CN107988267A - A kind of high throughput carries out embryonated egg the electric shifting method of gene editing - Google Patents
A kind of high throughput carries out embryonated egg the electric shifting method of gene editing Download PDFInfo
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Abstract
The present invention relates to medical biotechnology field, more particularly to a kind of high throughput carries out embryonated egg the electric shifting method of gene editing.Wherein, turn culture medium solution to the electricity emphatically and electricity turns parameter and adjusted.Method according to the present invention, turns experiment by electricity, it is found that mouse positive ratio significantly improves 2 times, while positive rate is improved, decreases the workload of later stage the care of animal and identification.
Description
Technical field
The present invention relates to medicine, biological technical field, more particularly to a kind of high throughput carries out gene editing to embryonated egg
Electric shifting method.
Background technology
CRISPR/Cas9 gene editings technology with its it is efficient, easy to operate, cost is low the features such as be widely used in gene
In knock-out mice experiment, but by the link of RNAs injection embryonated eggs, traditional microinjection is needed by the micro- of precision
The artificial carry out embryonated egg injection one by one of operating system, of high cost, more demanding to operator training, operation is time-consuming longer.
With the development of CRISPR/Cas9 gene editing technologies, there is electricity consumption and turn the traditional microinjection of embryonated egg replacement
Method.The technology is put processed embryonated egg and RNA using instrument and the system that fertilised non-human eggs electricity turns is suitable for jointly
Enter and shock treatment is carried out in electric field, can successfully realize the editor to embryonated egg gene.It will turn the zygote transplation of modification by electricity
Into replace-conceive mouse body, the later stage can obtain the mouse that genetic modification is crossed, and can stablize heredity.
But the electricity of current embryonated egg turns technology also in the more primary stage, in large fragment knockout, high throughput, stabilization
Property, also there is obvious deficiency in positive rate, birth rate.
The content of the invention
Electricity the present invention seeks to solve existing embryonated egg turn technology there are the defects of, improve embryonated egg carry out gene editing
Efficiency, improve once electricity turn ovum quantity, mouse birth positive rate.
In order to solve the above technical problems, the technical solution of the offer of the present invention is：A kind of high throughput carries out base to embryonated egg
Because of the electric shifting method of editor, it includes the following steps：
(1) draw the embryonated egg cultivated in right amount and be transferred in M2 nutrient solutions and cleans, then above-mentioned embryonated egg is transferred to desk-top
Acidifying solution is digested, and postdigestive embryonated egg is transferred to electricity turns to clean in buffer solution；
(2) draw a small amount of electricity being incubated to turn culture medium solution and be put into electricity to turn ware, embryonated egg then is transferred to electricity turns ware
Interior electricity turns culture medium solution,
(3) electricity is turned into ware positive and negative electrode immediately and is connected to beginning electricity turn operation on electroporation；
(4) after electricity turns, remove electricity and turn ware and take under microscope, ovum is transferred in M2 culture mediums and is cleaned, Ran Houfang
Enter CO2Continue culture observation in incubator；It is characterized in that,
The component that the electricity turns culture medium solution is as follows：
| Component | Concentration |
| L-Glutamine | 50-150mM |
| CaCl2 | 1-10mM |
| HEPES | 5-50mM |
| Tris-HCL | 0-15mM |
| EDTA | 0.01-0.2mM |
| NaCl | 10-80mM |
In currently preferred technical solution, the component that the electricity turns culture medium solution is as follows：
| Component | Concentration |
| L-Glutamine | 50-100mM |
| CaCl2 | 1-5mM |
| HEPES | 10-30mM |
| Tris-HCL | 1-10mM |
| EDTA | 0.1-0.2mM |
| NaCl | 20-50mM |
In currently preferred technical solution, in the step (3), the duration of shocking by electricity in electricity turn operation is 2-5ms, electricity
Number is hit as 3-5 times, embryonated egg digestion time is 18s, and RNA concentration is 500-600ng/ μ L.
In currently preferred technical solution, in the step (3), the duration of shocking by electricity in electricity turn operation is 2ms, electric shock
Number is 5 times, and embryonated egg digestion time is 18s, and RNA concentration is 550ng/ μ L.
The innovation of mouse manufacturing technology is as shown in table 1：
Fluctuate smaller between different genes knockout project of the present invention, can reach substantially more than 50% (can reach 90% individually)
The mouse of birth is the mouse after genetic modification.Both the stability and reproducibility of experiment can guarantee that, while also substantially reduced
Cost, realizes convenient, efficient, high throughput.
Technology is turned using the electricity, electricity turn after fertilization survival rate of ovum more than 95%, mouse birth rate is 20% after transplanting
Left and right, birth mouse positive rate can reach more than 50%；The limitation of personnel's technology is breached, reduces experimental cost, while greatly
Improve to amplitude the success rate of project.Once the technology extensive use, the more efficient promotion mice gene knockouts research of energy
Development.
Embodiment：
To further understand the present invention, preferred solution of the present invention is described with reference to specific embodiment, but should
Work as understanding, these descriptions are simply further explanation the features and advantages of the present invention, rather than the limit to the claims in the present invention
System.
Required apparatus：BEX electroporation electrodes ware, centrifuge, stereoscope, incubator, mouth suction pipe, hand-made glass needle.
Required reagent：M2 culture mediums, M16 culture mediums, desk-top acidifying solution, electricity turn buffer solution, distilled water, gRNA,
TracrRNA, Cas9, cross drainage (no RNase).
First, the electricity of mouse fertilized egg turns operation
1. the digestion of embryonated egg
1.1 make a desk-top acidifying solution drop of 200 μ L first of liquid-transfering gun in 60mm culture dishes, then do 3 on right side
The electricity of a 50 μ L or so turns the M of buffer solution drop and 3 50 μ L or so2Drop,
1.2 from CO2The embryonated egg cultivated in M16 is taken out in incubator, 100-150 fertilization is drawn with hand-made glass needle
Ovum is transferred to from M2Middle cleaning,
Then embryonated egg is transferred to the 200 desk-top acidifying solutions of μ L by 1.3 with hand-made glass needle is digested, and placing 10-20s will
Ovum suctions out,
Then ovum is transferred to electricity by 1.4 with hand-made glass needle turns to clean 3 times in buffer solution；
2. embryonated egg electricity turns
2.1 after electricity turns culture medium solution incubation 10min, and it is molten to turn culture medium with 10 μ L of the liquid-transfering gun absorption electricity being incubated
Liquid, is added to electricity and turns in ware, and embryonated egg is transferred to electricity with hand-made glass needle under the microscope and is turned in ware,
Electricity is turned ware positive and negative electrode by 2.2 immediately is connected to beginning electricity turn operation on electroporation,
After 2.3 electricity turn, remove electricity and turn ware and take under microscope, ovum is transferred to M with hand-made glass needle2Culture medium
In, clean 2-3 times, observe the state and death condition of ovum, ovum is transferred in M16 and is put into CO2Continue to cultivate in incubator, the
Two days situations for calculating and observing 2-cell,
2.4 are transplanted to 2cell the replace-conceive mouse of 0.5d, and mouse is born after 20d, carry out PCR to birth mouse and sequencing is reflected
It is fixed；Count mouse birth rate and positive rate.
2nd, operating method is turned according to the electricity of above-mentioned mouse fertilized egg, carries out following implementation：
Embodiment 1.
Same gene knockout project is selected, while on C57BL/6 Strains of Mouse embryonated eggs, it is micro- that protokaryon is respectively adopted
The electricity of injection and protokaryon embryonated egg turns operation contrast.
A groups：Microinjection operates, and RNA injection concentrations are 30ng/ μ L
B groups：Electricity turns operation, and it is 300ng/ μ L, voltage 30V that RNA electricity, which turns concentration, and electricity swivel is 10 μ L, and electricity turns the duration
3ms, electricity turn number 4 times.
Experiment is turned by electricity, it is found that mouse positive ratio significantly improves 2 times, while positive rate is improved, also reduces
Later stage the care of animal and the workload of identification, effect are as shown in table 2：
Table 2
In addition to it can improve positive rate, electricity turns operation, relative to microinjection, also has 3 advantages below of table：
Table 3
| Microinjection | Electricity turns | |
| Instrument expense | 400000 | 100000 |
| Training time | 1 year | 2 weeks |
| Operating time | 1-2h | 5min |
The electricity of embodiment 2. conversion culture medium and the processing optimization of embryonated egg
C groups：The Opti-MEM culture mediums of the prior art or Electroporation Buffer
D groups：Electricity turns liquid after present invention optimization, is more suitable for embryonated egg
The culture medium for being used for electricity and turning operation existing at present, primarily directed to cell design, due to cell culture medium
Not fully it is adapted to the culture of embryonated egg；Embryonated egg also needs to the protection of transparency, and extraneous RNA or DNA are turned in electricity
It is more difficult to than ES cell enter break-through in journey.
For such case, the culture medium turned for electricity is optimized, main optimization component is as shown in table 4：
Table 4
| Component | Concentration |
| L-glutamine | 50-150mM |
| CaCl2 | 1-10mM |
| HEPES | 5-50mM |
| Tris-HCL | 0-15mM |
| EDTA | 0.01-0.2mM |
| Nacl | 10-80mM |
| Phenol red | Be free of |
Culture medium after optimization, electricity turn after the fertilization survival rate of ovum and birth rate in later stage and positive rate higher, be more suitable for
Electricity for mouse fertilized egg turns, and Contrast on effect is as shown in table 5：
Table 5
The different electricity of embodiment 3. turn parameter comparison (table 6)
E groups：Former electricity turns parameter
F groups：Electricity turns parameter after modification
Table 6
| Sequence number | Parameter | It is existing | Parameter after Cyagen optimizations |
| 1 | Shock by electricity the duration | 1ms | 2ms |
| 2 | Number of shocks | 7 times | 5 times |
| 3 | Embryonated egg digestion time | 10s | 18s |
| 4 | RNA concentration | 300ng/μL | 550ng/μL |
Experimental result is as shown in table 7：
Table 7
Two groups turn culture medium using the electricity after optimization.
The electricity that can be explained by the data of upper table after adjustment turns parameter, the mouse after the survival rate of embryonated egg after electricity turns and transplanting
On Birth Situation, all have a clear superiority, whole efficiency but improves 2 times.
Embodiment 4 combines CRISPR/Cas9 and electricity turns the batch operation that technology carries out mice gene knockouts.
After the optimization of parameter, culture medium, operating method is turned by the screening to electroporation device and electricity, grasped at present
Make simple, effect stability, while the high efficiency method of high-volume mice gene knockouts can be carried out.
20 different projects are have selected, gene knockout experiment is carried out by the way of mouse fertilized egg electricity turns.
Electricity turns scheme, and the electric shock duration is 2ms, and number of shocks is 5 times, and embryonated egg digestion time is 18s, and RNA is dense
Spend for 550ng/ μ L.
8 disparity items electricity of table turns operational circumstances
Have selected 20 different projects, carry out high-volume experimental implementation, except end item be probably because gene or
Birth rate caused by operating reason and positive rate are relatively low outer, and the electric transfer efficient of most of project is all higher.
All items can obtain positive mice, and the positive rate of only 2 projects is below 5%, repeatability and stability
It is preferable.
Disposably only electricity turns 200 embryonated eggs to each scheme, and needs to note for general scheme of traditional microinjection
The embryonated egg of 300 is penetrated, later stage birth positive mice can be only achieved enough quantity；It is nothing like in stability and positive rate
Electricity turns operation.
Microinjection operation is also influenced by the injection needle and operating personnel's state of different batches at the same time, an operating personnel
The microinjection that 300-400 embryonated egg can only generally be carried out in one day, and turn for electricity, only ovum quantity is enough, one day can be with
Electricity turns the embryonated egg of thousands of.
In addition, the quality higher for the ovum that microinjection needs, so just can guarantee that ovum after the mechanical damage injected,
It can recover normal condition and the later stage can develop into individual mice.And turn for electricity, the screening of embryonated egg requires relatively low so that after
Phase quickly obtains a large amount of embryonated eggs by way of combining superfecundation and IVF, then carries out the mode that electricity turns and carry out genetic modification
Method feasibility higher.The usage quantity of mouse can be so reduced, while solves the quantity restricted problem of embryonated egg.
Conclusion：Mouse fertilized egg electricity after optimization turns technology, can it is easier, efficiently carry out gene editing.Existing skill
The electric shifting method of art once operable 50 embryonated eggs；And the electricity after Optimal Parameters of the present invention turn operation once operable 200~
300 embryonated eggs, positive rate is still in normal range (NR).
Claims (4)
1. a kind of high throughput carries out embryonated egg the electric shifting method of gene editing, it includes the following steps：
(1) embryonated egg that absorption is cultivated in right amount, which is transferred in M2 nutrient solutions, cleans, then above-mentioned embryonated egg is transferred to desk-top acidifying
Liquid is digested, and postdigestive embryonated egg is transferred to electricity turns to clean in buffer solution；
(2) draw a small amount of electricity being incubated to turn culture medium solution and be put into electricity to turn ware, embryonated egg is then transferred to electric turn in ware
Electricity turns culture medium solution,
(3) electricity is turned into ware positive and negative electrode immediately and is connected to beginning electricity turn operation on electroporation；
(4) after electricity turns, remove electricity and turn ware and take under microscope, ovum is transferred in M2 culture mediums and is cleaned, is then placed in CO2
Continue culture observation in incubator；It is characterized in that,
The component that the electricity turns culture medium solution is as follows：
2. electricity shifting method according to claim 1, it is characterised in that the component that the electricity turns culture medium solution is as follows：
3. electricity shifting method according to claim 1, it is characterised in that in the step (3), electricity turns to shock by electricity in operation and continues
Time is 2-5ms, and number of shocks is 3-5 times, and embryonated egg digestion time is 18s, and RNA concentration is 500-600ng/ μ L.
4. electricity shifting method according to claim 1, it is characterised in that in the step (3), electricity turns to shock by electricity in operation and continues
Time is 2ms, and number of shocks is 5 times, and embryonated egg digestion time is 18s, and RNA concentration is 550ng/ μ L.
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Citations (5)
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|---|---|---|---|---|
| CN105593369A (en) * | 2013-10-04 | 2016-05-18 | 内帕基因株式会社 | Mammalian gene modification method using electroporation |
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| CN107002098A (en) * | 2014-09-29 | 2017-08-01 | 杰克逊实验室 | Genetic modification mammal is produced by electroporation high efficiency, high flux |
| CN107406846A (en) * | 2015-02-19 | 2017-11-28 | 国立大学法人德岛大学 | Cas9 mRNA are imported into the method for the embryonated egg of mammal by electroporation |
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2017
- 2017-12-18 CN CN201711364817.7A patent/CN107988267A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105637093A (en) * | 2013-08-28 | 2016-06-01 | 荷兰皇家科学院 | Transduction buffer |
| CN105593369A (en) * | 2013-10-04 | 2016-05-18 | 内帕基因株式会社 | Mammalian gene modification method using electroporation |
| CN107002098A (en) * | 2014-09-29 | 2017-08-01 | 杰克逊实验室 | Genetic modification mammal is produced by electroporation high efficiency, high flux |
| CN107406846A (en) * | 2015-02-19 | 2017-11-28 | 国立大学法人德岛大学 | Cas9 mRNA are imported into the method for the embryonated egg of mammal by electroporation |
| CN106520838A (en) * | 2016-10-24 | 2017-03-22 | 湖北省农业科学院畜牧兽医研究所 | New method for gene injection for somatic cell nuclear transfer reconstructed embryo |
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| Title |
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| CALEGARI ET AL.: "Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture", 《DIFFERENTIATION》 * |
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Effective date of registration: 20190326 Address after: 215400 No. 162 Renmin South Road, Chengxiang Town, Taicang City, Suzhou, Jiangsu Applicant after: Race (Suzhou) Biological Technology Co., Ltd. Applicant after: Competition Model Biological Research Center (Taicang) Co., Ltd. Address before: 215400 No. 100 Yueyang Road, Taicang Economic Development Zone, Suzhou City, Jiangsu Province Applicant before: Race (Suzhou) Biological Technology Co., Ltd. |
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