CN102559752A - Method for transfection and implantation of pre-embryo by plasmid DNA (deoxyribonucleic acid) or antisense oligonucleotide - Google Patents
Method for transfection and implantation of pre-embryo by plasmid DNA (deoxyribonucleic acid) or antisense oligonucleotide Download PDFInfo
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- CN102559752A CN102559752A CN2011104037923A CN201110403792A CN102559752A CN 102559752 A CN102559752 A CN 102559752A CN 2011104037923 A CN2011104037923 A CN 2011104037923A CN 201110403792 A CN201110403792 A CN 201110403792A CN 102559752 A CN102559752 A CN 102559752A
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Abstract
The invention discloses a method for transfection and implantation of pre-embryo by plasmid DNA (deoxyribonucleic acid) or antisense oligonucleotide, which comprises the following steps of: (1) preparing an electroporation solution to be transfected; (2) weakening embryo zona pellucida; and (3) implanting the embryo into the prepared electroporation solution, standing at the room temperature, and then implanting the embryo together with the electroporation solution into an electroporation slot for electroporation. Because the method of electroporation transfection is adopted, two or more kinds of plasmid DNA or antisense oligonucleotide can also be simultaneously transferred into the implanted pre-embryo together; and if the exogenous fragment carried by the constructed plasmid DNA is a key gene or an interference fragment targeting a key gene, the concrete effect of the key gene in the development process of the implanted pre-embryo of a mouse can be concretely researched.
Description
Technical field
The invention belongs to embryo's transfection field, relate to a kind of DNA or Antisense OligodeoxynucleotideTransfection Transfection and attach the method for planting preceding embryo.
Background technology
The Mammals early embryonic development is controlled by the genetic mechanism of himself, and studying the basic skills that this genetic mechanism adopts is the expression of regulation and control key gene, observes follow-up developmental state and analyze the effect that this key gene is brought into play in growth course.At present, foreign gene being imported the expression that the embryo carried out expression or suppressor gene is the consistent and classic methods of research gene function.
Be transferred to foreign gene cell/embryo or be incorporated into mainly infection and the microinjection through transfection, virus vector of genome, and the expression of suppressor gene mainly rely on clpp gene low with the gene knockout technology.Yet, with regard to attach plant before with regard to the embryo, import very difficulty to foreign gene through the infection of transfection or virus vector because attach plant before the embryo outside wrap up by the layer of transparent band.Though the gene knockout technology is got well but can not be used widely, because this technology wastes time and energy and spends huge.
At present the sequence of foreign gene and down-regulation of gene expression comprises that dsRNA, siRNA and antisense oligonucleotide mainly attach through the microinjection importing and plants preceding embryo.This kind method efficient is very low because with foreign gene and dsRNA, siRNA and antisense oligonucleotide import later the attaching of 2-cell stage plant before the embryo need the injection of blastomere one by one, waste time and energy and can only operate an embryo at every turn.
The method of inhibition of gene expression is broadly divided into two types according to the principle difference: the first, utilize siRNA or dsRNA degraded said target mrna, and make key gene lose the template of translation; The second, utilize antisense oligonucleotide to combine, or suppress the translation of said target mrna, or change the shearing of said target mrna, and then show the phenotype of certain key gene inactivation with said target mrna.In the past, during the expression of suppressor gene, using maximum was siRNA and dsRNA; But recent years; The utilization of antisense oligonucleotide has the trend of rising, and said target mrna and difficult by RNase H degraded can not kept the long period in cell or embryo because antisense oligonucleotide is not degraded.At present, the developmental biology man method that adopts microinjection is injected antisense oligonucleotide the zygote and then the research fetal development of ovum or zebra fish, frog, Ascidian, sea urchin etc. more.This method can accurately be controlled the importing time of antisense oligonucleotide, but this method is very high to operator's technical requirements, and can only inject an embryo at every turn, and efficient is very low
Therefore, demand urgently seeking a kind of with foreign gene or distinguished sequence rapidly and efficiently import mouse attach plant before embryo's method, for further further investigation mouse early embryonic development mechanism provides technical support.
Electroporation technology mainly is to utilize instantaneous pulsed electrical field to change cytolemma lipid structure of two layers, makes cytolemma form temporary hole, and macromole gets into cell or embryo under effect of electric field.In the electricity of success changeed, electropermeabilization and electrophoretic action played a part very crucial, and in addition, macromolecular passive diffusion and cell endocytic have also played certain function.And utilize electroporation technology with DNA or antisense oligonucleotide import attach plant before embryo's report do not see so far.
Summary of the invention
The problem that the present invention solves is to provide a kind of DNA or Antisense OligodeoxynucleotideTransfection Transfection to attach plant before embryo's method, realize the embryo is carried out safe, easy, transfection efficiently.
The present invention realizes through following technical scheme:
A kind of DNA or Antisense OligodeoxynucleotideTransfection Transfection attach the method for planting preceding embryo, may further comprise the steps:
1) damping fluid is changeed in the DNA electricity consumption that will treat transfection, and to be diluted to concentration be 10～80 μ g/ml, obtains DNA electroporation solution;
Damping fluid is changeed in the antisense oligonucleotide electricity consumption that perhaps will treat transfection, and to be diluted to concentration be 0.2mM, obtains antisense oligonucleotide electroporation solution;
2) will treat attaching of transfection plant before the embryo place acid tyrode's solution, incubated at room 10～20s stops digestion then and cleans;
3) DNA electroporation solution or antisense oligonucleotide electroporation solution are added in the groove between two electrodes of electricity commentaries on classics ware; To attach and plant preceding embryo's linear array between two electrodes of electricity commentaries on classics ware; Under pulsed electrical field, utilize electroporation to attach plant before the embryo carry out transfection.
Described DNA also goes intracellular toxin to handle before transfection, damping fluid
is changeed in the DNA electricity consumption of going intracellular toxin to handle dilute.
Described will attach plant before the embryo also the embryo is cleaned with H-KSOM solution before acid tyrode's solution handles carrying out.
Described termination digestion is the embryo to be transferred to the H-KSOM solution from acid tyrode's solution clean, and then cleans with
solution.
The DNA of described transfection is the DNA that has fluorogene;
Described antisense oligonucleotide also carries out the lissamine mark before transfection, damping fluid
is changeed in the antisense oligonucleotide electricity consumption of lissamine mark dilute.
The DNA of described transfection comprises the DNA that one or more above sequences are different.
Described attach plant before the embryo comprise that being in attaching of different development stage plants preceding embryo.
The parameter that described electroporation carries out plasmid DNA transfection is controlled to be: voltage 20V～40V; Burst length 1～2ms; Pulse number 3～4 times.
Described attach plant before the embryo be that mouse attaches and plants preceding embryo, electricity changes parameter and is controlled to be: voltage 20V～40V; Burst length 1～2ms; Pulse number 3～4 times.
After described electroporation is accomplished, the embryo after the transfection cleaned and be transferred in the KSOMaa nutrient solution cultivate.
Compared with prior art, the present invention has following beneficial technical effects:
DNA provided by the invention or Antisense OligodeoxynucleotideTransfection Transfection attach the method for planting preceding embryo; 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 DNA or antisense oligonucleotide entering cell under the normal physiological situation.
Owing to adopted the method for electroporation transfection; The present invention can also change two or more DNAs together over to attach simultaneously and plant preceding embryo; If the entrained exogenous segment of DNA that makes up is the interference fragment of certain key gene or certain key gene of target, then can specifically studies certain key gene and attach the concrete effect that is play in embryo's the growth course before planting mouse.
Owing to adopted the method for electroporation transfection, the present invention can with designed, with the mRNA bonded Antisense OligodeoxynucleotideTransfection Transfection of different target genes in the embryo, can be used for studying goal gene and attach the concrete effect that is play in embryo's the growth course before planting mouse.
In order further to observe or follow the tracks of the transfection effect; The DNA of treating transfection has fluorogene; And the antisense oligonucleotide of treating transfection carries out lissamine (Liz amine, can rubescent look fluorescence) mark, behind electroporation transfection, can observe intuitively through fluorescent microscope like this.
The present invention with DNA or antisense oligonucleotide change over to attach plant before the embryo save time, laborsaving, cost less, simple to operate, to the embryo of operator and operation do not have toxicity, efficient is high, speed is fast.Once can operate 50～100 pieces of embryos, the electric commentaries on classics time is in 1min.Electricity changes back vitro culture 24h, the embryo is observed and carry out statistical analysis through fluorescent microscope to show that electric transfer efficient of the present invention is about 97%.For adopting gene or its function in the embryo of genetic transcription thing research that DNA or antisense oligonucleotide were directed against that effective transfection method is provided.
Description of drawings
Fig. 1 attaches the Photomicrograph of planting preceding embryo for DNA changes different development stage over to;
Fig. 2 attaches the Photomicrograph of planting preceding embryo for antisense oligonucleotide changes different development stage over to.
Embodiment
Attach the growth of planting preceding embryo and mainly experience three transformations (being respectively the activation of zygotic gene group, densification and blastaea forms) and twice pedigree differentiation (differentiation of trophectoderm and inner cell mass, inner cell mass are divided into primitive endoderm and ectoderm).The differentiation of transformation or pedigree all is accompanied by the great variety that embryonic gene is expressed at every turn, and therefore, research embryo expression in transformation or pedigree atomization changes bigger gene can further disclose the molecular regulation mechanism that early embryonic development is contained.Yet all there is certain defective in technology commonly used at present aspect the research gene function.Transfection provided by the invention attaches the method for planting preceding embryo, adopts the method transfection DNA of electroporation, has utilized electroporation hypotoxicity, high, the fireballing advantage of efficient.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 transfection attaches the method for planting preceding embryo, and the instantaneous micropore that under pulsed electrical field, forms through embryo's zona pellucida and cytolemma gets into the embryo, 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 the method that those skilled in the art can pass through to be provided is carried out the seldom experiment of number of times and is screened for these concrete parameters and optimize; Can confirm the optimized parameter of respective embryo, accomplish embryo's transfection.
DNA is respectively two kinds of different molecular tools of research gene function with GEM 132.Utilize plasmid DNA transfection to carry out the research expressing and suppress to express, several kinds of different plasmids of transfection simultaneously, and the expression that GEM 132 can only suppressor gene to key gene.Relative GEM 132, the molecule of DNA is bigger.
Following mask body with mammal particularly mouse attach plant before the embryo as objective for implementation; Specify plasmid DNA transfection and attach the method for planting preceding embryo; 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.
Embodiment 1
Plasmid DNA transfection attaches the method for planting preceding embryo, may further comprise the steps:
1, at first to DNA is carried out pre-treatment:
A, the concrete DNA that adopts are that pIRES2-AcGFP1-Nuc (Clontech) example describes; DNA can adopt different plasmid vectors to carry the different purpose gene as required; The plasmid vector general requirement of treating transfection has fluorescent mark gene or other marker gene, so that follow-up observation and screening;
Go intracellular toxin to handle DNA, going intracellular toxin to handle is for the intracellular toxin of eliminating in the plasmid growing of embryo to be impacted;
B, will go endotoxic DNA to be diluted in
solution; DNA is different; The concentration of dilution was also different when electricity changeed, and the concentration after the dilution of pIRES2-AcGFP1-Nuc DNA is controlled to be 40 μ g/ml; Though the expression of every kind of DNA all is not quite similar, through repeatedly can suitably adjusting after the experiment.
2, mice embryonic carries out the pre-treatment of zona pellucida reduction:
The mouse of a, different times that collection is obtained attach plant before the embryo in H-KSOM operation liquid, wash 3 times, to remove fragment of tissue and granulosa cell;
100ml H-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;
B, will collect good embryo insert acid tyrode's solution (sigma, the U.S., T1788) in 15s, carry out the zona pellucida reduction and handle; Different embryos' the concrete reduction time can obtain through repeatedly testing to screen;
C, the mice embryonic of handling that will weaken are washed 3 times in H-KSOM operation liquid, to remove acid tyrode's solution;
D, the embryo is washed 3 times in
solution, inserting dilution afterwards has in
solution of DNA;
After the pre-treatment of completion to the embryo, the embryo is carried out electroporation, concrete operations may further comprise the steps:
1) use electroporation apparatus (ECM 2001Electro Cell Manipulator, BTX, the U.S.) to carry out electroporation, plasmid DNA solution adds electricity changes ware: getting 30 μ l plasmid DNA solutions adding electricity changes the groove between two electrodes of ware;
2) mouse attach plant before the embryo insert electricity and change between two electrodes of ware: embryo's linear array was changeed between two electrodes of ware before attaching of mouse different times planted at electricity;
3) electricity of DNA changes: electricity changes parameter and is: voltage 30V, pw 1ms, pulse number 2 times, multiplicity 1 time;
4) electricity changes back embryo's cultivation: wash 3 times with embryo's sucking-off and in operation liquid H-KSOM, cultivate afterwards in the KSOMaa nutrient solution;
100ml KSOMaa 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.
5) under fluorescent microscope, observe the situation of embryo's green-emitting fluorescence: after electricity changes 24h, the embryo of different times placed observe photograph fluorescent microscope under and calculate electric transfer efficient.
The fluorescence microscope result is as shown in Figure 1; Wherein scheme the plasmid DNA transfection result that A, figure B, C, figure D, figure E and figure F carry out respectively when 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage and morula; The observations qualitatively of fluorescent brightness is shown, all reached good transfection effect in embryo's transfection of different times.
Embodiment 2
Antisense OligodeoxynucleotideTransfection Transfection attaches the method for planting preceding embryo, may further comprise the steps:
1, at first antisense oligonucleotide is carried out pre-treatment:
A, specifically to adopt the standard control antisense oligonucleotide be that example describes, and antisense oligonucleotide can design to the different purpose gene as required, treats that the antisense oligonucleotide of transfection generally has the fluorophor mark, so that follow-up observation and screening;
Specifically adopting the standard control antisense oligonucleotide (commercial) of lissamine mark, can not influence the function of any gene, only is a control group in normal experiment.Because the sequence of antisense oligonucleotide is very short, only about 25bp, by synthetic.As long as the standard control antisense oligonucleotide can transfection success, other antisense oligonucleotide to any gene can both change into.
B, the antisense oligonucleotide of lissamine mark is diluted in
solution; Antisense oligonucleotide is different; The concentration of dilution was also different when electricity changeed, and the concentration after the dilution of standard control antisense oligonucleotide is controlled to be 0.2mM; Though the target gene transcript that every kind of antisense oligonucleotide was directed against all is not quite similar, through repeatedly can suitably adjusting its transfection concentration after the experiment.
2, mice embryonic carries out the pre-treatment of zona pellucida reduction:
The mouse of a, different times that collection is obtained attach plant before the embryo in H-KSOM operation liquid, wash 3 times, to remove fragment of tissue and granulosa cell;
100ml H-KSOM Working solution prescription is with embodiment 1;
B, will collect good embryo and insert 15s in the acid tyrode's solution (sigma, the U.S.), and carry out the zona pellucida reduction and handle; Different embryos' the concrete reduction time can obtain through repeatedly testing to screen;
C, the mice embryonic of handling that will weaken are washed 3 times in H-KSOM operation liquid, to remove acid tyrode's solution;
D, the embryo is washed 3 times in
solution, insert afterwards in the antisense oligonucleotide diluent;
After the pre-treatment of completion to the embryo, the embryo is carried out electroporation, concrete operations may further comprise the steps:
1) use electroporation apparatus (ECM 2001Electro Cell Manipulator, BTX, the U.S.) to carry out electroporation, antisense oligonucleotide solution adds electricity changes ware: getting 30 μ l antisense oligonucleotide solution adding electricity changes the groove between two electrodes of ware;
2) mouse attach plant before the embryo insert electricity and change between two electrodes of ware: embryo's linear array was changeed between two electrodes of ware before attaching of mouse different times planted at electricity;
3) electricity of antisense oligonucleotide changes: electricity changes parameter and is: voltage 30V, pw 1ms, pulse number 2 times, multiplicity 1 time;
4) electricity changes back embryo's cultivation: wash 3 times with embryo's sucking-off and in operation liquid H-KSOM, cultivate afterwards in the KSOM nutrient solution;
100ml KSOM nutrient solution prescription is with embodiment 1;
5) under fluorescent microscope, observe the situation of embryo's green-emitting fluorescence: after electricity changes 24h, the embryo of different times placed observe photograph fluorescent microscope under and calculate electric transfer efficient.
The fluorescence microscope result is as shown in Figure 2; The antisense oligonucleotide electricity that figure A, figure B, figure C, figure D, figure E and figure F carry out when 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage and morula respectively changes the result; The observations qualitatively of fluorescent brightness is shown, all reached good transfection effect in embryo's transfection of different times.
Claims (10)
- DNA or Antisense OligodeoxynucleotideTransfection Transfection attach plant before embryo's method, it is characterized in that, may further comprise the steps:1) damping fluid is changeed in the DNA electricity consumption that will treat transfection, and to be diluted to concentration be 10～80 μ g/ml, obtains DNA electroporation solution;Damping fluid is changeed in the antisense oligonucleotide electricity consumption that perhaps will treat transfection, and to be diluted to concentration be 0.2mM, obtains antisense oligonucleotide electroporation solution;2) will treat attaching of transfection plant before the embryo place acid tyrode's solution, incubated at room 10～20s stops digestion then and cleans;3) DNA electroporation solution or antisense oligonucleotide electroporation solution are added in the groove between two electrodes of electricity commentaries on classics ware; To attach and plant preceding embryo's linear array between two electrodes of electricity commentaries on classics ware; Under pulsed electrical field, utilize electroporation to attach plant before the embryo carry out transfection.
- 2. plasmid DNA transfection as claimed in claim 1 attaches the method for planting preceding embryo; It is characterized in that; Described DNA also goes intracellular toxin to handle before transfection, damping fluidis changeed in the DNA electricity consumption of going intracellular toxin to handle dilute.
- 3. plasmid DNA transfection as claimed in claim 1 attach plant before embryo's method, it is characterized in that, described will attach plant before the embryo also the embryo is cleaned with H-KSOM solution before acid tyrode's solution handles carrying out.
- 4. plasmid DNA transfection as claimed in claim 1 attaches the method for planting preceding embryo; It is characterized in that; Described termination digestion is the embryo to be transferred to the H-KSOM solution from acid tyrode's solution clean, and then cleans withsolution.
- 5. plasmid DNA transfection as claimed in claim 1 attaches the method for planting preceding embryo, it is characterized in that the DNA of described transfection is the DNA that has fluorogene;Described antisense oligonucleotide also carries out the lissamine mark before transfection, damping fluidis changeed in the antisense oligonucleotide electricity consumption of lissamine mark dilute.
- 6. plasmid DNA transfection as claimed in claim 1 attaches the method for planting preceding embryo, it is characterized in that the DNA of described transfection comprises the DNA that one or more above sequences are different.
- 7. plasmid DNA transfection as claimed in claim 1 attach plant before embryo's method, it is characterized in that, described attach plant before the embryo comprise that being in attaching of different development stage plants preceding embryo.
- 8. plasmid DNA transfection as claimed in claim 1 attaches the method for planting preceding embryo, it is characterized in that the parameter that described electroporation carries out plasmid DNA transfection is controlled to be: voltage 20V～40V; Burst length 1～2ms; Pulse number 3～4 times.
- 9. plasmid DNA transfection as claimed in claim 1 attach plant before embryo's method, it is characterized in that, described attach plant before the embryo be that mouse attaches and plants preceding embryo, electricity changes parameter and is controlled to be: voltage 20V～40V; Burst length 1～2ms; Pulse number 3～4 times.
- 10. plasmid DNA transfection as claimed in claim 1 attach plant before embryo's method, it is characterized in that, after described electroporation is accomplished, the embryo after the transfection cleaned and is transferred in the KSOMaa nutrient solution cultivate.
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| US6284944B1 (en) * | 1997-08-29 | 2001-09-04 | Cephalon, Inc, | Gene-targeted non-human mammal with a human fad presenilin mutation and generational offspring |
| JP2004041023A (en) * | 2002-07-09 | 2004-02-12 | Japan Science & Technology Corp | Method for transfecting intracellular transfecting material into animal cell using electroinjection method and apparatus therefor |
| CN102127521A (en) * | 2010-12-25 | 2011-07-20 | 河南科技大学 | Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method |
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2011
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0919121A1 (en) * | 1996-07-08 | 1999-06-02 | Dnavec Research Inc. | In vivo electroporation method for early animal embryo |
| US6284944B1 (en) * | 1997-08-29 | 2001-09-04 | Cephalon, Inc, | Gene-targeted non-human mammal with a human fad presenilin mutation and generational offspring |
| JP2004041023A (en) * | 2002-07-09 | 2004-02-12 | Japan Science & Technology Corp | Method for transfecting intracellular transfecting material into animal cell using electroinjection method and apparatus therefor |
| CN102127521A (en) * | 2010-12-25 | 2011-07-20 | 河南科技大学 | Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method |
Non-Patent Citations (3)
| Title |
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| JOANNA B. GRABAREK等: "Efficient Delivery of dsRNA into Zona-Enclosed Mouse Oocytes and Preimplantation Embryos by Electroporation", 《GENESIS》, vol. 32, 31 December 2002 (2002-12-31) * |
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