind of method for preparing GOT1 gene knock-out pig, to utilize the CRISPR/
The knockout of Cas9 gene knockout carrier realization pig GOT1 gene.
6th aspect, the present invention provide the specific primer pair of detection pig GOT1 gene expression dose, sequence such as SEQ
Shown in ID NO.4-5.
7th aspect, the present invention provide a kind of method for detecting pig GOT1 gene expression dose comprising following steps:
(1) cell total rna to be measured is extracted, reverse transcription is at cDNA；
(2) fixed to, fluorescence using the specific primer as shown in SEQ ID NO.4-5 using the cDNA of step (2) as template
It measures PCR (qPCR) and expands GOT1 gene, detect the expression of GOT1 gene.
Preferably, the fluorescent quantitative PCR is using 18S rDNA as reference gene in above-mentioned steps (2).
It is further preferred that the fluorescent quantitative PCR primer sequence of 18S rDNA is as shown in SEQ ID NO.6-7.
Preferably, the reaction system of the qPCR is as follows in above-mentioned steps (2): 2 × GoTaq qPCR Master
10 μ L of Mix removes RNA enzyme water 7.4 μ L, 10 μM of upstream primers 0.3 μ L, 10 μM of 0.3 μ L, cDNA template of downstream primer, 2.0 μ L.
The response procedures of the qPCR are as follows: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, 45 recycle；
95 DEG C of 15s, 65 DEG C of 1min, 95 DEG C of 30min, 60 DEG C of 15s make melting curve.
Preferably, the amplification curve and melting curve obtain to qPCR real-time monitoring divides in above-mentioned steps (2)
Analysis.The table of GOT1 gene is calculated using control group as outer ginseng (the outer purpose joined of setting is to eliminate the test error of different batches)
Up to amount.GOT1 gene differential expression in different disposal is compared using independent sample T check analysis, as a result with average value ± standard
It accidentally indicates, P < 0.05 indicates that significant difference, P < 0.01 indicate that difference is extremely significant.All statistical analysis use SPSS version
20.0 software is completed.
Beneficial effects of the present invention include at least:
The present invention knocks out the knockout that system carries out pig GOT1 gene using CRISPR/Cas9.Pass through pig GOT1 gene target position
The special selection and the special design of the sgRNA for the target site of point, obtain the pig GOT1 gene that can efficiently practice shooting
SgRNA significantly improves the knockout efficiency of pig GOT1 gene.It is carried experiments verify that CRISPR/Cas9 provided by the invention is knocked out
Body can be realized higher pig GOT1 gene knockout efficiency, significantly improve GOT1 Knockout cells and GOT1 gene knock-out pig
Building efficiency.
The specific primer of detection pig GOT1 gene expression dose provided by the invention can be quickly and accurately to pig GOT1
The expression of gene carries out quantitative analysis.
The knockout technique of pig GOT1 gene provided by the invention and the detection method of pig GOT1 gene expression dose have very
Strong practicability provides effective ways and basis for the functional study and its application of pig GOT1 gene.
Detailed description of the invention
Fig. 1 is the map of pHS-CR054 carrier in the embodiment of the present invention 2；The position of BsmBI restriction enzyme site has been marked in figure
It sets, there are two BsmBI restriction enzyme sites at 3058bp and 3741bp.
Fig. 2 is the CRISPR/Cas9 base of the sgRNA containing 3 selectively targeted pig GOT1 genes in the embodiment of the present invention 2
Because of the sequencing result of the sgRNA sequence with BsmBI restriction enzyme site of knockout carrier insertion；Wherein, GOT1_KO1 be containing
The CRISPR/Cas9 gene knockout carrier of sgRNA-1, GOT1_KO2 are that the CRISPR/Cas9 gene knockout containing sgRNA-2 carries
Body, GOT1_KO3 are the CRISPR/Cas9 gene knockout carrier containing sgRNA-3.
Fig. 3 is the microscopy results figure that PK15 cell is transfected in the embodiment of the present invention 3；Wherein, GOT1_KO1,
GOT1_KO2 and GOT1_KO3 is respectively that the transfection of the pHS-CR054 carrier of carrying sgRNA-1, sgRNA-2 and sgRNA-3 is thin
Born of the same parents.
Fig. 4 is the mrna expression amount testing result of GOT1 gene after transfecting 48h in the embodiment of the present invention 3；Wherein, NC is sky
Carrier, GOT1_KO1, GOT1_KO2 and GOT1_KO3 are respectively the pHS-CR054 for carrying sgRNA-1, sgRNA-2 and sgRNA-3
Carrier；* significant difference (P < 0.05) is indicated, * * indicates that difference is extremely significant (P < 0.01).
Fig. 5 is the Western blotting testing result of GOT1 albumen after transfecting 48h in the embodiment of the present invention 3；Wherein,
Control is empty vector control, GOT1_KO1, GOT1_KO2 and GOT1_KO3 be respectively carry sgRNA-1, sgRNA-2 and
The pHS-CR054 carrier of sgRNA-3；* significant difference (P < 0.05) is indicated, * * indicates that difference is extremely significant (P < 0.01).
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The present invention by from database download pig GOT1 gene nucleotide sequence, based on to pig GOT1 gene order
Analysis identifies the design principle of target site according to CRISPR/Cas9, carries out the screening of the target site of pig GOT1 gene and be directed to be somebody's turn to do
The design and screening of the sgRNA of target site finally screens and obtains the knockout highest sgRNA-3 of efficiency (such as SEQ ID NO.3 institute
Show), and the CRISPR/Cas9 knockout carrier containing the sgRNA is constructed, realize the efficient knockout of pig GOT1 gene.Below
Effect explanation is carried out in embodiment by taking 3 in a large amount of candidate sgRNA for different target sites that the present invention designs as an example.
The acquisition of the sgRNA of the selectively targeted pig GOT1 gene of embodiment 1
SgRNA-1 targets pig GOT1 gene the 518th to the 537th sequence, and sgRNA-2 targets pig GOT1 gene the
919 to the 938th sequences, sgRNA-3 target pig GOT1 gene the 517th to the 536th sequence.sgRNA-1,
The sequence of sgRNA-2 and sgRNA-3 is as follows:
SEQ ID NO.1:sgRNA-1:5 '-CATTCGGTCCTATCGCTATT-3 '；
SEQ ID NO.2:sgRNA-2:5 '-AGAAGATCGTGCGAGTGACG-3 '；
SEQ ID NO.3:sgRNA-3:5 '-ACATTCGGTCCTATCGCTAT-3 '.
To connect convenient for subsequent with carrier, BsmBI restriction enzyme site sequence, obtained double-stranded DNA are added at the both ends of sgRNA
Sequence is as shown in table 1.
The corresponding double chain DNA sequence of 13 sgRNA of table
The building of the CRISPR/Cas9 gene knockout carrier of sgRNA of the embodiment 2 containing selectively targeted pig GOT1 gene
The present embodiment provides the CRISPR/Cas9 gene knockout carriers of the sgRNA containing selectively targeted pig GOT1 gene
And its construction method.
The construction method packet of the CRISPR/Cas9 gene knockout carrier of sgRNA containing selectively targeted pig GOT1 gene
Include following steps:
1, the building of linearized vector
Recover and expand culture carry pHS-CR054 carrier bacterial strain (pHS-CR054 Vector map as shown in Figure 1, purchase
From Beijing symphysis Gene Tech. Company Limited), according to OMEGA plasmid extraction kit specification, carry out plasmid extraction.
It extracts product and carries out digestion, the reaction system of digestion are as follows: restriction enzyme with restriction enzyme BsmBI
1 μ L, pHS-CR054 vector plasmid 500ng, NEB 3.1Buffer of BsmBI 1 μ L, ddH2O polishing is to 10 μ L.The reaction of digestion
Program are as follows: 55 DEG C of digestions 1h, 80 DEG C of inactivation 20min.
2, the annealing of sgRNA sequence
The segment for the sgRNA sequence that 3 pairs as shown in Table 1 carry BsmBI restriction enzyme site is annealed respectively, formation contains
There is the segment of BsmBI restriction enzyme site cohesive end.The reaction system of annealing is as follows: PNK buffer 2 μ L, 100 μ of upstream sequence
M, 100 μM of downstream sequence, ddH2O polishing is to 20 μ L.The response procedures of annealing are as follows: 95 DEG C, 5min；95 DEG C to 25 DEG C slowly cold
But, 2 DEG C are dropped per minute.
3, it connects
SgRNA annealed product obtained in step 2 is connect with the linearized vector pHS-CR054 that step 1 obtains.Connection
Reaction system is as follows: 1 μ L of annealed product, 1 μ L, T4DNA Ligase of digestion products 1 μ L, 10 × buffer 1 μ L, and ddH2O is mended
Together to 10 μ L, it is as follows to connect response procedures: 16 DEG C overnight.
4, it converts
It takes the 5 μ L of connection product in step 3 to be added in the 50 μ L DH5 α competent cells just to have thawed, mixes, ice bath
After 30min, 42 DEG C of heat shock 45s are put stand 2min on ice immediately, the LB liquid medium that 950 μ L of addition are preheated in 37 DEG C, and 37 DEG C
Bacterium solution low-speed centrifugal 2-3min is outwelled part supernatant by shake culture 45min, and remaining supernatant is blown and beaten with pipette tips to be precipitated, and is taken
100 μ L bacterium solutions are applied in the plate of ammonia benzyl antibiotic, after just putting 1h in 37 DEG C of constant incubators, are inverted culture 12h.
5, it screens positive bacterium colony and carries out sequencing identification
The single bacterium colony cultivated in picking step 4 carries out bacterium colony PCR, is detected with agarose gel electrophoresis.Qualified
Bacterium colony carries out shaking bacterium, send Huada gene company sequencing sequencing primer sequence as follows 1mL bacterium solution:
CAGGAAGAGGGCCTATTTCCC.To being sequenced, correct bacteria liquid sample carries out protecting bacterium and plasmid extracts.It will the correct difference of sequencing
PHS-CR054 carrier containing 3 sgRNA be respectively designated as GOT1_KO1 (the pHS-CR054 carrier containing sgRNA-1),
GOT1_KO2 (the pHS-CR054 carrier containing sgRNA-2), GOT1_KO3 (the pHS-CR054 carrier containing sgRNA-3),
Sequencing result is as shown in Figure 2.
The knockout and detection of 3 pig GOT1 gene of embodiment
The present embodiment provides the CRISPR/ of the sgRNA containing selectively targeted pig GOT1 gene constructed using embodiment 2
Cas9 gene knockout carrier GOT1_KO1, GOT1_KO2 and GOT1_KO3 carry out method and the pig of the knockout of pig GOT1 gene
The detection method of GOT1 gene expression dose.
1, the knockout of pig GOT1 gene
(1) cell culture and collection
Enter logarithmic growth phase after passing on 3 times using complete medium recovery PK15 cell to cell and transfected.Turn
Dye the previous day, by 5 × 105~8 × 1052mL antibiotic-free culture medium is added to 6 orifice plates in a cell inoculation in every hole, when
Cell density is transfected when reaching 60%-80%.
(2) it transfects
According to the requirement that Lipofectamine 2000 illustrates, contain selectively targeted pig GOT1 for what embodiment 2 constructed
CRISPR/Cas9 gene knockout carrier GOT1_KO1, GOT1_KO2 and GOT1_KO3 of the sgRNA of gene is used
Lipofectamine 2000 is transfected, and cell is collected after 48h；Using NC plasmid (empty plasmid) as control.It is thin to transfect PK15
The microscopy results of born of the same parents are as shown in Figure 3.
2, the qPCR detection of pig GOT1 gene expression dose
QPCR detection is carried out to the positive cell that step 1 obtains, is specifically comprised the following steps:
(1) cell RNA extracts and cDNA is synthesized
The total serum IgE of cell to be measured is extracted using TaKaRa RNAiso Plus kit, extracting method is said referring to kit
Bright book.The integrality of total serum IgE is detected by agarose gel electrophoresis and is determined；Total rna concentration passes through the micro purple of NanoDrop 1000
Outer spectrophotometric determination.
(2) design of primers
Detectable pig GOT1 is designed according to the pig GOT1 gene mRNA sequence (GenBank:EU822301.1) that NCBI is announced
The primer pair of gene knockout obtains can be realized the pair of primers GOT1-1 of efficient pig GOT1 genetic test through screening.Primer
It is as follows to the sequence of GOT1-1:
Upstream primer, GOT1-1F:5 '-CATCCTGCGAGTCCTTTC-3 '；
Downstream primer, GOT1-1R:5 '-CGGTCAGCCATTGTCTTC-3 '；
For the 18S gene order design primer announced according to NCBI to 18S, the sequence of primer pair 18S is as follows:
Upstream primer, 18S-F:5 '-ATGCCAGAGTCTCGTTCGTTAT-3 '；
Downstream primer, 18S-R:5 '-CGGACAGGATTGACAGATTGAT-3 '.
(3) qPCR is expanded
Using cell cDNA obtained in above-mentioned steps (1) as template, the primer pair GOT1-1 and primer pair of step (2) are utilized
18S carries out qPCR amplification.
The reaction system of qPCR are as follows: GoTaq qPCR Master Mix (2 ×) 10 μ L removes 7.4 μ L of RNA enzyme water, on 10 μM
Swim primer 0.3 μ L, 10 μM of 0.3 μ L, cDNA template of downstream primer, 2.0 μ L.The preparation of qPCR reaction system uses mixing sample-adding method,
That is the number of PCR needed for the quantity of various components and 1 secondary response according to needed for each reaction system reaction, calculates various
The total amount of reactive component is added to 1 and goes in the 1.5mL centrifuge tube of RNA enzyme, mixes well rear brief centrifugation, then be dispensed into glimmering
In dedicated 8 union of Fluorescent Quantitative PCR, it is then respectively adding template cDNA, then fluorescent quantitative PCR is carried out after brief centrifugation,
Whole operation process is protected from light as far as possible.
The response procedures of qPCR are as follows: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, 45 recycle；95℃
15s, 65 DEG C of 1min, 95 DEG C of 30min, 60 DEG C of 15s make melting curve.
(4) the data analysis of qPCR
The amplification curve and melting curve of qPCR result are analyzed.The result shows that each gene magnification curve is in " S "
Shape and arrival plateau, illustrate effectively to expand；And the melting curve peak value of each gene is single, illustrates that amplified production is special, nothing
Primer dimer and other non-specific amplifications can carry out the analysis of subsequent result.According to the CT value of different disposal group, with NC
The control group of plasmid (empty plasmid) is as outer ginseng (the outer ginseng of setting is for elimination different batches test error) using relative quantification side
Relative expression quantity=2 of method calculating pig GOT1 gene knockout-△△CT。
(5) it maps
According to the calculated result of relative quantification, significance test of difference is carried out using 20.0 software of SPSS version, and
It is mapped by GraphPad Prism 5, as a result as shown in figure 4, it is 4 that individual repeat number, which is the repetition of 3, qPCR loading techniques, in figure.
The results show that compared with the control group, knocking out GOT1 gene lowers its transcriptional level significantly；In the pig GOT1 that 3 sgRNA are mediated
In gene knockout, the transcriptional level for the GOT1 gene in knockout cell that sgRNA-3 is mediated is minimum, and it is excellent to show that sgRNA-3 has
Different pig GOT1 gene knockout efficiency, can be realized the efficient knockout of pig GOT1 gene.
3, the Western blotting detection of pig GOT1 gene expression dose
Pig GOT1 gene expression dose is further verified using Western blotting, the specific method is as follows:
The total protein for extracting knockout group and corresponding blank control group cell takes 200ng albumen loading, carries out SDS polypropylene
Acrylamide gel electrophoresis.It is exposed using after transferring film, closing, incubation primary antibody and secondary antibody using LICOR instrument, it is included to reuse instrument
Software I mage Studio calculates analysis band OD value.As a result as shown in figure 5, the pig GOT1 gene mediated in 3 sgRNA
In knockout, the expression for the GOT1 gene in knockout cell that sgRNA-3 is mediated is minimum, and it is excellent to show that sgRNA-3 has
Pig GOT1 gene knockout efficiency can be realized the efficient knockout of pig GOT1 gene.
Described in summary, using Idiotype provided by the invention targeting pig GOT1 gene sgRNA (SEQ ID NO.3) and
CRISPR/Cas9 gene knockout carrier containing the sgRNA can be realized efficient pig GOT1 gene knockout；This hair is utilized simultaneously
The specific primer of the pig GOT1 gene of bright offer can be realized the quick and precisely detection of GOT1 gene expression dose；The present invention is
The functional study and its application of pig GOT1 gene provide effective ways and basis.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Agricultural University Of Shanxi
<120>method of CRISPR/Cas9 system knock-out pig GOT1 gene is utilized
<130> KHP191113496.5
<160> 14
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<400> 2
agaagatcgt gcgagtgacg 20
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<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
acattcggtc ctatcgctat 20
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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Claims (10)
1. the sgRNA of selectively targeted pig GOT1 gene, which is characterized in that it targets the 517th to the 536th of pig GOT1 gene
The sequence of position.
2. the sgRNA of selectively targeted pig GOT1 gene according to claim 1, which is characterized in that its encoding gene
Nucleotide sequence is as shown in SEQ ID NO.3.
3. the CRISPR/Cas9 gene knockout of the sgRNA containing selectively targeted pig GOT1 gene of any of claims 1 or 2
Carrier.
4. CRISPR/Cas9 gene knockout carrier according to claim 3, which is characterized in that it has the right to want for connection
The pHS-CR054 carrier of the sgRNA of selectively targeted pig GOT1 gene described in asking 1 or 2.
5. CRISPR/Cas9 gene knockout carrier is in pig described in sgRNA of any of claims 1 or 2 or claim 3 or 4
Application in GOT1 gene knockout.
6. a kind of method for preparing GOT1 Knockout cells, which is characterized in that specificity as claimed in claim 1 or 2 will be contained
The CRISPR/Cas9 gene knockout carrier for targeting the sgRNA of pig GOT1 gene imports cell, knocks out GOT1 gene.
7. a kind of method for preparing GOT1 gene knock-out pig, which is characterized in that utilize CRISPR/ described in claim 3 or 4
The knockout of Cas9 gene knockout carrier realization pig GOT1 gene.
8. detecting the specific primer pair of pig GOT1 gene expression dose, which is characterized in that its sequence such as SEQ ID NO.4-5 institute
Show.
9. a kind of method for detecting pig GOT1 gene expression dose, which comprises the steps of:
(1) cell total rna to be measured is extracted, reverse transcription is at cDNA；
(2) using the cDNA of step (2) as template, using the specific primer as shown in SEQ ID NO.4-5 to, fluorescent quantitation
PCR amplification GOT1 gene detects the expression of GOT1 gene.
10. the method for detection pig GOT1 gene expression dose according to claim 9, which is characterized in that the fluorescence is fixed
The response procedures for measuring PCR are as follows: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, 45 recycle；95 DEG C, 15s, 65
DEG C, 1min, 95 DEG C, 30min, 60 DEG C, 15s makes melting curve；
And/or
The reaction system of the quantitative fluorescent PCR is as follows: 2 × GoTaq qPCR Master Mix, 10 μ L removes 7.4 μ of RNA enzyme water
L, 10 μM of upstream primers 0.3 μ L, 10 μM of 0.3 μ L, cDNA template of downstream primer, 2.0 μ L.
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