CN110029130A - Sidt2 genetic method is rejected in Beta-TC-6 cell strain based on CRISPR/Cas9 system - Google Patents
Sidt2 genetic method is rejected in Beta-TC-6 cell strain based on CRISPR/Cas9 system Download PDFInfo
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Abstract
The invention discloses Sidt2 genetic method is rejected in Beta-TC-6 cell strain based on CRISPR/Cas9 system, for the feature that the difficult transfection of mouse islets element tumor beta Cell of islet (Beta-TC-6) and CRISPR/Cas9 plasmid molecule are larger, by taking electroporation to carry out the delivering of Cas9 plasmid, and different perforation voltage gradient progress electricity is set and turns the optimization of parameter, CRISPR/Cas9 plasmid is efficiently delivered in mouse islets element tumor beta Cell of islet (Beta-TC-6) to realize；Simultaneously, by the way that CRISPR/Cas9 gene editing system and electroporation are combined, the characteristics of overcoming the transduction of CRISPR/Cas9 plasmid difficulty, carry out CRISPR/Cas9 gene editing system for conventional Molecular Biology Lab and provide practical ways, there is high application value.
Description
Technical field
The present invention relates to medical genetic engineering fields, more particularly to thin in Beta-TC-6 based on CRISPR/Cas9 system
Sidt2 genetic method is rejected in born of the same parents' strain.
Background technique
CRISPR/Cas9 gene editing system becomes after zinc finger protein (ZFPs), transcriptional activation increment effector nucleic acid
Third generation gene editing technology after enzyme (TALEs).Have the advantages that compared with traditional gene editing tool it is many, first
CRISPR/Cas9 gene editing system suitability and strong operability, come the Cas9 of self-produced Streptococcus pyrogenes and streptococcus thermophilus due to
PAM identifies that sequence is only 2 bases (GG), and a large amount of target spots can be almost found in all genes.Cas9 albumen is current
It is active in the almost all creatures and cell tested, including bacterium, yeast, plant, fish and mammalian cell；
Secondly, CRISPR/Cas9 gene editing system is easy to learn, there are many mature CRISPR/Cas9 gene editing systems at present
System plasmid vector, conventional Molecular Biology Lab can carry out building, it is only necessary to which the sgRNA for being about 25bp or so is bis-
For chain link into the carrier of linearisation, building difficulty is small relative to common expression vector and Reporter gene vector, and building is completed
Carrier only need to identify by bacterium colony PCR or sequencing, it is not necessary to as construction of expression vector worries that the base of long segment is prominent
Change problem.But to need to express cas9 albumen bigger than normal relative to other carriers for CRISPR/Cas9 plasmid, generally usually exists
10kbp or more, transfection cell difficulty is larger, is even more to make the matter worse particularly with hardly possible transfection cell.Although can be by success
The antibiotic-screening or fluorescent marker of the cell expression of transfection can be screened, but transfection efficiency is low for subsequent screening
Cause very big difficulty with Colony Culture, and cas9 albumen has certain cytotoxicity, subsequent antibiotic-screening and
Airflow classification also can also have an impact to successful cell is transfected.Therefore it is anxious for improving the transfection efficiency of CRISPR/Cas9 plasmid
Problem to be solved.
The method of current existing plasmid transfection mainstream includes liposome transfection, cationic non-liposomal transfection, calcium phosphate turn
The methods of dye.These methods have the advantages that respective, while there is also disadvantages.For example liposome transfection is high-efficient, but for
The cell transfecting efficiency of some primary cells or the secretory of difficult transfection is lower.Electroporation is also referred to as electrotransfection, is to pass through height
The electric field action of intensity makes to form reversible instant channel on cell membrane, and plasmid is entered under the action of electric field by channel thin
It is intracellular.Electroporation transfection compared to other transfection methods have it is easy to operate, high-efficient for primary cell and difficult transfection cell,
The advantages that reproducible.But electroporation transfection is larger for different cell line transfection parameter differences, it is therefore desirable to for not
With cell line carry out electrotransfection parameter optimization, at present the cell with electroporation of comparative maturity be all usual cell system or
Person's primary cell, it is less using electroporation to cell lines derived from pancreatic islets, electrotransfection parameter is not carried out yet to it and is optimized.In addition plasmid
Purity and the efficiency that electricity can all be turned such as concentration, the state of cell have an impact.Generally guaranteeing when electricity turns parameter optimization
Under the premise of transfected plasmids purity levels and cell state, by the fixed aperture time, grope different perforation voltage gradients, with
Grope the optimal perforation voltage of cell line.
According to above-mentioned, the present invention is for the difficult transfection of mouse islets element tumor beta Cell of islet (Beta-TC-6) and CRISPR/
The larger feature of Cas9 plasmid molecule takes electroporation to carry out the delivering of Cas9 plasmid, and different perforation voltage ladders is arranged
Degree carries out electricity and turns the optimization of parameter, efficiently delivers in mouse islets element tumor beta Cell of islet (Beta-TC-6) to realize
CRISPR/Cas9 plasmid；Meanwhile the present invention is overcome by combining CRISPR/Cas9 gene editing system and electroporation
The characteristics of CRISPR/Cas9 plasmid difficulty transduction, carries out CRISPR/Cas9 gene for conventional Molecular Biology Lab
Editing system provides practical ways.
Summary of the invention
Technical problem to be solved by the present invention lies in: it provides based on CRISPR/Cas9 system in Beta-TC-6 cell
Strain in reject Sidt2 genetic method, come overcome CRISPR/Cas9 plasmid difficulty transduce the characteristics of, to realize in mouse islets
CRISPR/Cas9 plasmid is efficiently delivered in plain tumor beta Cell of islet (Beta-TC-6), for conventional Molecular Biology Lab
Carry out CRISPR/Cas9 gene editing system and provides practical ways.
In order to solve the above technical problems, the technical scheme is that based on CRISPR/Cas9 system in Beta-TC-6
Sidt2 genetic method is rejected in cell strain, steps are as follows for specific experiment:
(1), sgRNA oligo DNA sequence dna synthesizes；Mouse Sidt2 gene is set using sgRNA Photographing On-line tool needle
The end of oligo DNA, sgRNAssDNA sequence F chain 5 ' that meter length is 20bp or so need to add CACC so as to and BbsI
Carrier after digestion is complementary, it should be noted that and first base of F chain must be G, if the first of the Guide sequence chosen
A base is not G, can add a G voluntarily to enhance the activity of U6 promoter, 5 ' end addition AAAC of R chain.In addition, need to be
Site upstream and downstream respectively designs a primer, is used for subsequent detection positive colony, and primer can expand the DNA fragmentation of about 300bp, upstream
Primer is away from mutational site about 100bp, and downstream primer is away from mutational site about 200bp.Sequence after design is synthesized, grade is purified
It Wei not PAGE；
(2), oligo DNA anneals: the sgRNA of synthesis is diluted to final concentration of 100uM, prepares annealing reaction system: dilute
The sgRNA released each 1ul, 2ulAnnealing Buffer for DNA Oligos (5X), supplies 10ul with ultrapure water.PCR
Room temperature cooling two hours are then taken out in 95 DEG C 10 minutes in instrument.Dilution 10 times after be stored in -20 DEG C it is spare；
(3), the building of px459 recombinant plasmid: px459 plasmid is the expression vector of the sgRNA skeleton containing U6 promoter.
(A) linearisation of px459 carrier: px459 carrier is linearized using BbsI restriction enzyme, is returned using plastic recovery kit
Receive linearisation px459 carrier.(B) sgRNA is connect with linearisation px459: reaction system is configured according to T4 ligase specification,
It is reacted 3 hours for 25 DEG C in PCR.(C) connection product transformed competence colibacillus cell DH5 α, 37 DEG C of overnight incubations.Next day picking colony shakes
Bacterium extracts plasmid；
(5), the verifying of px459 recombinant plasmid: plasmid send biotech firm to be sequenced, and sequencing primer, which is that U6 promoter is general, to be drawn
Object identifies whether sgRNA is connected correctly in carrier according to sequencing result.Successful plasmid is sequenced and is named as px459-Sidt2,
Using removing endotoxin plasmid a large amount of extracts kits extracting plasmids, it is desirable that plasmid concentration is greater than 1ug/ul and purity reaches electricity and turns
Dye requires；
(6), the culture of Beta-TC-6 cell: cell culture condition is the DMEM high glucose medium containing 15% fetal calf serum,
Cell confluency degree reaches 70% or so and is passed on；
(7), the electricity of px459-Sidt2 plasmid turns: cell fusion degree reaches 80% or so before electricity turns, after trypsin digestion cell
It is 1x106/90ul that Opti-MEM, which is added, and becomes single cell suspension and adjusts cell quantity, and it is outstanding to draw 90ul cell
Liquid, which is added after 10ul plasmid mixes gently, to be added in electric revolving cup.Setting imports electricity and turns parameter: pulse voltage 20V, burst length
For 20ms, pulse spacing 50ms, pulse number is 5 times, voltage attenuation 40%, and electroporation mode is positive direction.Perforation electricity
Turn parameter are as follows: fixed pulse time 5ms, pulse spacing 50ms, pulse number 2 times, voltage attenuation 10%, electroporation mode is
Positive direction, setting pulse voltage gradient are 125V, 150v, 175v, 200v.The cell for completing electrotransfection was cultivated in six orifice plates
Night；
(8), the screening of positive cell: 48 hours addition puromycins of electrotransfection to final concentration of 3ug/ml are screened
(need to carry out groping for puromycin screening gradient in advance, this laboratory mice insulinoma beta Cell of islet Beta-TC-6's
Screening concentration is 3ug/ml), regular culture conditions are changed to after 72 hours, amplification freezes；
(9), T7 enzyme and Western blot detection: wild-type cell and positive cell a part are extracted using kit
DNA, a part extract whole-cell protein using cell pyrolysis liquid.The DNA of extraction is carried out by target area of the PCR to sgRNA
Amplification, 54 DEG C of the annealing temperature of amplimer.(1) T7 restriction analysis: being operated to specifications, and the product after digestion passes through
2% agarose gel analysis.(2) Western blot is detected: being measured protein concentration using micro-spectrophotometer, is taken equivalent
Albumen albumen sample-loading buffer is added, SDS-PAGE electrophoresis is carried out after boiling, it is wet after the completion of electrophoresis to go on PVDF, with 5%
Skimmed milk power close a hour, primary antibody be incubated overnight, progresss secondary antibody incubation in second day, be added exposure substrate after use oversoul
Quick Multifunctional imaging instrument is exposed, using β-actin as internal reference；
(10), px459-Sidt2 clone building: px459 empty carrier is after BbsI linearization for enzyme restriction, from Ago-Gel
Linearized fragment after recycling digestion.The sgRNA double-strand of annealing is connected with the linearisation px459 carrier of recycling, is constructed
Px459-Sidt2 recombinant plasmid.The px459-Sidt2 of building shows that the position of insetion sequence, direction are correct through sequencing, recombinates
Plasmid px459-Sidt2 is constructed successfully；
(11), T7E1 enzyme and Western blot detection mouse Sidt2 gene knockout effect: T7E1 enzyme the results show that
The mixing clone cell of electrotransfection px459-Sidt2 has the band significantly cut, i.e., alkali occurs in mixing clone cell
Base mispairing shows that px459-Sidt2 realizes targeting knockout in Beta-TC-6 cell.Western blot detection knocks out effect,
Compare as the result is shown with wild-type cell, mixing clone cell Sidt2 protein expression significantly reduces (t=6.668, P=
0.0026).Further experiment needs to carry out Colony Culture, to obtain knocking out the clonal cell line that efficiency is 100%.
Further, material used in the step (1)-step (11) is plasmid vector pSpCas9 (BB) -2A-
Puro(PX459)(Addgene plasmid#62988)；Competent cell DH5 α；Beta-TC-6 mouse islets element tumor pancreas islet β
Cell.
Further, main agents and instrument used in the step (1)-step (11) are BbsI restriction enzyme
Enzyme, T4DNA Ligase, T7 endonuclease I；Opti-MEM subtracts blood serum medium；Fetal calf serum (standard level)；Annealing
Buffer for DNA Oligos (5X), β-actin antibody；Sidt2 rabbit polyclonal antibody；PCR instrument；Protein blot system；
21 high efficiency gene transfection system of NEPA；Electric revolving cup.
Further, antibiotic-screening after electrotransfection after described (7) step, when pulse voltage is 150V as the result is shown
Transfection efficiency highest and cell state is not substantially change, it is thus determined that Beta-TC-6 cell electrotransfection px459-Sidt2 is most
Excellent electricity turns perforation electricity and turns parameter are as follows: pulse voltage 150V, burst length 5ms, pulse spacing 50ms, pulse number 2 times, voltage declines
Subtract 10%, electroporation mode is positive direction.
Sidt2 genetic method should be rejected in Beta-TC-6 cell strain based on CRISPR/Cas9 system, for mouse pancreas
The difficult feature transfected and CRISPR/Cas9 plasmid molecule is larger of island element tumor beta Cell of islet (Beta-TC-6), by taking electricity
Perforation carries out the delivering of Cas9 plasmid, and different perforation voltage gradient progress electricity is arranged and turns the optimization of parameter, to realize
CRISPR/Cas9 plasmid is efficiently delivered in mouse islets element tumor beta Cell of islet (Beta-TC-6)；Meanwhile pass through by
The characteristics of CRISPR/Cas9 gene editing system and electroporation combine, and overcome the transduction of CRISPR/Cas9 plasmid difficulty, is normal
The Molecular Biology Lab of rule carries out CRISPR/Cas9 gene editing system and provides practical ways, has
High application value.
Detailed description of the invention
Fig. 1 is that successfully building px489-Sidt2 is cloned；
Fig. 2 is that the positive cell after pulse voltage gradient is pressurizeed screening 72 hours is arranged；
Fig. 3 is the knockout effect of T7E1 enzyme and W estem b lot detection mouse Sidt2 gene；
Fig. 4 is mouse Sidt2 sgRNA and verifying primer sequence.
The following detailed description will be further explained with reference to the above drawings.
Specific embodiment
Hereinafter, a variety of specific details are elaborated, in order to provide to the saturating of the concept for constituting described embodiment basis
Thorough understanding.However, it will be apparent to those skilled in the art that described embodiment can be in these no specific details
In some or all situations get off practice.In other cases, well-known processing step is not specifically described.
Embodiment 1
As shown in Figure 1, Figure 2, Figure 3 and Figure 4, it is rejected in Beta-TC-6 cell strain based on CRISPR/Cas9 system
Sidt2 genetic method, steps are as follows for specific experiment:
The synthesis of sgRNA oligo DNA sequence dna；Mouse Sidt2 gene is designed using sgRNA Photographing On-line tool needle and is grown
The end of oligo DNA, sgRNAssDNA sequence F chain 5 ' that degree is 20bp or so need to add CACC so as to BbsI digestion
Carrier afterwards is complementary, it should be noted that first base of F chain must be G, if first alkali of the Guide sequence chosen
Base is not G, can add a G voluntarily to enhance the activity of U6 promoter, 5 ' end addition AAAC of R chain.In addition, need to be on site
A primer is respectively designed in downstream, is used for subsequent detection positive colony, and primer can expand the DNA fragmentation of about 300bp, upstream primer away from
Mutational site about 100bp, downstream primer is away from mutational site about 200bp.Sequence after design is synthesized, purifying rank is
PAGE.Oligo DNA annealing: the sgRNA of synthesis is diluted to final concentration of 100uM, prepares annealing reaction system: diluted
SgRNA each 1ul, 2ulAnnealing Buffer for DNA Oligos (5X), supplies 10ul with ultrapure water.In PCR instrument
95 DEG C 10 minutes, then take out room temperature cooling two hours.Dilution 10 times after be stored in -20 DEG C it is spare.Px459 recombinant plasmid
Building: px459 plasmid be the sgRNA skeleton containing U6 promoter expression vector.(A) it the linearisation of px459 carrier: uses
BbsI restriction enzyme linearizes px459 carrier, recycles linearisation px459 carrier using plastic recovery kit.(B)sgRNA
It is connect with linearisation px459: configuring reaction system according to T4 ligase specification, reacted 3 hours for 25 DEG C in PCR.(C) it connects
Product transformed competence colibacillus cell DH5 α, 37 DEG C of overnight incubations.Next day picking colony shakes bacterium extracting plasmid.Px459 recombinant plasmid
Verifying: plasmid send biotech firm to be sequenced, and sequencing primer is U6 promoter universal primer, whether identifies sgRNA according to sequencing result
It is connected correctly in carrier.Successful plasmid is sequenced and is named as px459-Sidt2, using going endotoxin plasmid largely to extract reagent
Box extracts plasmid, it is desirable that plasmid concentration is greater than 1ug/ul and purity reaches electrotransfection requirement.The culture of Beta-TC-6 cell:
Cell culture condition is the DMEM high glucose medium containing 15% fetal calf serum, and cell confluency degree reaches 70% or so and passed on.
The electricity of px459-Sidt2 plasmid turns: cell fusion degree reaches 80% or so before electricity turns, and Opti-MEM is added after trypsin digestion cell
Becoming single cell suspension and adjusting cell quantity is 1x106/90ul, draws 90ul cell suspension and 10ul plasmid is added
It is added after mixing gently in electric revolving cup.Setting imports electricity and turns parameter: pulse voltage 20V, burst length 20ms, pulse spacing
For 50ms, pulse number is 5 times, and voltage attenuation 40%, electroporation mode is positive direction.Perforation electricity turns parameter are as follows: fixed arteries and veins
Time 5ms, pulse spacing 50ms are rushed, pulse number 2 times, voltage attenuation 10%, electroporation mode is positive direction, setting pulse electricity
Pressing gradient is 125V, 150v, 175v, 200v.The cell of completion electrotransfection overnight incubation in six orifice plates.The sieve of positive cell
Choosing: 48 hours addition puromycins of electrotransfection are screened to final concentration of 3ug/ml (to need to carry out puromycin screening in advance
Gradient is groped, the screening concentration of this laboratory mice insulinoma beta Cell of islet Beta-TC-6 is 3ug/ml), 72 hours
After be changed to regular culture conditions, amplification freezes.T7 enzyme and Western blot detection: wild-type cell and positive cell one
DNA is extracted using kit in part, and a part extracts whole-cell protein using cell pyrolysis liquid.The DNA of extraction passes through PCR pairs
The target area of sgRNA is expanded, 54 DEG C of the annealing temperature of amplimer.(1) it T7 restriction analysis: is grasped to specifications
Make, the product after digestion passes through 2% agarose gel analysis.(2) Western blot is detected: being surveyed using micro-spectrophotometer
Determine protein concentration, takes the albumen of equivalent that albumen sample-loading buffer is added, SDS-PAGE electrophoresis is carried out after boiling, it is wet after the completion of electrophoresis
It goes on PVDF, closes a hour with 5% skimmed milk power, primary antibody is incubated overnight, and progress secondary antibody incubation in second day is added and exposes
It is exposed after light substrate using hypersensitive Multifunctional imaging instrument, using β-actin as internal reference.Px459-Sidt2 clone's building:
Px459 empty carrier after BbsI linearization for enzyme restriction (Figure 1A), the linearized fragment after recycling digestion in Ago-Gel.It will move back
The sgRNA double-strand of fire is connected with the linearisation px459 carrier of recycling, constructs px459-Sidt2 recombinant plasmid.The px459- of building
Sidt2 is sequenced, and the position for showing insetion sequence, direction are correct (Fig. 1 C), and recombinant plasmid px459-Sidt2 constructs successfully (figure
1B).The knockout effect of T7E1 enzyme and Western blot detection mouse Sidt2 gene: T7E1 enzyme is the results show that electrotransfection
The mixing clone cell of px459-Sidt2 has the band significantly cut (Fig. 3 A), i.e., alkali occurs in mixing clone cell
Base mispairing shows that px459-Sidt2 realizes targeting knockout in Beta-TC-6 cell.Western blot detection knocks out effect
Fruit is compared with wild-type cell as the result is shown, and mixing clone cell Sidt2 protein expression significantly reduces (t=6.668, P=
0.0026) (Fig. 3 B, C).Further experiment needs to carry out Colony Culture, so that it is thin to obtain the clone that knockout efficiency is 100%
Born of the same parents' strain, material used in the step (1)-step (11) are plasmid vector pSpCas9 (BB) -2A-Puro (PX459)
(Addgene plasmid#62988)；Competent cell DH5 α；Beta-TC-6 mouse islets element tumor beta Cell of islet, it is described
Main agents used in step (1)-step (11) and instrument are BbsI restriction enzyme, T4DNA Ligase, T7 nucleic acid
Restriction endonuclease I；Opti-MEM subtracts blood serum medium；Fetal calf serum (standard level)；Annealing Buffer for DNA
Oligos (5X), β-actin antibody；Sidt2 rabbit polyclonal antibody；PCR instrument；Protein blot system；21 high efficiency gene of NEPA
Transfection system；Electric revolving cup, antibiotic-screening after electrotransfection after described (7) step, pulse voltage is 150V as the result is shown
When transfection efficiency highest and cell state do not substantially change, it is thus determined that Beta-TC-6 cell electrotransfection px459-
The optimal electricity of Sidt2 turns perforation electricity and turns parameter are as follows: pulse voltage 150V, burst length 5ms, pulse spacing 50ms, pulse number 2
Secondary, voltage attenuation 10%, electroporation mode is positive direction (Fig. 2).
Sidt2 genetic method should be rejected in Beta-TC-6 cell strain based on CRISPR/Cas9 system, for mouse pancreas
The difficult feature transfected and CRISPR/Cas9 plasmid molecule is larger of island element tumor beta Cell of islet (Beta-TC-6), by taking electricity
Perforation carries out the delivering of Cas9 plasmid, and different perforation voltage gradient progress electricity is arranged and turns the optimization of parameter, to realize
CRISPR/Cas9 plasmid is efficiently delivered in mouse islets element tumor beta Cell of islet (Beta-TC-6)；Meanwhile pass through by
The characteristics of CRISPR/Cas9 gene editing system and electroporation combine, and overcome the transduction of CRISPR/Cas9 plasmid difficulty, is normal
The Molecular Biology Lab of rule carries out CRISPR/Cas9 gene editing system and provides practical ways, has
High application value.
The present invention is not limited to above-mentioned specific embodiment, those skilled in the art from the above idea,
Without creative labor, the various transformation made are within the scope of the present invention.
Claims (4)
1. rejecting Sidt2 genetic method in Beta-TC-6 cell strain based on CRISPR/Cas9 system, specific experiment step is such as
Under:
(1), sgRNA oligo DNA sequence dna synthesizes；Mouse Sidt2 gene is designed using sgRNA Photographing On-line tool needle and is grown
The end of oligo DNA, sgRNAssDNA sequence F chain 5 ' that degree is 20bp or so need to add CACC so as to after BbsI digestion
Carrier it is complementary, it should be noted that first base of F chain must be G, if first base of the Guide sequence chosen
It is not G, a G can be added voluntarily to enhance the activity of U6 promoter, 5 ' end addition AAAC of R chain.In addition, need to be above and below site
One primer of each design is swum, subsequent detection positive colony is used for, primer can expand the DNA fragmentation of about 300bp, and upstream primer is away from prominent
Displacement point about 100bp, downstream primer is away from mutational site about 200bp.Sequence after design is synthesized, purifying rank is
PAGE；
(2), oligo DNA anneals: the sgRNA of synthesis is diluted to final concentration of 100uM, prepares annealing reaction system: diluted
SgRNA each 1ul, 2ulAnnealing Buffer for DNA Oligos (5X), supplies 10ul with ultrapure water.In PCR instrument
95 DEG C 10 minutes, then take out room temperature cooling two hours.Dilution 10 times after be stored in -20 DEG C it is spare；
(3), the building of px459 recombinant plasmid: px459 plasmid is the expression vector of the sgRNA skeleton containing U6 promoter.(A)
The linearisation of px459 carrier: px459 carrier is linearized using BbsI restriction enzyme, uses plastic recovery kit exhausting line
Property px459 carrier.(B) sgRNA connect with linearisation px459: according to T4 ligase specification configuration reaction system, in PCR
25 DEG C are reacted 3 hours.(C) connection product transformed competence colibacillus cell DH5 α, 37 DEG C of overnight incubations.Next day picking colony shakes bacterium extracting
Plasmid；
(5), the verifying of px459 recombinant plasmid: plasmid send biotech firm to be sequenced, and sequencing primer is U6 promoter universal primer, root
Whether it is connected correctly in carrier according to sequencing result identification sgRNA.Successful plasmid is sequenced and is named as px459-Sidt2, uses
Remove a large amount of extracts kit extracting plasmids of endotoxin plasmid, it is desirable that plasmid concentration is greater than 1ug/ul and purity reaches electrotransfection and wants
It asks；
(6), the culture of Beta-TC-6 cell: cell culture condition is the DMEM high glucose medium containing 15% fetal calf serum, cell
Convergence degree reaches 70% or so and is passed on；
(7), the electricity of px459-Sidt2 plasmid turns: cell fusion degree reaches 80% or so before electricity turns, and is added after trypsin digestion cell
It is 1x106/90ul that Opti-MEM, which becomes single cell suspension and adjusts cell quantity, draws 90ul cell suspension and is added
10ul plasmid is added in electric revolving cup after mixing gently.Setting imports electricity and turns parameter: pulse voltage 20V, burst length 20ms,
Pulse spacing is 50ms, and pulse number is 5 times, and voltage attenuation 40%, electroporation mode is positive direction.Perforation electricity turns parameter
Are as follows: fixed pulse time 5ms, pulse spacing 50ms, pulse number 2 times, voltage attenuation 10%, electroporation mode is positive direction,
It is 125V, 150v, 175v, 200v that pulse voltage gradient, which is arranged,.The cell of completion electrotransfection overnight incubation in six orifice plates；
(8), the screening of positive cell: 48 hours addition puromycins of electrotransfection to final concentration of 3ug/ml are screened (in advance
Need to carry out groping for puromycin screening gradient, the screening of this laboratory mice insulinoma beta Cell of islet Beta-TC-6 is dense
Degree is 3ug/ml), regular culture conditions are changed to after 72 hours, amplification freezes；
(9), T7 enzyme and Western blot detection: wild-type cell and positive cell a part use kit to extract DNA,
A part extracts whole-cell protein using cell pyrolysis liquid.The DNA of extraction is expanded by target area of the PCR to sgRNA,
54 DEG C of the annealing temperature of amplimer.(1) T7 restriction analysis: being operated to specifications, and the product after digestion passes through 2% fine jade
Sepharose analysis.(2) Western blot is detected: being measured protein concentration using micro-spectrophotometer, is taken the albumen of equivalent
Albumen sample-loading buffer is added, SDS-PAGE electrophoresis is carried out after boiling, it is wet after the completion of electrophoresis to go on PVDF, with 5% degreasing
Milk powder closes a hour, and primary antibody is incubated overnight, progress secondary antibody incubation in second day, uses the more function of hypersensitive after exposure substrate is added
Energy imager is exposed, using β-actin as internal reference；
(10), px459-Sidt2 clone building: px459 empty carrier is recycled from Ago-Gel after BbsI linearization for enzyme restriction
Linearized fragment after digestion.The sgRNA double-strand of annealing is connected with the linearisation px459 carrier of recycling, constructs px459-
Sidt2 recombinant plasmid.The px459-Sidt2 of building shows that the position of insetion sequence, direction are correct through sequencing, recombinant plasmid
Px459-Sidt2 is constructed successfully；
(11), the knockout effect of T7E1 enzyme and Western blot detection mouse Sidt2 gene: T7E1 enzyme is the results show that electricity turns
The mixing clone cell of dye px459-Sidt2 has the band significantly cut, i.e., base mistake occurs in mixing clone cell
Match, shows that px459-Sidt2 realizes targeting knockout in Beta-TC-6 cell.Western blot detection knocks out effect, as a result
Display is compared with wild-type cell, and mixing clone cell Sidt2 protein expression significantly reduces (t=6.668, P=0.0026).Into
The experiment of one step needs to carry out Colony Culture, to obtain knocking out the clonal cell line that efficiency is 100%.
2. rejecting the gene side Sidt2 in Beta-TC-6 cell strain based on CRISPR/Cas9 system as described in claim 1
Method, which is characterized in that material used in the step (1)-step (11) is plasmid vector pSpCas9 (BB) -2A-Puro
(PX459)(Addgene plasmid#62988)；Competent cell DH5 α；Beta-TC-6 mouse islets element tumor beta Cell of islet.
3. rejecting the gene side Sidt2 in Beta-TC-6 cell strain based on CRISPR/Cas9 system as described in claim 1
Method, which is characterized in that main agents used in the step (1)-step (11) and instrument be BbsI restriction enzyme,
T4 DNA Ligase, T7 endonuclease I；Opti-MEM subtracts blood serum medium；Fetal calf serum (standard level)；Annealing
Buffer for DNA Oligos (5X), β-actin antibody；Sidt2 rabbit polyclonal antibody；PCR instrument；Protein blot system；
NEPA21 high efficiency gene transfection system；Electric revolving cup.
4. rejecting the gene side Sidt2 in Beta-TC-6 cell strain based on CRISPR/Cas9 system as described in claim 1
The cultural method of method, which is characterized in that antibiotic-screening after electrotransfection after described (7) step, pulse is electric as the result is shown
Pressure be 150V when transfection efficiency highest and cell state do not substantially change, it is thus determined that Beta-TC-6 cell electrotransfection
The optimal electricity of px459-Sidt2 turns perforation electricity and turns parameter are as follows: pulse voltage 150V, burst length 5ms, pulse spacing 50ms, pulse
Number 2 times, voltage attenuation 10%, electroporation mode is positive direction.
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