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CN107841481A - A kind of culture medium for unicellular culture and its preparation method and application - Google Patents

A kind of culture medium for unicellular culture and its preparation method and application Download PDF

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CN107841481A
CN107841481A CN201610831445.3A CN201610831445A CN107841481A CN 107841481 A CN107841481 A CN 107841481A CN 201610831445 A CN201610831445 A CN 201610831445A CN 107841481 A CN107841481 A CN 107841481A
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culture medium
culture
unicellular
immortalized cells
cell
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向熙
李聪慧
洪伟芳
李勇
陈茜
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Shenzhen Huada gene agriculture Holding Co., Ltd.
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BGI ARK BIOTECHNOLOGY Co Ltd SHENZHEN
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Abstract

This application discloses a kind of culture medium for unicellular culture and its preparation method and application.The culture medium for unicellular culture of the application, including ordinary culture medium and additive, additive is the immortalized cells secretion for extracting from immortalized cells cultured products, and immortalized cells is the unicellular species identical immortalized cells for being cultivated used in the culture medium of unicellular culture.The culture medium of the application, add immortalized cells secretion, make primary cell at a low density, also can proper splitting propagation, solve the problems, such as that fibroblast low-density can not meet that monoclonal cell is selected.Using the culture medium of the application, without marker gene, the risk that marker gene causes endogenous gene function to be destroyed is avoided, is laid a good foundation for gene editing cell commercial applications.Due to being screened without drug resistance, reagent cost is reduced, shortens screening time, avoided medicine to cellular damage, wild type state and multiple fission ability are kept beneficial to cell.

Description

A kind of culture medium for unicellular culture and its preparation method and application
Technical field
The application is related to field of cell culture, more particularly to a kind of culture medium for unicellular culture and its preparation side Method and application.
Background technology
Gene editing refers to by accurately nuclease gene shearing technique, according to the wish of the mankind, to particular organisms Genomic dna sequence is modified, and conventional technology has Zinc finger nuclease (ZFN), activating transcription factor sample effector nuclease (TALENs) and the short palindrome in rule recurrence interval repeats (Clustered Regularly Interspersed Short Palindromic Repeats, CRISPR/cas9) system.Wherein CRISPR/cas9 is because build simple, DNA shear efficiencies It is high and be rapidly widely used in each field, such as agricultural, medical science, environmental protection, there are huge scientific research and business application to develop Potentiality.
A kind of adaptive immunity defence that CRISPR/Cas9 is bacterium and archeobacteria is formed during long-term evolution, can For resisting the virus of invasion and exogenous DNA.System is made up of two parts, as shown in figure 1, one is to carry U6 promoter energy tables Up to guidRNA carrier, one is the carrier for expressing spCas9 albumen, and two parts can also be configured to coexpression vector.Design The guidRNA carriers of targeting are built, import that animal or plant is intracellular jointly with spCas9 carriers, orientable modification animals and plants Gene, reach the purpose of genetic modification.
The gene editing of cell is carried out using CRISPR/Cas9 or other technologies, there is huge market application value.Embody , (1) is oriented transformation to the cytogene of the herding class animal such as pig, ox, sheep, and by animal cloning technology, it is available Herding class animal with fixed merit, such as the CD163 genes of knock-out pig, obtains the pig of anti-blue otopathy, can save every year Save substantial amounts of vaccine expense；The MSTN genes of knock-out pig, it can obtain the preferable bacon hogs of meat；Fixed point is transferred to UCP1 genes Pig, the freezing tolerance of its piglet can be remarkably reinforced.(2) gene site-directed transformation is carried out to the industrialization cell such as CHO, can obtain certain Kind specific protein or antibody high yield or safer cell line, as knocked out the FUT8 genes in Chinese hamster ovary celI, CHO productions can be eliminated The glycosylation phenomenon of obtained immunoglobulin, reduce cytotoxicity when immunoglobulin is used for human body.
At present, the positive cell strain of gene editing cell line is mainly fibroblast, and positive cell strain screening generally needs Introduce drug selectable marker gene, such as neomycin resistance gene NeoR, puromycin resistance gene PuroR.These resistances Gene integration enters in the genome of target cell, and after forming stable expression, positive cell strain could be obtained by drug screening.Such as When doing the screening of CRISPR/Cas9 Knockout cells strains, need while guidRNA expression vectors are imported in cell, SpCas9 expression vectors and the expression vector with NeoR resistant genes；Wherein the first two is circular vectors, will not be integrated into thin Born of the same parents' genome, and NeoR expression vectors, then for linearized vector, it is necessary to random integration enters in cellular genome, it could carry out G418 drug screenings, its cell screening process are as shown in Figure 2.However, the medicine of non-target gene is introduced in cellular genome Resistant gene, these resistance gene fragments radom insertion, copy number in genome are failed to understand, while may destroy endogenous work( Can gene, add gene editing technology be used for eat, medicinal risk, be unfavorable for later stage commercial application and popularization；Therefore, People be more desirable to only target gene is introduced in cell, and be not inserted into other independent basises because.
If while target gene is imported, riddled basins are not introduced, i.e., do not pass through medicine or other mark bases Because of the method selected clone of screening, then need to divide disk culture after being diluted cell, select unicellular identified.Into fibre Tie up ability of cell proliferation it is relatively weak, it is necessary to compared with many cells culturing in groups, pass through intercellular constantly material and signaling molecule Exchange, can just have preferable multiple fission ability.Fibroblast is cooked into unicellular culture in the state of very dilute, then breeds energy Power is very poor, and the single cell clone that can not form densification or the number for forming monoclonal are seldom.Fibroblast typically need to be closeer Cell concentration under, gradually apply the pressure of drug screening, the single cell clone that can be just stablized, and these clones are bands There is drug resistant markers' gene.Also, for fibroblast, if not introducing drug resistance gene, dividing disk in 10cm During Tissue Culture Dish, if cell density is inadequate, easily cause single cell clone growth ability poor, can not be met and select gram Grand cell density；If dividing cell density during disk excessive, single cell clone easily links again forms non-monoclonal, final The cell clone impure to genotype, is also not used to the research and production in later stage.
Therefore, the gene editing primary cultured cell line of screening marker-free how is obtained at present, is still scientific research and production Difficult point.
The content of the invention
The purpose of the application is to provide a kind of new culture medium for unicellular culture, and its preparation method and application.
To achieve these goals, the application employs following technical scheme：
This application discloses a kind of culture medium for unicellular culture, including ordinary culture medium and additive, additive To extract from the immortalized cells secretion of immortalized cells cultured products, immortalized cells is the culture for unicellular culture The unicellular species identical immortalized cells of the used culture of base.
It should be noted that the key of the application makes in the addition of immortalized cells secretion in ordinary culture medium Primary cell in the case where density is relatively low, also can normal division growth, until being formed slender available for genotype identification Born of the same parents clone；It is weak to solve fibroblast proliferation ability, the problem of monoclonal cell is selected can not be met at a low density.This Shen In a kind of implementation please, fibroblast, also can be just under the low density cell culture of 400-600 cell/10cm culture dishes Normal division growth, form the single cell clone available for genotype identification.
It should also be noted that, wherein ordinary culture medium is for existing in general culture medium, such as compare Common ordinary culture medium is the DMEM culture mediums added with hyclone, or adds hyclone, growth factor, glutamy The DMEM culture mediums of amine and NEAA.Wherein, NEAA is non-essential amino mixture acid, is that several nonessential amino acid combine , in ordinary culture medium, it can be selected to use NEAA according to specific use demand, or can not also add；But this There is addition in the preferred scheme of application.DMEM culture mediums are dulbecco's modified eagle medium abbreviations, are A kind of culture medium containing various amino acid and glucose, is developed on the basis of MEM culture mediums.
In the preferable scheme of the application, ordinary culture medium is added with hyclone, growth factor, glutamine and NEAA DMEM culture mediums.
It should be noted that in the application, immortalized cells is for being cultivated used in the culture medium of unicellular culture Unicellular species identical immortalized cells, in particular to, if for example, to prepare culture the fibroblastic unicellular training of pig Foster culture medium, then using pig immortalized cells secretion, i.e. PK15 immortalized cellses secretion；If prepare culture The culture medium of the unicellular culture of human fibroblasts, then using people immortalized cells secretion, as 293T immortalize it is thin Intracrine thing；If the culture medium of the unicellular culture of culture mouse fibroblast cell is prepared, using the immortalized cells of mouse Secretion, such as Hepa immortalized cells secretion.
It should also be noted that, ordinary culture medium is for common cell culture medium, wherein hyclone and Growth factor is its most common and most basic component.Growth factor can carry out adding for selectivity according to different culture cells Add.
Preferably, growth factor bFGF.
It should be noted that the positive cell strain of gene editing cell line is mainly fibroblast, and therefore, growth factor For bFGF.Wherein, bFGF is a kind of material that can promote fibroblastic growth, because it is to sour and thermo-responsive, isoelectric point About 9.6, in alkalescence, therefore referred to as basic FGF, abridge bFGF.
Preferably, hyclone accounts for the 5-20% of culture medium cumulative volume, and growth factor accounts for the 2-5% of culture medium cumulative volume, Glutamine and NEAA account for the 0-5% of culture medium cumulative volume respectively.
It should be noted that wherein NEAA and glutamine can select to use as the case may be or without using, therefore, Its dosage is 0-5%；But both there is addition in the preferred scheme of the application.
The another side of the application discloses the preparation method of the culture medium for unicellular culture of the application, including to forever Hyclone, growth factor, glutamine and NEAA are added in OEG cell cultured products, forms the training for unicellular culture Support base；Wherein, immortalized cells secretion is contained in immortalized cells cultured products；The preparation side of immortalized cells cultured products Method is to be cultivated immortalized cells in immortalized cells culture medium, treats that immortalized cell line grows to 50%-80% remittances When right, nutrient solution is collected, obtains filtrate with membrane filtration, the filtrate is immortalized cells cultured products.
It should be noted that the immortalized cells secretion of the application, is that immortalized cell line grows to 50%-80% remittances When right, by the secretion extracted in its culture medium.The application studies have shown that growth converge spend it is low, then may collect simultaneously The various factor quantity secreted in the culture medium of filtering by immortalized cells are inadequate, are not suitable for fibroblastic unicellular training Support；Height is spent if converged, such as more than 90%, then the nutritional ingredient consumption in possible culture medium is excessive, and immortalized cells Metabolism discharges some harmful substances, is also unfavorable for the unicellular culture in later stage.50-80% degree of converging is immortalized cells growth Vigorous period is just started, and has not been many to the nutrient consumption of the culture medium in former ware；Therefore, during 50%-80% degree of converging Its secretion is extracted, effect is preferable, and degree of converging is too high or too low, may all influence unicellular culture effect.
The culture medium for unicellular culture for simultaneously disclosing the application again of the application is compiled in the gene of pig, people or mouse Collect the application in cell line positive cell strain screening.
It should be noted that the fibroblast of mouse and pig, the condition of culture of immortalized cells are one during experiment Sample, the fibroblast of people, immortalized cells condition of culture are also close, therefore, the training for unicellular culture of the application Foster base can apply to the gene editing cell line positive cell strain screening of pig, people or mouse.
It is appreciated that in the application of some comparative maturities, can also be by the culture for unicellular culture of the application Base is prepared into kit or the special equipment dedicated for unicellular culture, the gene editing cell for pig, people or mouse It is positive cell strain screening.
The application's simultaneously discloses a kind of kit for unicellular culture again, wherein being just used for containing the application The culture medium of unicellular culture.
The application's simultaneously discloses a kind of culture medium for the unicellular culture of pig fibroblast, including common training again It is the PK15 immortalized cells secretion for extracting from PK15 immortalized cells cultured products to support base and additive, additive.
Preferably, in the culture medium of the unicellular culture of pig fibroblast, its ordinary culture medium to include accounting for culture medium Cumulative volume 5-20% hyclone, 2-5% bFGF growth factors, 0-5% glutamine and 0-5% NEAA.
Due to being using the beneficial effect of above technical scheme, the application：
The culture medium for unicellular culture of the application, with the addition of immortalized cells secretion wherein, make primary thin Born of the same parents in the case where density is relatively low, also can normal division growth, formed available for genotype identification single cell clone；Solve Fibroblast proliferation ability is weak, can not meet the problem of monoclonal cell is selected at a low density；Realize into fiber finer The positive cell strain screening of the gene editing cell line of the screening marker-free of born of the same parents.Using the training for unicellular culture of the application Base is supported, without introducing drug resistance gene or other marker gene, will not be produced due to marker gene in genome with the machine transplanting of rice Endogenous gene function is destroyed caused by entering, and reduces risk cost, and base has been established for the commercial applications of gene editing cell Plinth.Also, due to drug resistance screening need not be carried out, reagent cost is both reduced, shortens whole gene editor's cell again Screening time, it is often more important that, damage of the medicine to cell is avoided, is advantageous to form and propagation point that cell keeps wild type Split ability.
Brief description of the drawings
Fig. 1 is CRISPR/Cas9 system architectures and effect schematic diagram in the application background technology；
Fig. 2 is the positive cell strain of drug resistance gene screening-gene editor's cell line traditional in the application background technology Schematic flow sheet；
Fig. 3 is that PK15 cells are cultivated in PK15 culture mediums to the microscope of 50-60% degree of converging in the embodiment of the present application Observe result figure；
Fig. 4 is the Screening of Media gene editing cell line sun of the unicellular culture using the application in the embodiment of the present application The schematic flow sheet of property cell line；
Fig. 5 is the medium culture microscopy results of 6 days and 8 days that unicellular culture is used in the embodiment of the present application Figure, wherein (a) figure is that two figures in left and right are the result figures cultivated 6 days above in figure, (b) figure is that two figures in left and right are below in figure The result figure of culture 8 days；
Fig. 6 is the electrophoresis result figure of T7 digestions PCR primer in the embodiment of the present application, wherein (a) figure is monoclonal PCR primer The running gel figure of T7 digestions is directly carried out, (b) figure is the electrophoresis of T7 digestions after monoclonal PCR primer mixing wild type PCR primer Glue figure；
Fig. 7 is sequencing comparison result figure in the embodiment of the present application, is compared wherein (a) figure is No. 2 cell clone sequencing results Figure, (b) figure is No. 3 cell clone sequencing result comparison charts, and (c) figure is No. 7 cell clone sequencing result comparison charts, and (d) figure is 9 Number cell clone sequencing result comparison chart, (e) figure is No. 11 cell clone sequencing result comparison charts.
Embodiment
Gene editing cell line it is more commonly used be fibroblast, the positive cell strain screening of gene editing cell line, It is generally necessary to drug selectable marker gene, i.e. drug resistance gene are introduced in fibroblast；But drug resistance gene Introduce for purely screening, not other purposes, or even security risk be present.Therefore, present inventor attempts Drug resistance gene is not introduced directly carries out positive cell strain screening.Positive cell strain screening is directly carried out, it is necessary to transfection Fibroblast be diluted after unicellular culture, obtain single cell clone, then filtered out by identified for genes positive thin Born of the same parents' strain.But find under study for action, fibroblastic multiplication capacity it is weaker, it is necessary to compared with many cells culturing in groups, by thin The continuous material of intercellular and exchanging for signaling molecule, can just there is preferable multiple fission ability.That is, the concentration of dilution culture It is too low, because multiplication capacity is weak, the single cell clone of densification can not be formed, follow-up single cell clone gene mirror can not be carried out It is fixed, it also can not just screen positive cell strain；And diluting the excessive concentration of culture, then be readily attached together can not for each cell clone Obtain single cell clone.
Studied based on more than and understanding, the application investigated a kind of culture medium dedicated for unicellular culture, that is, exist On the basis of existing culture medium, the immortalized cells secretion of corresponding species is added so that even if fibroblast is relatively low Concentration under, can also have preferable multiple fission ability, so as to formed densification single cell clone, can pick out and be used for The single cell clone of genotype identification.
Using the culture medium for unicellular culture of the application, due to drug resistance screening need not be carried out, examination is reduced Agent cost, the screening time of whole gene editor's cell is shortened, in a kind of implementation of the application, it is only necessary to be within 6-8 days The screening of positive cell strain can be completed, and the method for conventional drug resistance gene screening at least needs 8-12 days.
It should be noted that being related to the culture medium of three concepts in the application altogether, it is respectively：Ordinary culture medium, it is used for Culture medium, the immortalized cells culture medium of unicellular culture.Wherein, ordinary culture medium refers to the training in general cell culture Support base；Culture medium for unicellular culture is the slender of the positive cell strain screening for gene editing cell line of the application The culture medium of born of the same parents' culture；Immortalized cells culture medium refers to the culture medium for cultivating immortalized cells.
The application is described in further detail below by specific embodiments and the drawings.Following examples are only to the application It is further described, should not be construed as the limitation to the application.
Embodiment
The gene editing cell line positive cell strain screening of this example does not use any marker gene, as shown in figure 4, directly right The fibroblast for turning core is diluted, and the culture medium for unicellular culture prepared using this example, carries out at a low density Colony Culture, genotype identification directly then is carried out to monoclonal, the monoclonal expansion for being accredited as positive cell strain is cultivated, Preservation.For whole process without using drug resistance gene or other marker gene, experiment in detail is as follows.
First, prepared by culture medium
The culture medium for unicellular culture of this example is for pig Fibroblast cell-culture, therefore, immortalized cells Using PK15 cells, for the culture medium of unicellular culture, hereinafter referred to as unicellular culture medium, its preparation method is specific as follows：
Prepare PK15 culture mediums, i.e. immortalized cells culture medium：10%FBS+90%DMEM, specifically, the FBS by 10mL It is added in 90mL DMEM, and 15-30 minutes is preheated in 37 DEG C of water-baths, is configured to 100mL PK15 culture mediums.
Then recovery immortalized cell line PK15 cells, i.e., the PK15 cells frozen is taken out from liquid nitrogen, are immediately placed in 37 In DEG C water-bath, cryopreservation tube is constantly rocked, until the cell fast melt in cryopreservation tube；By the PK15 cells in cryopreservation tube, and Former frozen stock solution, it is transferred in the centrifuge tube of 1.5mL sterilizings, is placed in a centrifuge with liquid-transfering gun, 1500 revs/min centrifuge 5 minutes, Supernatant is abandoned in suction, leaves and takes cell precipitation；According to cell concentration, gently cell is suspended with 0.5-1mL PK15 culture mediums, mixed； It is transferred in 12 orifice plates or 6 orifice plates and is cultivated with liquid-transfering gun, changes a PK15 culture medium within every 2 days.This example specifically uses 1mL culture mediums Suspension cell, it is placed in 12 orifice plate cultures.Until when PK15 cell growths are to degree of converging 50-90%, had digestive transfer culture is to a diameter of Cultivated in 15cm Tissue Culture Dish；Concretely comprise the following steps：Culture medium in 12 orifice plates is inhaled and abandoned totally, with 1mL PBS cell 3 times, add 100 μ L 0.25%EDTA-trypsin, be placed in 37 DEG C digestion 3-5 minutes, micro- Microscopic observation cell rounding and After suspension, 900 μ L PK15 culture mediums are added in the orifice plate, and gently piping and druming is mixed, and the cell that 15cm is transferred to liquid-transfering gun is trained Support in ware, and add 80mL PK15 culture mediums, after gently mixing, be placed in 37 DEG C, 5%CO2Incubator in cultivate.See daily The degree of converging to 50-60% up to PK15 cell growths is examined, as shown in figure 3, this example is specifically, growth reaches 50- after 2 days 60% density.The degree of converging drawn with 50mL syringes in 15cm culture dishes reaches 50-60% PK15 cell cultures, so One 0.8 μm of filter membrane is plugged in injector head afterwards, by the culture medium in syringe slowly by press filtration to 50mL scales from In heart pipe.When the culture medium of filtering has 45mL amounts, supplement 5mL FBS, and supplement addition cumulative volume 1% glutamine and 1% NEAA, the bFGF of supplement addition 2%, that is, obtain the unicellular culture medium of this example, store standby to 4 DEG C of refrigerators.
Wherein, NEAA is original-pack solution (article No. M7145) of the purchase from Sigama Aldrich.Glutamine be 100 × Glutamine, its compound method are：2.922g glutamine is dissolved in tri-distilled water, and moisturizing is made into 200mmol/L's to 100mL Solution, after being sufficiently stirred dissolving, 30 DEG C of heating, filtration sterilization in whipping process, it is packed as mono- bottle of 50mL, -20 DEG C of preservations.
Tested as a comparison in addition, this example employs ordinary culture medium, the preparation of ordinary culture medium includes：By 15mL FBS be added in 81mL DMEM, thereto add cumulative volume 1% glutamine, 1% NEAA and 2% bFGF, so 15-30 minutes are preheated in 37 DEG C of water-baths afterwards, that is, are configured to 100mL ordinary culture medium.
2nd, core is turned
This example is with slender after the Wuzhi Mountain primary fibroblast system LDLR (i.e. LDL receptor) gene knockout Born of the same parents' culture is tested.The guidRNA expression vectors of the pLDLR genes prepared (are purchased from CMV-Cas9 carriers Addgene), imported by way of consideration convey in the fibroblast of the Wuzhi Mountain.Comprise the following steps that：
A) pLDLR-gRNA is designed:Seq ID No.1:GTTTGTCTGTGACTCGGACCGGG
B) gRNA primers are synthesized (by Beijing, six directions Hua Da Gene Tech. Company Limited synthesizes)
pLDLR-sgRNA-F:Seq ID No.2:ACCGTTTGTCTGTGACTCGGACCG
pLDLR-sgRNA-R:Seq ID No.3:AAAACGGTCCGAGTCACAGACAAA
After gRNA synthesis is returned, primer is dissolved into 10 μM with water, upstream and downstream respectively adds 10 μ L to be well mixed, 95 DEG C, 10min, Room temperature places 30min, is allowed to be formed the gRNA double-stranded DNAs with cohesive end.
C) skeleton connects
Skeleton is to have connected U6 promoters and gRNAtail carrier T, restriction enzyme site BsaI.After skeleton is carried out, connected with T4 Meet 16 DEG C of connection 4h of enzyme.The μ L of reaction system 20 are specifically included：μ L of 10X T4DNA Ligase Buffer 2, skeleton template 50ng, The μ L of gRNA double-strands 50ng, T4DNA Ligase 1, no enzyme water is supplemented to 20 μ L.
D) consideration convey
Consideration convey reagent uses the Basic Fibroblasts Nucleofector Kit (25RCT) of lonza companies of the U.S. Kit, it is specific as follows：
WillSupplement is all added(4 DEG C of stable guarantors in Solution Deposit 3 months)；
6well plates are arrived into primary fibroblast recovery, cell dissociation got off when cell concentration grows to 90%-95%, Culture medium is removed after centrifugation, PBS is washed once；
Prepare a 12well plate and add 500 μ L culture mediums, 37 DEG C of preheatings；
With 100 μ L SupplementCell is resuspended in Solution, and is mixed into total amount and is no more than 5ug Plasmid amount, the guidRNA expression vectors of pLDLR genes and the ratio of CMV-Cas9 carriers are 3:1；
The mixed liquor of cell and plasmid is carefully added into the consideration convey cup of offer, after program U-023 consideration conveys, adds 500 μ L The culture medium of preheating, cell is transferred in preheated 12well plates, shakes up rear quiescent culture at least 24h；
Next day is observed.
3rd, unicellular culture
Fibroblast after consideration convey, first it is placed in 12 orifice plates and cultivates 1-2 days, the list of this example preparation is respectively adopted in culture medium Cell culture medium and ordinary culture medium, are compared.Condition of culture is identical, is 37 DEG C, 5%CO2Incubator in quiescent culture. During the degree of converging to be grown to 60-90%, by the inoculum concentration of 400-600 cell/10cm culture dishes, each control is inoculated in 4-5 In individual 10cm culture dishes, 10mL culture mediums are added in culture dish.This example when growing to 60-90% degree of converging specifically, will turn Nucleus, by Trypsin Induced, after cell counting count board calculates cell quantity, cell is pressed into 400/culture dish, respectively It is passaged in 10cm Tissue Culture Dish, the culture for turning nucleus and adding three unicellular culture mediums of unicellular medium culture In ware, the consideration convey cell of ordinary culture medium culture is added in the culture dish of three ordinary culture mediums, amounts to six culture dishes.
Culture can need not change culture medium in first 4 days, change a subculture within every 2 days later, the amount for changing culture medium is 10mL。
After culture 6-8 days, cell clonal formation situation is observed, and picked clones form is preferably cloned, and is trained in 96 orifice plates Support.This example carries out micro- sem observation to the cell clonal formation situation of culture 6 days and culture 8 days respectively, as a result as shown in figure 5, In figure, (a) figure is that two figures in left and right are the observation result figures cultivated 6 days above, and i.e. two figures in left and right are cultures 8 to (b) figure below It observation result figure.
At the same time, this example is after culture 5-6 days, and statistics can choose the number of cell clones of clone, final system per ware daily It is as shown in table 1 to count result.
Table 1 can choose monoclonal cell quantity
Culture medium No. 1 ware (individual) No. 2 wares (individual) No. 3 wares (individual)
Ordinary culture medium 4 3 6
Unicellular culture medium 19 24 22
Shown by the result of table 1, the unicellular culture medium of this example can pick out more monoclonals, be more suitable for cell list The growth and shaping of clone；Averagely clone that number is ordinary culture medium using the unicellular culture medium of this example 5 times, it is seen then that this example Unicellular culture medium ability of cell proliferation be remarkably reinforced.
In 96 orifice plates during cell growth to 80-90% degree of converging, cell dissociation is passed in 48 orifice plates.Specifically, 96 holes Cell growth refers to the 80- for observing cell attachment covering bottom area under the microscope to the judgement of 80-90% degree of converging in plate 90%.Cell dissociation is passed in 48 orifice plates, abandoned totally specifically, the culture medium in 96 orifice plates is inhaled, the PBS with 100 μ L is clear Wash cell 3 times, add 20 μ L 0.25%EDTA-trypsin, be placed in 37 DEG C of digestion 3-5 minutes, micro- Microscopic observation cell becomes After circle and suspension, 100 μ L PK15 culture mediums are added in the orifice plate, and gently piping and druming is mixed, and 48 hole cells are transferred to liquid-transfering gun In culture plate, and 80 μ L corresponding culture medium is added, gently mixed.Wherein correspond to culture medium to refer to, what is used before is unicellular Culture medium then adds unicellular culture medium, then adds ordinary culture medium using ordinary culture medium before.
In 48 orifice plates during cell growth to 80-90% degree of converging, cell dissociation half is got off, cracks and obtains after centrifugation DNA profiling, genotype identification is done, be left the cell foramen primum culture of half.The specific steps that cracking obtains DNA profiling include, will The cell centrifugation digested, removes supernatant, lysate is added into cell, mixed with cell precipitation, and 55 DEG C of incubations 2 are small When, subsequent 95 DEG C of incubation half an hour, that is, obtain the DNA profiling for being used directly for PCR.During PCR, in 20 μ L PCR reactants In system, the DNA profiling of the 1 foregoing preparations of μ L is added.Wherein, the formula of lysate is to add 1 μ L's in 19 μ L deionized waters Proteinase K so that the final concentration of 1mg/mL of Proteinase K.
All cultures that this example is related to, all it is in 37 DEG C, 5%CO2Incubator in quiescent culture.
4th, genotype identification
The genotype identification of this example, mixed using PCR primer with wild type PCR primer and carry out T7 digestion methods.Specifically It is as follows：
1.PCR is expanded
A) primer information
This example carries out the PCR primer of genotype identification, as shown in table 2, amplified fragments 607bp.
The active primer information table of table 2
B) PCR reaction systems
This example uses the rTaq enzyme reaction solutions of TAKARA companies.The μ L of overall reaction system 20, including：The μ of rTaq enzyme reaction solutions 10 L, the μ L of 10mM LDLR-iden-F 0.4, the μ L of 10mM LDLR-iden-R 0.4, DNA 100ng, supplement sterilizing ddH2O water is to 20 μL。
C) PCR reaction conditions
After having configured PCR reaction solutions, the enterprising performing PCR reaction of PCR instrument is placed on, the reaction condition of this example is：94 DEG C pre- 5min is denatured, subsequently into 35 circulations：94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C of 1min, circulation terminate after 72 DEG C extension 5min, 25℃1min.Reaction terminate after 4 DEG C it is standby.
This example specifically employs 48 orifice plate cultured products of 13 monoclonal cells of unicellular medium culture ware picking Tested, and design 1 water blank control, therefore 14 PCR reactions altogether.
The wild type PCR of this example, primer, reaction system, the reaction condition of use are same as above；The wild type of this example is not Do the PK15 cells of any gene editing processing, template is the base of the PK15 cells not processed used by wild type PCR Because of a group DNA sample.
2.T7 digestions
This example carries out digestion to the PCR primer of 13 monoclonal cells of picking respectively, meanwhile, by part PCR primer with The mixing of wild type PCR primer carries out digestion.It is specific as follows：
The PCR primer digestion system of 13 monoclonal cells is：μ L of T7 enzymes Buffer 2, μ L of monoclonal PCR primer 8, ddH2μ L of O 8, the μ L of T7 enzymes 0.2.In 37 DEG C of isothermal reaction 45min.
Mixing PCR primer digestion system is：μ L of T7 enzymes Buffer 2, μ L of monoclonal PCR primer 8, the μ of wild type PCR primer 8 L, the μ L of T7 enzymes 0.2.In 37 DEG C of isothermal reaction 45min.
After digestion terminates, observed using 2% agarose gel electrophoresis, as a result as shown in fig. 6, (a) figure is in figure Monoclonal PCR primer directly carries out the running gel figure of T7 digestions, and (b) figure is after monoclonal PCR primer mixes wild type PCR primer The running gel figure of T7 digestions.The result of comparison diagram 6 shows that 2,3,6,7, No. 9 monoclonals may be the homozygosis of LDLR gene knockouts Genotype, 11,13 may be the heterozygous genotypes knocked out, it is seen that using the monoclonal cell of the unicellular medium culture of this example 50% positive rate is had more than, the result and the positive rate that drug resistance gene screens are similar.
5th, sequencing compares
Sanger sequencing comparisons are carried out to the LDLR gene locis of 2,3,7,9, No. 11 monoclonal cells.Specifically, using Primer and PCR reaction systems, condition in " four, genotype identification ", the DNA of 2,3,7,9, No. 11 monoclonal cells is carried out PCR is expanded.Pcr amplification product is connected into pMD-18T cloning vectors, then each clone chooses 4 sub- carrier clonings and is Sanger Sequencing, and sequence alignment is carried out using Taqman softwares.PMD-18T cloning vectors are connected and sub- carrier cloning is selected using normal The test method of rule is carried out, not tired herein to state.Sanger sequencings are carried out by Hua Da gene.
Comparison result is sequenced as shown in fig. 7, in figure, (a) is No. 2 cell clone sequencing results, and display genotype is heterozygosis Type (- 54bp, -38bp)；(b) it is No. 3 cell clone sequencing results, display genotype is heterozygous (- 6bp, -12bp)；(c) it is No. 7 cell clone sequencing results, display genotype are heterozygous (- 54bp, -38bp)；(d) it is No. 9 cell clone sequencing results, Show that genotype is homozygous (- 6bp)；(e) be No. 11 cell clone sequencing results, display genotype be heterozygous (- 5bp ,- 7bp).The result further confirms that and illustrates the unicellular culture medium of this example, has filtered out the qualified sun for not adding any mark Property clone.
Above content is to combine the further description that specific embodiment is made to the application, it is impossible to assert this Shen Specific implementation please is confined to these explanations.For the application person of an ordinary skill in the technical field, do not taking off On the premise of conceiving from the application, some simple deduction or replace can also be made, should all be considered as belonging to the protection of the application Scope.

Claims (10)

  1. A kind of 1. culture medium for unicellular culture, it is characterised in that：Including ordinary culture medium and additive, the additive To extract from the immortalized cells secretion of immortalized cells cultured products, the immortalized cells is used for unicellular training to be described The unicellular species identical immortalized cells of the used culture of foster culture medium.
  2. 2. the culture medium according to claim 1 for unicellular culture, it is characterised in that：The ordinary culture medium be containing There are the DMEM culture mediums of hyclone；
    Preferably, growth factor, glutamine and NEAA are also contained in the ordinary culture medium.
  3. 3. the culture medium according to claim 2 for unicellular culture, it is characterised in that：The growth factor is bFGF。
  4. 4. the culture medium for unicellular culture according to Claims 2 or 3, it is characterised in that：The hyclone accounts for The 5-20% of culture medium cumulative volume, growth factor account for the 2-5% of culture medium cumulative volume, and glutamine and NEAA account for culture medium respectively The 0-5% of cumulative volume.
  5. 5. the preparation method of the culture medium for unicellular culture according to claim any one of 1-4, it is characterised in that： Including adding hyclone into immortalized cells cultured products, the culture medium for being used for unicellular culture is formed；Wherein, forever Contain the immortalized cells secretion in OEG cell cultured products；
    The preparation method of the immortalized cells cultured products is to be trained immortalized cells in immortalized cells culture medium Support, when immortalized cell line grows to 50%-80% degree of converging, collect nutrient solution, filtrate, the filtrate are obtained with membrane filtration That is immortalized cells cultured products；
    Preferably, in addition into immortalized cells cultured products growth factor, glutamine and NEAA are added.
  6. 6. the culture medium for unicellular culture according to claim any one of 1-4 is in the gene editing of pig, people or mouse Application in cell line positive cell strain screening.
  7. 7. the culture medium for unicellular culture according to claim any one of 1-4 is preparing the gene of pig, people or mouse Edit the application in cell line positive cell strain screening reagent box or equipment.
  8. A kind of 8. kit for unicellular culture, it is characterised in that：Contain any one of claim 1-4 in the kit The described culture medium for unicellular culture.
  9. A kind of 9. culture medium for the unicellular culture of pig fibroblast, it is characterised in that：Including ordinary culture medium and addition Agent, the additive are the PK15 immortalized cells secretion for extracting from PK15 immortalized cells cultured products.
  10. 10. the culture medium according to claim 9 for the unicellular culture of pig fibroblast, it is characterised in that：It is described Ordinary culture medium includes the paddy ammonia for accounting for culture medium cumulative volume 5-20% hyclone, 2-5% bFGF growth factors, 0-5% The NEAA of acid amides and 0-5%.
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