CN102329819B - Electroporation method for transfection of mammal embryo with siRNA (small interfering Ribose Nucleic Acid) - Google Patents
Electroporation method for transfection of mammal embryo with siRNA (small interfering Ribose Nucleic Acid) Download PDFInfo
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- CN102329819B CN102329819B CN 201110300097 CN201110300097A CN102329819B CN 102329819 B CN102329819 B CN 102329819B CN 201110300097 CN201110300097 CN 201110300097 CN 201110300097 A CN201110300097 A CN 201110300097A CN 102329819 B CN102329819 B CN 102329819B
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Abstract
The invention discloses an electroporation method for transfection of mammal embryo with siRNA (small interfering Ribose Nucleic Acid). The electroporation method comprises the following steps: (1) weakening an embryo zona pellucid; (2) preparing electroporation solution containing siRNA; and (3) conveying the embryo into the prepared electroporation solution containing siRNA, standing at the room temperature, and then conveying the embryo and the electroporation solution into an electric turn trough for electroporation. The electroporation method comprises the steps of weakening the embryo zona pellucid and complexing the siRNA, and then placing the embryo under the action of a pulsed electric field to increase the permeability and the membrane conductance of the embryo instantaneously, and thus the siRNA which is difficult to penetrate through a cell membrane under the normal physiological condition enters the cell. Statistic observation and analysis prove that the stable positive transfection efficiency of survival embryos exceeds 95%, and the practical effective utilization rate of the collected total embryo exceeds 70%. The electroporation method is a safe, simple and efficient transfection technology of mammal embryo with siRNA.
Description
Technical field
The invention belongs to embryo's transfection field, the RNA that relates to the siRNA mediation disturbs particularly a kind of siRNA transfection mammal embryo's electroporation method.
Background technology
RNA by siRNA (small interfering RNA) mediation disturbed (RNAi) to become the strong instrument of research gene function gradually in recent years.Traditional method of siRNA being transduceed to the cell has viral mediated method, microinjection.Virus-mediated method can be used for difficult cells transfected, but requires cell need be in division stage, and has safety-problems.Microinjection is to realize transfection accurately, but needs expensive micrurgy instrument, and complicated operation, and bigger to embryo's wound, transfection quantity is also limited.Therefore seek a kind of safety, easy, embryo's transfection method becomes current fetology research field urgent problem efficiently.
Summary of the invention
The problem that the present invention solves is to provide a kind of siRNA transfection embryo's electroporation method, realizes the embryo is carried out safe, easy, the transfection of siRNA efficiently.
The present invention realizes through following technical scheme:
1, a kind of siRNA transfection embryo's electroporation method may further comprise the steps:
The embryo that 1) will treat transfection places tyrode's solution, and incubated at room 5～15s stops digestion then and cleans;
2) be that electricity changes damping fluid and dilutes with opti-MEM, with dilution good treat transfection siPORT
TMMix the back room temperature with siRNA 1: 1 in molar ratio～2 and leave standstill 10～15min, obtain containing the electroporation solution of siRNA, wherein the final concentration of siRNA is 0.1～1 μ mol/L;
3) embryo is placed the electroporation solution that contains siRNA, under pulsed electrical field, utilize electroporation that the embryo is carried out the siRNA transfection.
Described the embryo is being carried out before tyrode's solution handles, also to the embryo with the cleaning of PBS-PVA solution, described PBS-PVA solution is to contain the PBS solution that volumetric concentration is 0.2～0.5%PVA.
Described termination digestion is the embryo to be transferred to from tyrode's solution stop digestion the embryo's manipulation in vitro liquid that contains serum, then the embryo is cleaned with opti-MEM solution.
Described siRNA is for having carried out fluorescently-labeled siRNA.
The parameter that described electroporation carries out the siRNA transfection is controlled to be: voltage 20V～40V; Burst length 1～2ms; Pulse number 3～4 times.Further be optimized for to mice embryonic: voltage 30V, burst length 1ms, pulse number 3 times.
After described electroporation is accomplished, the embryo is cleaned and be transferred to balance in advance cultivate in the embryo medium of 30min at least.
Compared with prior art, the present invention has following beneficial technical effects:
The electroporation method of siRNA transfection mammal embryo provided by the invention; At first embryo's zona pellucida structure is used the tyrode's solution processing that weakens; And then the embryo after will handling places under the pulsed electrical field effect; Form instantaneous micropore on the cell membrane lipid bilayer, cause the instantaneous increase of its permeability and membrane conductance, can make that cytolemma is difficult to penetrating siRNA entering cell under the normal physiological situation.And if not to the zona pellucida processing that weakens, low voltage is not easy to penetrate zona pellucida, high-voltage can make embryo's mortality ratio increase again.Therefore the control to digestion process, the especially digestion time of embryo's zona pellucida is one step of key of transfection.
Next uses siPORT
TMCombine siPORT with siRNA
TMCan form complex compound with siRNA, when the instantaneous micropore of electroporation occurring, promote that siRNA is efficient must get into the embryo.
In order further to observe or to follow the tracks of the transfection effect, the siRNA that can also treat transfection carries out fluorescent mark, behind electroporation transfection, can observe intuitively through fluorescent microscope like this.
Through the fluorescence inverted microscope embryo being carried out the statistics observation analysis shows; The present invention embryo of surviving stablizes positive transfection efficiency and is about 95%; The actual effective rate of utilization of total embryo of gathering surpasses 70%, for adopting the function of RNA perturbation technique research gene in the embryo by the siRNA mediation effective transfection method is provided.
Description of drawings
Fig. 1 be the mouse fertilized egg zona pellucida through different digestions time, behind the negative control siRNA of electroporation transfection Cy3 mark, the red fluorescence level of embryo under the identical time shutter (200ms);
Fig. 2 is for using transfection mouse different development stage embryo's of the present invention Photomicrograph.
Embodiment
The present invention provides a kind of siRNA transfection embryo's electroporation method, is particularly useful for embryo's transfection of mammal, comprises the complexing processing with siRNA is handled in the reduction of embryo's zona pellucida.Below in conjunction with concrete electroporation process and transfection results the present invention is done further detailed description, said is to explanation of the present invention rather than qualification.
Described siRNA transfection is based on siRNA and gets into the embryo through the instantaneous micropore that embryo's zona pellucida and cytolemma form under pulsed electrical field, is a kind of transfection method of biophysics, does not have special kind requirement so treat the embryo of transfection.Because reduction time of the zona pellucida the when embryo that morphological structure differs greatly carries out electroporation and electroporation parameter possibly have some difference; Yet thereby those skilled in the art can carry out the seldom experiment of number of times through the method that this patent provided to screen for these concrete parameters and optimize; Can confirm the optimized parameter of respective embryo, accomplish embryo's transfection.
Following mask body with mammal particularly mice embryonic as objective for implementation; Specify siRNA transfection mammal embryo's electroporation method; This type of embryo morphology difference is little, and the zona pellucida constituent is similar, thus transfection conditions and electroporation parameter be provided with change little.
The electroporation method of siRNA transfection mice embryonic may further comprise the steps:
At first mice embryonic is carried out the pre-treatment of zona pellucida reduction:
A, the mice embryonic of gathering is shifted 3-5 time each solution that more renews in PBS-PVA solution; Described PBS-PVA solution is for containing the PBS solution that volumetric concentration is 0.2%PVA (polyvinyl alcohol, a Z 150PH);
B, mice embryonic transferred in the tyrode's solution (Sigma, the U.S.) leave standstill 10s, carry out the zona pellucida reduction and handle in room temperature; And the selection of the time of handling for reduction follow-uply further specifies with the control experiment data;
Immediately the embryo is transferred to after c, timing finish and stop digestion in the mice embryonic manipulation in vitro liquid Hepes-KSOM solution;
100ml Hepes-KSOM Working solution prescription is: NaCl, 0.5552g; KCl, 0.0186g; KH
2PO
4, 0.0047; D-glucose, 0.0036; NaHCO
3, 0.0336g; Sodium.alpha.-ketopropionate, 0.0022g; L-glutaminate, 0.0146g; EDTA-Na
2, 0.0014g; CaCl2H
2O, 0.0250g; MgSO
4, 0.0024g; HEPES, 0.2384g; Indispensable amino acid (EAA, invitrogen, 50 *), 2ml; Non-essential amino acid (NEAA, invitrogen, 100 *, 1ml; 60% Sodium.alpha.-hydroxypropionate, 350 μ l; Bovine serum albumin (BSA), 0.6g;
D, the embryo is shifted 3-5 time in opti-MEM (Invitrogen, the U.S.) solution, the solution that at every turn more renews is accomplished before the electroporation pre-treatment to the embryo.
After the pre-treatment of completion to the embryo, the embryo is carried out electroporation, concrete operations may further comprise the steps:
1) preparation comprises the electroporation solution of siRNA (#AM4621, ambion, the U.S.) of Cy3 mark: be that electricity changes damping fluid and dilutes (with opti-MEM with siPORT with opti-MEM
TMBe diluted to suitable concentration with siRNA), with the siPORT after the dilution
TMMix with 1: 1 in molar ratio～2 of dilution siRNA, room temperature leaves standstill 10min; The siRNA of dilution is that 50 μ l opti-MEM add 5 μ l siRNA, the siPORT of dilution
TM(#AM4502, introgen, the U.S.) is that 50 μ l opti-MEM add 3 μ lsiPORT
TM, final siRNA weaker concn is 0.5 μ mol/L in the electroporation solution; Because different siRNA performances disturb the concentration of effect to be not quite similar, so when carrying out the dilution of siRNA concentration, will adjust extension rate as the case may be;
For the ease of the situation behind the observation siRNA transfection embryo; Therefore it is carried out mark with fluorescent mark Cy3; And the siRNA that is selected for use is a kind of negative control siRNA, and it is made up of the 19bp stochastic sequence, with known mouse; Therefore rat and people's gene group do not have homology, change that the normal development to the embryo can not impact behind the embryo over to.
Because the siRNA that adopts is the fluorescently-labeled siRNA of Cy3, therefore dilution and the electroporation process lucifuge of all need trying one's best is gone out in order to avoid cause fluorescence to come together, and in the time need not carrying out fluorescent mark to siRNA, is so just operated without lucifuge.
2) embryo who pre-treatment is accomplished moves into the electroporation solution of the siRNA that contains the Cy3 mark for preparing; This step requires the least possible liquids; In order to avoid the electroporation solution that dilution prepares, the embryo moves into back room temperature lucifuge and hatches 1～3min, and this can make siRNA be attached to the embryo surface; Be beneficial to the entering of siRNA when micropore produces in the electroporation process; But be exposed to the degraded that causes siRNA in the environment easily for a long time, and the embryo to descend for a long time the disadvantageous effect to fetal development at room temperature environment be significant, so incubation time is unsuitable long;
3) embryo is moved into (interelectrode distance is 1mm) in the electric turn trough together with electroporation solution, use electroporation apparatus (ECM 2001Electro Cell Manipulator, BTX, the U.S.) to carry out electroporation, the electroporation parameter is voltage 20～40V; Burst length 1～2ms; Pulse number 3～4 times; Through grouping experiment parameter is optimized, the optimum electroporation parameter that obtains to mice embryonic is voltage 30V; Burst length 1ms; Pulse number 3 times;
4) after electroporation is accomplished, from electric turn trough, shift the embryo and to Hepes-KSOM, clean, shift the solution that at every turn more renews 3-5 time;
5) will clean the back embryo and move into balance in advance and cultivate in the KSOM nutrient solution of 30min at least and observe, carry out the subsequent interference test experience.100ml KSOM nutrient solution prescription is following: NaCl, 0.5552g; KCl, 0.0186g; KH
2PO
4, 0.0047g; D-glucose, 0.0036g; NaHCO
3, 0.21g; Sodium.alpha.-ketopropionate, 0.0022g; L-glutaminate, 0.0146g; EDTA-Na
2, 0.0014g; CaCl2H
2O, 0.0250g; MgSO
4, 0.0024g; Indispensable amino acid (EAA, Invitrogen, 50 *), 2ml; Non-essential amino acid (NEAA, Invitrogen, 100 *, 1ml; 60% Sodium.alpha.-hydroxypropionate, 350 μ l; Bovine serum albumin (BSA), 0.6～0.8g.
For electroporation transfection embryo's result better is described, specifically combine statistic data and accompanying drawing that the transfection effect is described.
The zona pellucida digestion time is to the influence of electroporation efficiency:
According to above-mentioned transfection method, as contrast, be evaluation index with digestion 0s with the transfection effect, digestion 10s, 20s, 30s experimental group are carried out statistical study, concrete result is as shown in table 1:
Table 1 zona pellucida digestion time is to the influence of electroporation efficiency
Statistical analysis adopts SPSS 13.0 softwares to carry out one-way analysis of variance (ANOVA), does not contain on the identical lowercase to have significant difference between the target data, p＜0.05.
Data presentation digests 30s in tyrode's solution after, embryo survival significantly descends, and undigested embryo survival is very high; But the fluorescence photo after the Fluirescence observation transfection (Fig. 1) shows when embryo that zona pellucida is handled without reduction carries out electroporation; SiRNA forms aggregated particles on zona pellucida, seldom get in the embryonic cell, and digestion 10s and 20s embryo's positive transfection efficiency does not have significant difference; But digestion 20s embryo survival is lower than digestion 10s experimental group; And significant difference, thus confirm that digestion 5～15s is the more excellent time, and 10s is a Best Times.
Fig. 1 is mice embryonic zona pellucida embryo's red fluorescence level under the identical time shutter (200ms) behind the siRNA of electroporation transfection Cy3 mark after the different digestions time.Horizontal side by side three pictures, first be the embryo under the light field, and second is the red fluorescence photo, and the 3rd is the result of embryo and fluorescence embryo merging under the light field; Vertically be respectively the result who handles 0s, 10s, 20s, 30s.Fig. 1 has shown that intuitively siRNA gets into embryo's practical situation, explains that siRNA gets into the embryo and obviously gets a promotion after the suitable reduction processing to embryo's zona pellucida.
Fig. 2 is for using transfection mouse different times embryo's of the present invention fluorescence micrograph; Horizontal side by side three pictures, first be the embryo under the light field, and second is the red fluorescence photo, and the 3rd is the result of embryo and fluorescence embryo merging under the light field; Be the different development stage embryo vertically, be followed successively by 2 cell stages, 4 cell stages, 8 cell stages, morula and blastaea from top to bottom.The result shows, all reached good transfection effect in embryo's transfection of different times.
The RNA perturbation technique of siRNA mediation can be implemented in the experimental plan time period, the mRNA of genetic transcription is carried out the specificity degraded, and then the variation of back fetal development morphological change and mRNA or protein level was disturbed in monitoring.The present invention provides effective transfection method for adopting the function of RNA perturbation technique research gene in the embryo by the siRNA mediation.
Claims (6)
1. a siRNA transfection mammal embryo electroporation method is characterized in that, may further comprise the steps:
The embryo that 1) will treat transfection places tyrode's solution, and incubated at room 5～15s stops digestion then and cleans;
2) be that electricity changes damping fluid and dilutes with opti-MEM, with dilution good treat transfection siPORT
TMRoom temperature left standstill 10～15min after mixed 1:1～2 in molar ratio with siRNA, obtained containing the electroporation solution of siRNA, and wherein the final concentration of siRNA is 0.1～1 μ mol/L;
3) embryo is placed the electroporation solution that contains siRNA, under pulsed electrical field, utilize electroporation that the embryo is carried out the siRNA transfection.
2. siRNA transfection mammal embryo's as claimed in claim 1 electroporation method; It is characterized in that; The embryo before carrying out the tyrode's solution processing, is also cleaned with PBS-PVA solution the embryo, and described PBS-PVA solution is to contain the PBS solution that volumetric concentration is 0.2～0.5%PVA.
3. siRNA transfection mammal embryo's as claimed in claim 1 electroporation method; It is characterized in that; Described termination digestion is the embryo to be transferred to from tyrode's solution stop digestion the embryo's manipulation in vitro liquid that contains serum, then the embryo is cleaned with opti-MEM solution.
4. siRNA transfection mammal embryo's as claimed in claim 1 electroporation method is characterized in that described siRNA is for having carried out fluorescently-labeled siRNA.
5. siRNA transfection mammal embryo's as claimed in claim 1 electroporation method is characterized in that the parameter that electroporation carries out the siRNA transfection is controlled to be: voltage 20～40V; Burst length 1～2ms; Pulse number 2～4 times.
6. siRNA transfection mammal embryo's as claimed in claim 1 electroporation method is characterized in that, after electroporation is accomplished, the embryo is cleaned and is transferred to balance in advance cultivate in the embryo medium of 30min at least.
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| WO2016054032A1 (en) | 2014-09-29 | 2016-04-07 | The Jackson Laboratory | High efficiency, high throughput generation of genetically modified mammals by electroporation |
| CN107708796B (en) * | 2015-06-25 | 2021-10-26 | 新南创新私人有限公司 | Electroporation system for controlled local therapeutic agent delivery |
| CN106754348B (en) * | 2016-12-23 | 2024-02-09 | 南京农业大学 | Electroactivated equipment and RNA interference method of oocyte or embryo using same |
| CN108546683B (en) * | 2018-03-23 | 2021-09-21 | 广西壮族自治区水牛研究所 | Method for efficiently producing transgenic buffalo embryos by adenovirus mediation |
| CN115044618A (en) * | 2022-07-29 | 2022-09-13 | 福建省妇幼保健院 | Method for non-invasive gene transfection of human embryo |
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| Mismatched siRNAs downregulate mRNAs as a function of target site location;Scott E. Martin et al.;《FEBS Letters》;20060605(第580期);3694–3698 * |
| Scott E. Martin et al..Mismatched siRNAs downregulate mRNAs as a function of target site location.《FEBS Letters》.2006,(第580期),3694–3698. |
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