CN108070617A - A kind of exopalaemon carinicauda embryo micro-injection method and Application way structure mRNA are overexpressed model - Google Patents
A kind of exopalaemon carinicauda embryo micro-injection method and Application way structure mRNA are overexpressed model Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention belongs to gene editing technical field, specifically a kind of exopalaemon carinicauda embryo micro-injection method and utilization this method structure mRNA are overexpressed model.Under the conditions of the microinjection system of hyperosmosis and specific microinjection microinjection is carried out to exopalaemon carinicauda one cell stage.Using in-vitro transcription mRNA as injection material, using the exopalaemon carinicauda embryo micro-injection method, structure is overexpressed the exopalaemon carinicauda model of mRNA.Expression of the in-vitro transcription EGFPmRNA in exopalaemon carinicauda embryo is successfully realized using the present invention.The foundation of exopalaemon carinicauda embryo's micro-injection method and its application being overexpressed in structure mRNA in model all have most important theories meaning and practice significance for the research and its gene editing for carrying out the crustaceans functional genes such as shrimps.
Description
Technical field
The invention belongs to gene editing technical field, specifically a kind of exopalaemon carinicauda embryo micro-injection method and utilization should
Method structure mRNA is overexpressed model.
Background technology
Mariculture industry is the important component of blue agriculture.How more and better sea-farming kind one is obtained
It is directly the important content that domestic and international scientist is directed to research.Meanwhile the development of social economy, to the demand of product diversification
Requirements at the higher level are proposed to marine organisms Genetic improvement.By taking prawn as an example, prawn is the important kind of China's sea-farming, in sea
It is occupied an important position in foreign fishery.China scientist by the method for traditional breeding, brought out Chinese prawn " Huanghai No.1 ",
The new varieties such as " Huanghai Sea 2 ", litopenaeus vannamei " section sea 1 ", and part new varieties have realized industrialization.But conventional herd breeding side
Method cycle length, slowly effect；It is developed in recent years by the orientation of production traits key gene, efficiently molecular breeding technology
It will be the developing direction in biological production of hybrid seeds future.At present, also underway, the substantial amounts of prawn function of prawn genome sequencing work
Gene and is exploited out, analyses in depth the function of these genes so as to illustrate its relation with the production traits, can
Reliable basis are provided with the selection for breeding key gene and evaluation.Although bioinformatics can carry out gene function pre-
It surveys, but is limited be subject to cell line and transgenic technology bottleneck, the experimental biology research of shrimps functional gene also only stops
Stay in gene level (gene expression diagramatic technology, quantitative technique, assignment of genes gene mapping etc.) and protein level (in-vitro recombination expression,
Western blot, immunohistochemistry, protein-protein interaction and protein-dna interaction etc.) on.
Transgenic technology is then a very important ring in gene function verification technique system, and falls within new show
For molecular breeding technology.And the microinjection of embryo is the important experimental implementation in transgenic research.Pass through the side of microinjection
A series of allogenic material can be imported into cell or embryo by formula；By way of microinjection, can also to embryo into
Row clpp gene subtracts with gene editing to study functional gene etc..However, for the Decapodas such as shrimps biology, function
The research means of gene are quite deficient.This causes application of the microinjection technique in shrimps embryo to seem particularly critical.
Exopalaemon carinicauda (Exopalaemon carinicauda), belongs to subphylum crustacea Decapoda (Decapoda), is
A kind of higher marine products shrimps of economic value.Exopalaemon carinicauda is strong to environmental suitability, is easy for workers to raise.Meanwhile breed energy
Power is strong, and female shrimp can continuously lay eggs.According to more than advantage, exopalaemon carinicauda E.carinicauda can be studied as new crustacean
Model, this economic crust species in particular for Decapoda, for disclosing this kind of species development, growth, metabolism and breeding
Etc. vital movements rule.But the shrimps lack effective allogenic material and import platform always, so exopalaemon carinicauda embryo is micro-
The structure of injection platform is of great significance.
It is one kind in indigo plant in addition, green fluorescent protein (Enhanced Green Fluorescent Protein, EGFP)
Color is to the albumen that under ultraviolet range light, can be inspired green fluorescence.Because this optical characteristics of EGFP but also
It is often applied to as a reporter gene in the researchs such as cytology and the molecular biology of biology.
The content of the invention
Present invention aims at solving the problems, such as that the research of crustacean gene editing is deficient, a kind of exopalaemon carinicauda embryo is provided
Micro-injection method and its Application way structure in-vitro transcription model.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of exopalaemon carinicauda embryo micro-injection method, by the microinjection system of hyperosmosis in specific microinjection
Under the conditions of to exopalaemon carinicauda unicellular period (before 8 cells) embryo carry out microinjection.
The microinjection system of the hyperosmosis for microinjection buffer and microinjection liquid wherein, delay by microinjection
Fliud flushing：The final concentration of 100mM HEPES and 1.5M NaCl solutions that no enzyme water is prepared；
Microinjection liquid is injection material and indicator；Wherein, injection material is through the water-reducible in-vitro transcription of no enzyme
EGFP mRNA, mRNA are diluted to 400ng/ μ L；Indicator is phenol red solution.
Exopalaemon carinicauda unicellular period, (before 8 cells) embryo was in the high sepage of microinjection after above-mentioned microinjection.Institute
It is the 5% ficoll Ficoll400 prepared with fresh seawater to state the high sepage of microinjection.
The microinjection condition is that pressure is 80Pa；The amount of microinjection is 0.5nL；The time of microinjection is
0.7s；Fertilized eggs culture environment after microinjection is ex vivo incubation in the fresh seawater filtered through 0.22 μm of filter membrane.
A kind of exopalaemon carinicauda embryo micro-injection method builds in-vitro transcription model, using in-vitro transcription mRNA as injection material
Material using the exopalaemon carinicauda embryo micro-injection method, is overexpressed the exopalaemon carinicauda model of mRNA outside construct.
Specifically, using the EGFP mRNA of in-vitro transcription synthesis as injection material, shown using the exopalaemon carinicauda embryo
Microinjection method, construct are overexpressed the exopalaemon carinicauda model of EGFP mRNA outside.
Advantage for present invention：The present invention establishes a kind of exopalaemon carinicauda embryo micro-injection method, and utilizes aobvious
Microinjection method is successfully realized expression of the EGFP mRNA of in-vitro transcription synthesis in exopalaemon carinicauda fertilized eggs, is shrimps etc.
The research of crustacean functional gene provides important research means.The specifically micro- note of exopalaemon carinicauda fertilized eggs embryo of the present invention
Shooting method all has important theory for carrying out the overexpression of exopalaemon carinicauda gene and the editor of exopalaemon carinicauda genome etc.
Meaning and practice significance.
Description of the drawings
Fig. 1 is the EGFP mRNA testing results of in-vitro transcription provided in an embodiment of the present invention synthesis.
Fig. 2 is the fluoroscopic examination result of embryo after EGFP mRNA microinjections provided in an embodiment of the present invention；Wherein A, B,
C and D is not inject control group；E, F, G and H inject experimental group for EGFP mRNA.A, C, E and G are to be imaged picture under dark field；
B, D, F and H are the fluorescence imaging under blue light excitation.To be imaged picture under 4 times of object lens, black scale is 500 μm by A, B, E and F；
To gather picture under 10 times of object lens, black scale is 200 μm by C, D, G and H.
Specific embodiment
The invention will be further elaborated in example below, however, the present invention is not limited thereto.
Embodiment 1：Exopalaemon carinicauda embryo's micro-injection method
1) selection of exopalaemon carinicauda and fertilized eggs
In experiments Microinjection, shrimp used is healthy adult exopalaemon carinicauda (E.carinicauda), and average body is a length of
5.5±0.5cm.The continuous charge culture in nature seawater of shrimp used in experiment changes water and feeds bait daily, and water temperature maintains 24
DEG C, the exopalaemon carinicauda of gonadal maturation is selected for testing.The characteristics of developmental stage each according to exopalaemon carinicauda fertilized eggs, knot
Experiments Microinjection success rate is closed, injection success rate is higher before 8 cell stages, wherein the injection survival rate highest of one cell stage；8 is thin
Injections difficult after born of the same parents' phase, injection needle are easy to fracture；So the fertilized eggs in unicellular period are selected as shrimp ovum used in microinjection
Optimal selection.
2) in microinjection system correlative factor optimization
Microinjection buffer is arrived involved in microinjection system：It is stable environment inside exopalaemon carinicauda fertilized eggs,
Itself only has very small-scale ability of regulation and control, so having to rely on the adjusting of buffer solution for each ingredient of microinjection to tie up
Hold with similar in fertilized eggs internal environment in the range of pH, present invention selection has stronger buffer capacity in near-neutral pH
HEPES buffer solutions maintain to have higher infiltration hydraulic pressure after injecting in embryo by adding certain density NaCl；It is micro-
Parenteral solution：To each ingredient of microinjection and the mixture of microinjection indicator solution, through the EGFP mRNA to various concentration
Microinjection is carried out, it turns out that the EGFP mRNA of 400ng/ μ L have preferable effect；The high sepage of microinjection：Spine end is white
For ovum content is prevented excessively to be lost in after the microinjection of shrimp fertilized eggs, need to be placed in high sepage to maintain, and not
Shaking table in the fresh seawater filtered through 0.22 μm of filter membrane is transferred in time more than 3h to be incubated.
The microinjection system of hyperosmosis is microinjection buffer and microinjection liquid；
(1) microinjection buffer：No enzyme water prepares the solution containing final concentration of 100mM HEPES and 1.5MNaCl.
(2) microinjection liquid：The EGFP mRNA concentration dilutions of synthesis to 400ng/ μ L are obtained into EGFP with no enzyme water
MRNA solution, and add final concentration of 0.5% phenol red (mass concentration) be used as indicator.
Above-mentioned (1) microinjection buffer of its 0.1 times of volume will be added in above-mentioned acquisition EGFP mRNA solution, and added
Final concentration of 0.5% phenol red (mass concentration) is added to be used as indicator, and then obtains the microinjection system of hyperosmosis
(3) the high sepage of microinjection：Fresh seawater prepares the ficoll Ficoll400 that mass concentration is 5%.
3) selection of microinjection condition
The correlated condition of microinjection includes the pressure selected during microinjection：80Pa is not to be exceeded in pressure, otherwise can rush
Substance arrangement inside random fertilized eggs；The amount of microinjection：The amount that microinjection enters fertilized eggs should be moderate, very few to be not enough to
Gene editing is carried out, excessive that again fertilized eggs are generated with toxic action, the present invention uses injection volume as 0.5nL；Microinjection when
Between：The time of microinjection and the amount of microinjection are shown there are positive correlation, therefore in order to ensure certain microinjection amount
0.7s is not to be exceeded in the time of microinjection；Fertilized eggs culture environment after microinjection：Ex vivo incubation is taken out through 0.22 μm of filter membrane
In the fresh seawater of filter.
Embodiment 2：Model is built using exopalaemon carinicauda embryo micro-injection method
(1) structure of SP6-EGFP DNA profilings
By the multiple cloning sites EcoR I of EGFP sequences insertion commercialization plasmid pIZT/V5-His (Invitrogen, USA)
Between Not I, pIZT/V5-EGFP recombinant plasmids are built.And according to the sequence design forward primer SP6- of pIZT/V5-EGFP
EGFP-F and reverse primer SP6-EGFP-R adds in SP6 promoters site in forward primer.
Primer sequence is as follows：
SP6-EGFP-F：gcATTTAGGTGACACTATAGAAACAG(single underscore is ATGGTGAGCAAGGGCGAGGA
SP6 promoter sequences)
SP6-EGFP-R：CGCGCTTGAAAGGAGTGTGTA
The EGFP sequences in pDHSP70-EGFP-His plasmids are expanded using primer SP6-EGFP-F and SP6-EGFP-R,
PCR reaction systems are：
PCR cycle program is such as：98 DEG C of pre-degeneration 1min；98 DEG C of 10sec, 59 DEG C of 5sec, 72 DEG C of 20sec, 35 Xun Huans；72
DEG C extension 10min；4 DEG C of heat preservations.
After PCR product detects SP6-EGFP amplified fragments sizes with 1% agarose gel electrophoresis, PCR product direct Sequencing,
It obtains that in-vitro transcription of the correct SP6-EGFP segments for EGFP mRNA is sequenced.
(2) in-vitro transcription of EGFP mRNA and purifying
According to SP6mMESSAGEKit (Ambion) specification, with gained SP6-EGFPDNA segments into
The in-vitro transcription of row EGFP mRNA, in-vitro transcription system are as follows：
After 37 DEG C are incubated 2hr；Add in 1 μ L TURBO DNase and 37 DEG C incubation 15min, remove DNA profiling, using phenol/
The mRNA that chloroform obtains transcription with isopropanol precipitating mode is purified, and is synthesized with 1% Ago-Gel detection
Whether mRNA is complete, and the results are shown in Figure 1.
(3) microinjection of EGFP mRNA
With no enzyme water prepare 20 μ L microinjection liquid, with no enzyme water by the EGFP mRNA concentration dilutions of above-mentioned synthesis extremely
400ng/ μ L add 2 μ L microinjection buffers, while the 0.5% of 2 μ L of addition is phenol red as indicator.To exopalaemon carinicauda I
Cell stage embryo carries out microinjection.Embryo after injection is placed in the culture dish for filling antiseptic sea water, and room temperature is incubated on shaking table
It educates, replaces antiseptic sea water sooner or later daily respectively once.After microinjection 20h, fluorescence microscopy Microscopic observation gathers control group respectively
With image of the experimental group embryo after injection under dark field and ultraviolet light excitation, and experimental group is analyzed with the presence or absence of green fluorescence
It generates, the results are shown in Figure 2.
From Figure 2 it can be seen that after when injection EGFP mRNA 20 are small, in fluorescence microscope the result shows that being excited in blue light
Under, experimental group has apparent green fluorescence compared with blank group.Experimental group exopalaemon carinicauda fertilized eggs green fluorescence shows body
The EGFP mRNA of outer synthesis are being injected into after exopalaemon carinicauda fertilized eggs the successful accurate translation in its embryo as active
EGFP。
The foundation of effective exopalaemon carinicauda embryo's micro-injection method in the present invention, for carrying out the mistake of exopalaemon carinicauda gene
Editor of expression and exopalaemon carinicauda genome etc. has very important theory significance and practice significance.
SEQUENCE LISTING
<110>The Institute of Oceanology of the Chinese Academy of Sciences
<120>A kind of exopalaemon carinicauda embryo micro-injection method and Application way structure mRNA are overexpressed model
<130>
<160> 2
<170> PatentIn version 3.1
<210> 1
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<213>Engineer
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<221> gene
<222> (1)..(46)<223>
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gcatttaggt gacactatag aaacagatgg tgagcaaggg cgagga 46
<210> 2
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<212> DNA
<213>Engineer
<220>
<221> gene
<222> (1)..(21)
<223>
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cgcgcttgaa aggagtgtgt a 21
Claims (5)
1. a kind of exopalaemon carinicauda embryo micro-injection method, it is characterised in that：By the microinjection system of hyperosmosis specific
Microinjection under the conditions of to exopalaemon carinicauda unicellular period (1 cell) embryo carry out microinjection.
2. by exopalaemon carinicauda embryo micro-injection method described in claim 1, it is characterised in that：The hyperosmosis it is micro-
Injection system is microinjection buffer and microinjection liquid；Wherein, microinjection buffer：No enzyme water is prepared containing final concentration of
The solution of 100mM HEPES and 1.5M NaCl；
Microinjection liquid is injection material and indicator；Wherein, injection material is through the water-reducible in-vitro transcription EGFP of no enzyme
MRNA, mRNA are diluted to 400ng/ μ L；Indicator is phenol red solution.
3. by exopalaemon carinicauda embryo micro-injection method described in claim 1, it is characterised in that：The microinjection condition is
Pressure is 80Pa；The amount of microinjection is 0.5nL；The time of microinjection is 0.7s；Fertilized eggs culture ring after microinjection
Border is ex vivo incubation in the fresh seawater filtered through 0.22 μm of filter membrane.
4. a kind of existed using exopalaemon carinicauda embryo micro-injection method structure in-vitro transcription model, feature described in claim 1
In：Using in-vitro transcription mRNA as injection material, using the exopalaemon carinicauda embryo micro-injection method, it is overexpressed outside construct
The exopalaemon carinicauda model of mRNA.
5. the utilization exopalaemon carinicauda embryo micro-injection method structure in-vitro transcription model as described in claim 4, special
Sign is：The EGFP mRNA synthesized using in-vitro transcription are as injection material, using the exopalaemon carinicauda embryo microinjection side
Method, construct are overexpressed the exopalaemon carinicauda model of EGFP mRNA outside.
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