CN106929534A - It is a kind of with slow virus as instrument in body gene silencing methods - Google Patents
It is a kind of with slow virus as instrument in body gene silencing methods Download PDFInfo
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Abstract
The invention belongs to molecular genetic biology and Developmental Biology field, disclose it is a kind of with slow virus as instrument in body gene silencing methods, the including in body gene silencing methods with slow virus as instrument：Using microsyringe injecting virus；2 days chicken embryos, burrow in air chamber, visually determine air chamber film location, by under the film of injector insertion air chamber center, virus are injected.The present invention establish with slow virus as instrument in body gene silencing methods, and be successfully established PPARalpha in body silence chicken embryo model.The method of the present invention is relatively simple, and visually lower to complete operation, low cost is practical, and survival rate is high in heart transfection efficiency up to 80%, in body silence efficiency up to 70% or so.In the complete development signal transduction process for remaining complexity of body transfection, the powerful that can be acted on played in development as goal in research gene.
Description
Technical field
It is the invention belongs to molecular genetic biology and Developmental Biology field more particularly to a kind of with slow virus as instrument
In body gene silencing methods.
Background technology
Genetic manipulation is to study the necessary method that specific gene is acted on played in vital movement, and classical method is gene
Knock out (Knock out), it can effectively remove target gene, but relative cost is high and time-consuming more long, simultaneously as its opposite
The change of thing genome, the side effect that some may be brought unknown reduces the reliability of research.
SiRNA be discovered in recent years it is a kind of can specific recognition and suppress, degrade specific mRNA, so as to not change
On the premise of DNA reduce target gene protein expression level microRNA, its be widely used in silence genes of interest with
Study its effect played in particular organisms activity.Common has electroporation, liposome by the method that siRNA imports cell
Method and viral method etc..Wherein viral method has the duration long, the advantages of efficiency high, but, typically with lentivirus mediated
With isolated cells as target, virus needs directly contact host cell to reach transfection to siRNA gene silencings, if note
Penetrate in whole animal, then due to the limitation of applicable virus quantity, can only localized transfection organize on a small quantity, and uniform turning can not be reached
Dye, the application value of method is little.
Isolated cells are classical research tools, but it has certain limitation, are only made up of one or several cells, are not had
There are microenvironment complicated in real organism, neurohumor regulation and control etc., therefore being studied in mechanism body for complexity can not be met
Need, the process for particularly needing various kinds of cell to be carried out jointly under complicated signal path as development, it is impossible to in vitro
Cell model is simulated, therefore current development research is difficult using slow virus silent technology, and multiplex traditional clpp gene division.
In sum, the problem of prior art presence is：The existing genetic manipulation method in overall biology is with high costs
And it is time-consuming more long, easily cause unknown side effect, and the siRNA silences of lentivirus mediated are substantially served only for isolated cells
Model, it is impossible to meet complicated integrated mechanism research needs, it is necessary to develop one kind can be with existing lentivirus mediated, in overall life
The method for playing gene silencing in thing.
The content of the invention
For prior art exist problem, the invention provides it is a kind of with slow virus as instrument in body gene silencing side
Method.
The present invention is achieved in that limited slow virus injection can not possibly be effectively if being model using adults
A large amount of cells of ground transfection whole body, and can then overcome this problem using chicken embryo, early stage chick embryo development, whole chicken embryo it is thin
Born of the same parents' quantity is simultaneously few, as long as appropriate slow virus is injected into chicken embryo in reasonable time point, will be enough to transfect most chicken embryos thin
Born of the same parents, simultaneously because the characteristics of slow virus, the expression of siRNA will be handed on permanently with the division of daughter cell, and order is incubated
The chicken after chick or even adult after change all carries the characteristic of gene silencing always.
It is a kind of with slow virus as instrument in body gene silencing methods, it is described with slow virus as instrument in body gene silencing
Method includes：
Step one, using microsyringe injecting virus；
Step 2,2 days chicken embryos, burrows in air chamber, visually determines air chamber film location, by injector insertion air chamber center film
Under, virus is injected.
Further, using the microsyringe injecting virus of 5ul in the step one.
Further, injection volume is set as every 20g egg sizes injection 1ul in the step one, and virus titer unification is
3x108TU/ml。
Further, the hole of diameter 1.5mm~2mm is opened in air chamber in the step 2.
Further, 1mm~2mm under the film of injector insertion air chamber center is injected virus in the step 2.
Another object of the present invention is to provide it is a kind of using it is described with slow virus as instrument in body gene silencing methods
The PPARalpha of foundation is in body silence chicken embryo model.
Advantages of the present invention and good effect are：Method is relatively simple, visually lower to complete operation, only needs asepsis ring
Border, thin awl one, the domestic microsyringe less than 100 yuans and common adhesive tape can be carried out, and be built
Stood with slow virus as instrument in body gene silencing methods, and be successfully established PPARalpha in body silence chicken embryo model,
At 15 days, chicken embryo survival rate is determined, tables of the PPAR alpha in 15 days chicken embryo hearts up to about 80% through western blot
Up to efficiency compared with control group reduction up to about 70%, and the slow virus silence efficiency in isolated cells model of other research reports
The data overwhelming majority illustrates that this method has reached in whole animal model and is no less than between 50%-70%, sometimes even excellent
Silencing efficiency in existing isolated cells model.
At present, do not have in the world generally acknowledged PPARalpha knock out or silence chicken embryo model, and only PPARalpha
In the knockout model of mouse, the present invention has filled up a technical blank.In the complete development letter for remaining complexity of body transfection
Number transductive process, can turn into the instrument that strong analysis target gene be acted on played in growth course.(example：Analysis PPAR
Whether alpha works in the development cardiac toxic of something induction, you can in body silence PPAR alpha, reapply analysis
Material, observe in the case of PPARalpha silences, its development cardiac toxic whether also exist).
Brief description of the drawings
Fig. 1 be it is provided in an embodiment of the present invention with slow virus as instrument in body gene silencing methods flow chart.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is explained in detail below in conjunction with the accompanying drawings.
As shown in figure 1, it is provided in an embodiment of the present invention with slow virus as instrument body gene silencing methods include it is following
Step：
S101：Using the microsyringe injecting virus of 5ul, injection volume is set as every 20g egg sizes injection 1ul, virus drop
It is 3x10 that degree is unified8TU/ml；
S102：2 days chicken embryos, the hole of diameter about 1.5-2mm is opened in air chamber, visually determines air chamber film location, carefully will
About 1-2mm under the film of injector insertion air chamber center, virus is injected.
Application principle of the invention is further described with reference to experiment.
Slow virus is imported in overall chicken embryo, using the microsyringe injecting virus of 5ul, injection volume is set as every 20g
Egg size injects 1ul, and virus titer unification is 3x108TU/ml.Four kinds of infusion protocols of Preliminary design：1,0 day chicken embryo (before development),
The hole of diameter about 3mm is carefully opened in air chamber, cameral mantle is torn, blastodisc is exposed, directly virus is injected visually lower.2,0
Its chicken embryo (before development), the hole of diameter about 1.5-2mm is opened in air chamber, air chamber film location is visually determined, carefully by injector
About 1-2mm under the film of insertion air chamber center, virus is injected.3,2 days chicken embryos, operate with scheme 1, are directly exposed 2 days after tearing film
Chicken embryo, visually lower injection is viral.4,2 days chicken embryos, operate with scheme 2.
All equal strict aseptic techniques of injecting method, after finally being sealed with adhesive tape, normal culture.First preliminary experiment
Carry out to six days, it is found that survival rate is：Scheme 1,30%, scheme 2,70%, scheme 3,0%, scheme 4,80% illustrates to open straight
Footpath 3mm holes are simultaneously torn the operation of cameral mantle and cannot ensure that chicken embryo is survived, and robin scheme 1,3 carries out the pre- reality of the second wheel with scheme 2,4
Test.At the same time, under 6 days chicken embryos being placed in into gel imaging instrument blue light is excited, and tentatively confirms to transfect green fluorescence after slow virus
In the presence of.2. the screening of transfection efficiency：Second wheel preliminary experiment is tested with scheme 2,4, and culture part chicken embryo was taken out to 6 days
It is imaged under gel imaging machine, partly to 15 days, takes out heart freezing microtome section, the image checking green fluorescence under fluorescence microscope, knot
Fruit is pointed out, and two schemes have green fluorescent protein to express, but scheme 2 expression efficiency far away from scheme 4, thus it is speculated that may be with 0 day
When embryo not yet develop, be available for the cell for infecting very little, blood supply is undeveloped, it is impossible to virus is delivered into embryo's whole body has in time
Close, thus it is final determine to carry out subsequent experimental with scheme 4, i.e., in 2 days chicken embryos, air chamber is opened a duck eye and is injected under the film of air chamber center about
1-2mm.3. the screening of gene silencing efficiency：The design construction of silence ppar alpha slow virus is carried out by the lucky triumphant gene in Shanghai,
Three kinds of slow virus (labeled as V1, V2, V3) are obtained, as it was previously stated, being injected in two days chicken embryos, is cultivated to 15 days, taken out
Chicken embryo heart, freezing microtome section determines that green fluorescent protein expresses (result sees appendix), as a result shows, three kinds of viruses can effectively make
Green fluorescent protein is expressed, and V3 efficiency is slightly lower.With western blot detect ppar alpha expressions, as a result show, v1 without
Obvious silence, v3 silence efficiencies only 33.4%, and v2 silence efficiencies reach 69.8%, are the diseases of preferable silence ppar alpha
Poison, ppar alpha genes embryos may be used to research ppar alpha in normal and abnormal hair in the success of body silence model construction
Effect played in educating.
Ppar alpha silence slow virus is changed into other slow virus should be still feasible, can be widely used for specific in growth course
The research of gene role.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (6)
1. it is a kind of with slow virus as instrument in body gene silencing methods, it is characterised in that it is described with slow virus as instrument
Body gene silencing methods include：
Step one, using microsyringe injecting virus；
Step 2,2 days chicken embryos, is burrowed in air chamber, visually determines air chamber film location, and injector insertion air chamber is hit exactly under film, will
Virus injection.
2. it is as claimed in claim 1 with slow virus as instrument in body gene silencing methods, it is characterised in that the step one
The microsyringe injecting virus of middle utilization 5ul.
3. it is as claimed in claim 1 with slow virus as instrument in body gene silencing methods, it is characterised in that the step one
Middle injection volume is set as every 20g egg sizes injection 1ul, and virus titer unification is 3x108TU/ml。
4. it is as claimed in claim 1 with slow virus as instrument in body gene silencing methods, it is characterised in that the step 2
In the hole of diameter 1.5mm~2mm is opened in air chamber.
5. it is as claimed in claim 1 with slow virus as instrument in body gene silencing methods, it is characterised in that the step 2
1mm~2mm under the middle film by injector insertion air chamber center, virus is injected.
6. described in a kind of utilization claim 1~5 any one with slow virus as instrument body gene silencing methods set up
PPARalpha is in body silence chicken embryo model.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110749728A (en) * | 2019-08-22 | 2020-02-04 | 青岛大学 | Chicken embryo diesel engine tail gas contamination method |
| CN111763691A (en) * | 2020-07-16 | 2020-10-13 | 扬州大学 | Method for introducing novel lentiviral vector into chick embryo |
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| CN1568366A (en) * | 2001-08-13 | 2005-01-19 | 恩布里克斯公司 | Methods for injecting avian eggs |
| CN101503697A (en) * | 2009-03-09 | 2009-08-12 | 中山大学 | Preparation of recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192A |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110749728A (en) * | 2019-08-22 | 2020-02-04 | 青岛大学 | Chicken embryo diesel engine tail gas contamination method |
| CN110749728B (en) * | 2019-08-22 | 2023-06-30 | 青岛大学 | Method for contaminating tail gas of chick embryo diesel engine |
| CN111763691A (en) * | 2020-07-16 | 2020-10-13 | 扬州大学 | Method for introducing novel lentiviral vector into chick embryo |
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