jove-protocol-3024.pdf

Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseAugust 2011|54|e3024|Page 1 of 6Video ArticleMouse in Utero Electroporation:Controlled Spatiotemporal Gene TransfectionAsuka Matsui1,Aya C.Yoshida1,Mayumi Kubota1,Masaharu Ogawa1,Tomomi Shimogori11Lab for Molecular Mechanisms of Thalamus Development,RIKEN Brain Science InstituteCorrespondence to:Tomomi Shimogori at tshimogoribrain.riken.jpURL:http:/ 54,In utero,electroporation,transfection,CNS,gene expression,gain of function,loss of function,neuron,axonDate Published:8/15/2011Citation:Matsui,A.,Yoshida,A.C.,Kubota,M.,Ogawa,M.,Shimogori,T.Mouse in Utero Electroporation:Controlled Spatiotemporal GeneTransfection.J.Vis.Exp.(54),e3024,doi:10.3791/3024(2011).AbstractIn order to understand the function of genes expressed in specific region of the developing brain,including signaling molecules and axonguidance molecules,local gene transfer or knock-out is required.Gene targeting knock-in or knock-out into local regions is possible to performwith combination with a specific CRE line,which is laborious,costly,and time consuming.Therefore,a simple transfection method,an in uteroelectroporation technique,which can be performed with short time,will be handy to test the possible function of candidate genes prior to thegeneration of transgenic animals 1,2.In addition to this,in utero electroporation targets areas of the brain where no specific CRE line exists,andwill limit embryonic lethality 3,4.Here,we present a method of in utero electroporation combining two different types of electrodes for simple andconvenient gene transfer into target areas of the developing brain.First,a unique holding method of embryos using an optic fiber optic light cablewill make small embryos(from E9.5)visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targetedbrain area 5,6.The patterning of the brain such as cortical area occur at early embryonic stage,therefore,these early electroporation from E9.5make a big contribution to understand entire area patterning event.Second,the precise shape of a capillary prevents uterine damage by makingholes by insertion of the capillary.Furthermore,the precise shape of the needle electrodes are created with tungsten and platinum wire andsharpened using sand paper and insulated with nail polish 7,a method which is described in great detail in this protocol.This unique techniqueallows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated.This will help to,open anew window for many scientists who are working on cell differentiation,cell migration,axon guidance in very early embryonic stage.Moreover,this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus,wherenot many region-specific CRE lines exist for gain of function(GOF)or loss of function(LOF)analyses.Video LinkThe video component of this article can be found at http:/ of capillary and DNA solution for injection1.Purify plasmid DNA with a Maxi-prep or equivalent method,and make a final concentration of 2-3 g/l.Aliquot 100 g of DNA,add 10lof fast green dye(1%stock)and TE(10mM Tris Base,1mM EDTA Solution,pH 8.0)and adjust a volume to 100 l to make the final DNAconcentration for injection mix to 1g/l.The concentration of plasmid DNA can be changed depends on the size of plasmid,and also itstransfection efficiency,which needs to be tested individually.2.Spin the DNA solution for 5 minutes at 14,000 RPM at room temperature and collect supernatant to remove any residue.3.Pull a glass capillary tubea glass capillary tubes using a micropipette puller.Pinch off the tip with forceps to make the tip has a 2030 mdiameter.Measure 800 m-1 mm from the tip and check the external diameter is less than 60 m(Fig.1A).4.Connect the capillary to the micromanipulator,and fill with mineral oil.To fill the capillary with the DNA solution,submerge the tip into thesolution and suck up the DNA solution.2.Stick platinum electrodesHere we used stick platinum electrodes(Nepe gene,CUY611P2-1)to electroporate into superficial region of brain,such as the cortex(Fig.1F).3.Preparation of micro-electrodesUse micro-electrodes to electroporated into deep parts of the brain(i.e.,thalamus and hypothalamus)(Fig.2B-E).1.One end of the tungsten(for negative electrodes)and platinum(for positive electrodes)wires is coiled on gold-plated pins and fixed withparafilm.Insert the wire to the stem of a cotton swab and wrap both ends with parafilm(Fig.1B).Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseAugust 2011|54|e3024|Page 2 of 62.Evenly sharpen the tip of the wire with sandpaper until it reaches 2030 m external diameter and 60 m from 800 m-1 mm of the tip(Fig.1C and D).3.Apply a thin coat of nail polish to the wire for insulation.After the nail polish has dried,use an acetone-soaked cotton swab to remove it fromthe tip(about 200 m from the tip).Important note:In order to prevent damage to the mouses uterus,the portion within 800 m-1 mm of thetip should be no more than 70 m in diameter(Fig.1E).4.Preparation of the surgery1.Anesthetize a pregnant mouse(E9.5-E15.5).We demonstrate anesthetization with pharmaceutical grade sodium pentobarbital(Table II;50g per gram body weight,inject intraperitoneally and wait for 5 min).Alternatives to sodium pentobarbital as an anesthetic include Ketamine/Xylazine injection or gas anesthetics(gas is preferable for short duration procedures).The injection should be done with attention to avoidany organs,otherwise it will cause to damage the uterus or kill animal.When successful IP is done,the survival rate is more than 95%.Alsothe surgery can be performed in open-air environment.Before started the operation,test for a lack of response by the animal to pinching theirtoe to confirm anesthetic.2.Shave the hair from the abdomen using a razor blade and 50%EtOH.Prepare the skin with alternating scrubs of betadine and alcohol(70%).3.Make an incision at the abdominal midline with fine scissors and pull out all uterine horns carefully onto a 37 C pre-warmed phosphate-buffered saline(PBS)-moistened cotton gauze,which is placed around the wound.Keep the uterus moist with PBS all of the time.4.Hold a flexible fiber optic cable between the index and middle fingers and place it under the uterine horn.Clean cable with 70%EtOH priorto use.No microscope or magnifier is required for visualization.Position uterus between the optic fiber light optic cable and the thumb,andsqueeze gently to push up the embryo closer to the uterine wall.5.Injection of DNA and electroporation1.When embryo is positioned,insert glass capillary carefully into target ventricle and inject approx 1 l of DNA solution(Fig.2A).2.For electroporation to superficial part of the brainIf using with stick platinum electrodes for electoporation,place the electrodes outside of theuterus and apply suggested square-wave current pulses according to manufactures instruction(Table I).If usingFor electroporation to deeperpart of the brain,nmicro electroporation,eedle type electrodes are used.Iinsert a fine tungsten negative electrode into DNA injected ventricleand a platinum electrode into the uterus and place target region between two electrodes,then apply suggested square-wave current pulses(Table I)(Fig.2B).6.Post electroporation1.Place the uterine horn back in its original location with and add 500 L of 37 C pre-warmed PBS.2.Suture the inner layer with surgical suture and close outer layer with a 9 mm autoclipauto clip.3.Place animals on a heating pad for 2 hours to allow recovery from anesthesia.Analgesics such as carprofen or buprenorphine can beadministered for post operative pain.7.Representative results:Some examples of cortical electroporation of pCAG-EYFP construct using stick platinum electrodes at different time points are shown in figureFigure 32.Brains are electroporated at E13.5,E14.5 and E15.5,and harvested at postnatal day(P)6.AThe ntibody staining of RORc revealsposition of layer 4 and position of EYFP transfected cells are revealed by GFP antibody staining,and both images are super imposed(Fig.3A and B).Labeled cortical cells are layer of cortical cells electroporated is clearly different in each experiment(Fig.32),which suggests time-dependent electroporation makes cortical layer-specific GOF/LOF possible.The viability of embryos and efficiency of cell transfection varies depending on the age of embryo and the location of electroporation.Samplesare listed on Table 1,however,conditions should be optimized for each electroporation depending on the stage and part of the brain.Electroporation into a deep tissue such as thalamus and hypothalamus with using stick platinum electrodes often give low efficiency(Table 1).This problem can be solved by using micro-electrodes whichelectrodes,which will reach to deep parts of the brain.Figure 3 4 shows exampleof pCAG-EYFP electroporation into the developing diencephalon.Plasmid DNA solution is injected into the 3rd ventricle at E11.5 and followedby micro electroporation(Fig.3C4C).The electroporated area is restricted in the thalamus where most of the axons project to the cortex(Fig.3B4B).Axon projection to layer 4 of the cortex can be also visualized by EYFP,which fillesfills up the entire neuron.(Fig.3A4A).Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseAugust 2011|54|e3024|Page 3 of 6 Figure 1.Cartoon schematic demonstrating the correct shape of the capillary.(A)A glass capillary tube is pulled using a micropipette pullerto make certain shape.A tip is pinched off by forceps to make it 2030 m.Measure 800 m-1mm from the tip(red bracket)and make surethat external diameter is less than 60 m(green bracket).(B)One end of the tungsten or platinum wires is coiled on gold-plated pins and fixedwith parafilm.Then use sand paper to make their tip to become 20-30 m external diameters and 60 m from 1 mm of the tip.(C)Image of thetungsten wire before shaping.(D)Image of the tungsten wire after shaping.(E)Image of the tungsten wire after applying thin coat of nail polish.Scale bar in E is 10 m(smallest scale)for C-E.(F)Stick platinum electrodes(Nepa gene.CUY611P2-1).Scale bar in F is 2 mm.Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseAugust 2011|54|e3024|Page 4 of 6 Figure 2.Cartoon schema of surgical procedure.(A)Diagram of injection into lateral ventricle or large embryos(left side)and into 3rd ventricle orsmall embryos(right side).(B)Diagram of electrporation by platinum stick electrode(left side)and by needle type electrode(right side).Figure 3.Developing mouse telencephalon was electroporated with pCAG-EYFP at E13.5(A),E14.5(B)and E15.5(C)and harvested at P6.Brains were sectioned coronal and stained with GFP antibody or RORc antibody separately and images are merged.(A)Electroporation ofpCAG-EYFP plasmid at E13.5 transfect many layer 4 neurons.which overlap with layer 4 markers RORc.(B)Electroporation of pCAG-EYFPplasmid at E14.5 transfect cells,which are more superficial than layer 4 neurons.(C)E15.5 electroporation label many superficial neurons suchas layer 2/3.Scale bar 200 m.Journal of Visualized ECopyright 2011 Creative Commons Attribution-NonCommercial LicenseAugust 2011|54|e3024|Page 5 of 6 Figure 4.Transfection of EYFP into thalamo-cortical axons(TCA).(A)TCA terminus in cortical layer 4 is revealed by GFP antibody staining.RORc antibody staining is used to reveal a position of layer 4.Both images are taken from same section and merged.(B)Position ofelectroporation into the thalamus is also shown by GFP antibody staining and RORc staining,which expression is restricted in the thalamus 11.(C)Cartoon schematic for thalamic electroporation and TCA projection are shown.Scale bar 200 m.DiscussionIn this protocol,we only described benefits of the technique of the pCAG-EYFP transfection into the restricted area of a small embryonic brainusing different shaped electrodes.For comparison of transfection efficiency by different promoter is described previously,which shows cell typespecificity 1.However,there are limitations for the technique,such as the transfection is only transient and varies depend on the plasmid,itdoes not have any cell specificity and it is hard to control numbers of transfected cells.ThereforeHowever,combining this protocol with othertechniques will further provide possibilities for different manipulation methods.First,incorporating cell specific promoter into plasmid DNA willallow cell-type specific transfection,such as neurons,glial cells and astrocytes.Second,since the transfection is transient,it is not suitable forscientists who want to analyze the function of the gene in later life.Therefore,combination with a transposase to make plasmid DNA integrateinto the genome will convert it into a stable transfection 8.Next,adoption of the tet-on or tet-off system will make it possible to manipulate thetiming or cell type-specific gene expression in neural cells 8.Alternatively,one can combine this protocol with tamoxifen-inducible Cre-ER(T)recombinases 9 and reporter mice 10.This broad accessibility of tissues will drastically change experimental designs used in neuroscience.DisclosuresNo conflicts of interest declared.AcknowledgementsThis work was funded by RIKEN Brain Science Institute(BSI),Human Frontier Science Program(HFSP)(awarded to T.S.)and RIKEN JuniorResearch Associate Program(JRA,awarded to M.K.)RIKEN Brain Science Institute(BSI),Human Frontier Science Program(HFSP)(awarded toT.S.)and RIKEN Junior Research Associate Program(JRA,awarded to M.K.)funded this work.References1.Tabata,H.&Nakajima,K.Efficient in utero gene transfer system to the developing mouse brain using electroporation:Visualization ofneuronal 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