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1. as monitored within the system High quality electrodes Effective electroporation of various tissues Voltages can be selected easily Intracel place High cost however 1 this system does not require the investigator to make modifica tions Inconsistent current delivery from pulse to pulse 4 Electrode quality questionable RECOMMENDATIONS Disadvantages outweigh advantages Buy another system if possible tech www protechinternational com 3 Intracel TSS20 Ovodyne electroporator www intracel co uk and 4 Grass SD9 square pulse stim ulator www grass telefactor com If you are initiating in ovo electropora tion experiments in your laboratory investigate these electroporators first by discussing their pros and cons with investigators with expertise in electroporation Table 1 These dis cussions ahead of your purchase will save your resources including money and effort Electrodes Electrodes can be built easily and at low cost by investigators or their lo cal instrument shops or purchased commercially As one example Pro tech sells high quality electrodes for use with their CUY 21 electroporator To construct your own electrodes you ll need the following equip ment supplies for electrode prepa ration cost 25 per pair solder ing iron lead free solder hair dryer or heat shrink gun black stranded 22 The Cadillac of electroporators Cost i2. tion Nat Neurosci 4 Suppl 1156 1158 ltasaki N Bel Vialar S Krumlauf R 1999 shocking developments in chick embry ology electroporation and in ovo gene expression Nat Cell Biol 1 E203 E207 Kos R Reedy MV Johnson RL Erickson CA 2001 The winged helix transcrip tion factor FoxD3 is important for estab lishing the neural crest lineage and re pressing melanogenesis in avian embryos Development 128 1467 1679 Kos R Tucker RP Hall R Duong TD Erick son CA 2003 Methods for introducing morpholinos into the chicken embryo Dev Dyn 226 470 477 Lee S K Pfaff SL 2003 Synchronization of neurogenesis and motor neuron spec ification by direct coupling of OHLH and homeodomain transcription fac tors Neuron 38 731 745 Martinez CY Hollenbeck PJ 2003 Trans fection of primary central and periph eral nervous system neurons by electro poration Methods Cell Biol 71 339 351 Momose T Tonegawa A Takeuchi J Ogawa H Umesono K Yasuda K 1999 IN OVO ELECTROPORATION IN CHICK 439 Efficient targeting of gene expression in chick embryos by microelectropora tion Dev Growth Differ 41 335 344 Muramatsu T Mizutani Y Okumura J 1996 Live detection of the firefly lucif erase gene expression by biolumines cence in incubating chicken embryos Ann Sci Technol 67 906 909 Muramatsu T Mizutani Y Onmori Y Oku mura J 1997 Comparison of three non viral transfection methods for foreign gene expression in early chic
3. Biotechniques 28 94 96 98 100 Arber S Han B Mendelsohn M Smith M Jessell TM Sockanathan S 1999 Re quirement for the homeobox gene Hb9 in the consolidation of motor neu ron identity Neuron 23 659 674 Audic Y Boyle B Slevin M Hartley RS 2001 Cyclin E morpholino delays em bryogenesisin Xenopus Genesis 30 107 109 Bach A Lallemand Y Nicola MA Ramos C Mathis L Maufras M Robert B 2003 Msx1 is required for dorsal diencepha lon patterning Development 130 4025 4036 Becker DL McGonnel Makarenkova HP Patel K Tickle C Lorimer J Green CR 1999 Roles for aloha 1 connexin in morphogenesis of chick embryos using a novel antisense approach Dev Genet 24 33 42 Bellairs R Osmond M 1998 The atlas of chick development San Diego Aca demic Press Bel Vialar S Ifasaki N Krumlauf R 2002 Initiating Hox gene expression in the early chick neural tube differential sen sitivity to FGF and RA signaling subdi vides the HoxB genes into two distinct groups Development 129 5103 5115 Bronner Fraser M Editor 1996 Methods in avian embryology San Diego Aca demic Press Eberhart J Swartz ME Koblar SA Pasquale EB Krull CE 2002 EonA4 constitutes a population specific guidance cue for motor neurons Dev Biol 247 89 101 Eberhart J Barr J O Connell S Flagg A swartz ME Cramer KS Tosney KW Pas quale EB Krull CE 2004 Ephrin AS ex erts positive or inhibitory effects on dis tinct subsets o
4. dm and the presumptive distal limb mesoderm asterisk at this stage E DNA is microin jected into the extracellular soace between the forming dermomyotome dm and scle rotome scl and will travel into cells in the lateral half dm upon current passage F Transverse section through embryo at forelimb level 1 day after electroporation as shown in E EGFP positive muscle precursors green enter the limb from the lateral dermomyotome dm In this embryo the dermomyotome of a single somite was electroporated C merlin protein red marks the myotome m whereas Pax7 protein purple binds to muscle precursors prominent in the dermomyotome place your electrodes on embryonic tissue to prevent injury and morpho logic defects Pass five 10 to 15 volt pulses each of 50 msec duration The DNA will be driven into the neu ral tube cells that lie adjacent to the positive electrode anode To trans fect dorsal neural tube cells reverse the polarity of your electrodes Re move the electrodes and apply a few drops of Ringer s to cool and hydrate the embryo Seal window in the eggshell with tape and reincu bate the egg until the desired stage of development is achieved THE CHALLENGE TARGETING MESENCHYME WITH IN OVO ELECTROPORATION Many investigators new to in ovo electroporation have been frus trated by their attempts to transfect mesenchyme or mesenchyme de rived tissues including limb meso derm and somites This frust
5. the addi tion of a GFP antibody to restore this signal may be required 6 Antibody staining or in situ hy bridization should be performed to determine whether one s gene of in terest is expressed and the duration of expression after in ovo electropo ration Western blot analysis serves as an excellent alternative ap proach COMMON PROBLEMS AND TROUBLESHOOTING A few of the typical problems en countered when using in ovo elec troporation are described below as well as troubleshooting strategies Problem 1 None or few cells are transfected and gene expression is short lived Solutions a Adjust elec trode placement to widen the elec tric field and target more cells b A different promoter may be useful To target gene expression more pre cisely c Replace electrodes if older than 3 months d Consider that the normal regulatory controls of the embryo may influence the expres sion of your gene of interest e Check that plasmid concentration is 2 pQ pl minimum f Test constructs for expression in primary cell cultures or cell lines g Increase voltage slightly and monitor cell death h In crease volume of DNA injected and i Apply Ringer s solution to enhance Current conduction Problem 2 Embryos do not sur vive after in ovo electroporation So lutions a Replace electrodes if older than 3 months b Determine whether electrodes are touching embryonic tissues during the electro poration damaging
6. the embryo c Confirm that DNA is in solution in wa ter or PBS not TE Prepare fresh DNA if impurities or contaminants are sus pect d Fast Green can interfere with survival of young embryos Try phenol red 0 1 final concentra tion as an alternative and e Re duce voltage pulse width and or number of pulses Problem 3 Embryos have major unexpected morphologic defects Solutions a Electrodes are old and must be replaced b Electrodes are in contact with embryonic tissues when current is being passed Avoid contact with embryonic tissues by placing electrodes in a pool of Ring er s solution Electrodes placed ven tral to the embryo should lie in yolk c Use lower voltages to reduce cell damage OTHER REFERENCES several good reviews and technical papers have been published about using in ovo electroporation to ana lyze gene function in chickens Itasaki et al 1999 Momose et al 1999 Atkins et al 2000 Yasuda et al 2000 Haas et al 2001 Swartz et al 2001a Inoue and Krumlauf 2001 Osumi and Inoue 2001 Nakamura and Funahashi 2001 Martinez and Hollenbeck 2003 Investigators are referred to these publications for ad ditional details FUTURE DIRECTIONS In ovo electroporation is a relatively new approach that has been used primarily to misexpress or overex press genes of interest during chick embryonic development Several in vestigators have used this approach in chickens for their ga
7. D E dermomyotome Fig 2 In ovo electroporation targets cells in the neural tube limb mesoderm and the somitic dermomyotome A C E Schematic diagrams showing how to perform in ovo electroporation including DNA microinjection micropipette and electrode placement and electrodes for successful transfection of a ventral quadrant of the neural tube limb mesoderm and lateral half of the somitic dermomyotome In each diagram one electrode is positioned ventral to the embryo by inserting it carefully at the interface between the embryo blastoderm and yolk A DNA is microinjected into the neural tube nt lumen and will travel into ventral neural tube cells upon current passage som somite no notochord B Transverse section through embryo at hindlimb level 2 5 days after electropo ration as shown in A EGFP signal green localizes to several motor neuron cell bodies in the ventral neural tube nt and the floor plate fp Islet antibody stains postmitotic neurons red C DNA is microinjected into the coelom co that lies ventral to the somatopleure sop which will generate limb mesoderm Injected DNA will travel into sop cells upon current passage to transfect limb mesoderm spl splanchnic mesoderm D Transverse section through embryo at forelimb level 1 day after electroporation as shown in C Limb mesoderm cells that lie at the base of the limb express EGFP green EohA4 protein red labels cells in the dermomyotome
8. DEVELOPMENTAL DYNAMICS 229 433 439 2004 A Primer on Using In Ovo Electroporation to Analyze Gene Function Catherine E Krull The chicken embryo has served as a classic model system for developmental studies due to its easy access for surgical manipulations and a wealth of data about chicken embryogenesis Notably the mechanisms controlling limb development have been explored best in the chick Recently the method of in ovo electroporation has been used successfully to transfect particular cells tissues during embryonic development without the production or infectivity associated with retroviruses With the sequencing of the chicken genome near completion this approach will provide a powerful opportunity to examine the function of chicken genes and their counterparts in other species In ovo electroporation has been most effectively used to date for ectopic or overexpression analyses However recent studies indicate that this approach can be used successfully for loss of function analyses including protein knockdown experiments with morpholinos and RNAi Here will discuss parameters for using in ovo electroporation successfully to study developmental processes Developmental Dynamics 229 433 439 2004 2004 Wiley Liss Inc Key words transfection EGFP chicken Received 28 August 2003 Revised 17 September 2003 Accepted 30 September 2003 BASIC PRINCIPLES Electroporation has served as an ef fective method for introducing
9. DNA into bacteria yeast and mammalian cells Neumann et al 1982 Potter 1988 This approach uses electric field pulses To disrupt plasma membrane stability transiently creating pores in cell membranes through which DNA is driven due to its negative charge Usually this tyoe of electroporation uses high voltage and short duration electrical pulses resulting in en hanced cell death The application of electroporation to chicken embryos in ovo was made feasible by altering voltage and current parameters square wave pulses of low voltage and longer duration are used to achieve DNA delivery in the embryo with minimal cell death and excellent survival The first published account of electroporation in chicken tissue in volved the transfection of plasmid DNA into retinal explants Pu and Young 1990 Shortly thereafter this approach was applied to chicken embryos in ovo Muramatsu ef al 1996 1997 Since then several inves tigators have made modifications to the original procedure to enhance embryo survival increase transfection efficiency and target gene manipu lations to particular cell types MAKING IN OVO ELECTROPORATION WORK THE HARDWARE REQUIREMENTS Over the past 4 years my col leagues and have optimized in ovo electroporation to transfect several Biological Sciences University of Missouri Columbia Columbia Missouri Grant sponsor National Institutes of Mental Health Muscular Dystrophy Associatio
10. ap propriate stage after in ovo electro poration confirm the success of transfection in ovo using a fluo rescence dissecting microscope equipped with GFP optics Embryos with poor transfections can be dis carded embryos that require further development before harvesting can be reincubated 2 The percentage of transfected cells will vary with electroporation conditions In our hands 50 85 of targeted cells are transfected Im portantly this percentage is not consistent from embryo to embryo Therefore if requires that investiga tors plan to transfect at least 2 4x as many embryos as needed for their functional analysis 3 Investigators should examine whether each construct used in in ovo electroporation results in en hanced cell death by using terminal deoxynucleotidyl transferase medi ated biotinylated UTP nick end label ing MUNEL labeling or Nile blue or acridine orange stains 4 Investigators should examine embryos carefully for nonspecific morphologic defects that are likely due to the electroporation tech nique Any contact by electrodes with embryonic tissue during Current passage will create damage These nonspecific morphologic defects will complicate your analysis postelec troporation and should be avoided 5 Embryos can be fixed and viewed as whole mounts or pre pared for cryostat paraffin or Vi bratome sectioning Fixation and ex posure to organic solvents dampens or destroys GFP signal so
11. cess 7 Voltage size and pulse number three to five should be altered sys tematically when first attempting in ovo electroporation Pulse duration remains typically at 50 msec al though longer pulses of 100 msec may be helpful to introduce other reagents RNA proteins pharmaco logic inhibitors into embryonic cells 8 Electrode placement will di rectly impact your transfection suc cess with in ovo electroporation Al tering electrode placement by a few microns can offen improve suc cess Again consider the geometry of the Tissue you want to transfect and whether it possesses a luminal surface place your electrodes so that the cells you want to transfect lie within your electric field 9 Connect your electroporator to an oscilloscope to check current readings and verify consistency in current delivery LOSS OF FUNCTION EXPERIMENTS USING IN OVO ELECTROPORATION Many investigators have routinely used in ovo electroporation for gain of function analyses overexpressing or ectopically expressing their gene of interest and EGFP or a variant Ar ber ef al 1999 Grapin Botton et al 2001 Bel Vialar et al 2002 Eberhart et al 2002 Bach et al 2003 William IN OVO ELECTROPORATION IN CHICK 437 ef al 2003 However a few recent studies have shown that in ovo elec troporation can be used effectively in loss of function experiments Three approaches are available thus far 1 proteins that act as compet
12. essful DNA will be confined to the lumen of the neural tube and soread rostrally and caudally from the injection site If DNA instead soreads over the top of the embryo you have not pierced the neural tube and entered its lumen Reposi tion your micropipette and embryo and try again If DNA spreads below the embryo the micropipette has penetrated ventral to the embryo Discard this egg and prepare a new embryo for microinjection Electrode Placement and Current Application shortly after microinjection of DNA into the lumen of the neural tube place two platinum electrodes see hardware requirements section lat eral and parallel to the embryo on the area opaca approximately 5 6 mm apart This electrode placement will result in the labeling of dorsal and ventral neural tube cells on one side of the embryo To target a ven tral quadrant of the neural tube place the positive electrode below the embryo see Fig 2A and the negative electrode dorsal to the neural tube and on the contralat eral side It is essential that your elec trodes remain parallel to each other with the same distance between them throughout your electropora tions to maintain consistency of cur rent delivery The territory in the mid dle of your electrodes should include the region of the neural tube to be electroporated and basically comprises your electric field Do not 436 KRULL o te q b pa wU w Q z
13. f EohA4 positive motor neurons J Neuroscience in press Ebert PJ Timmer JR Nakada Y Helms AW Parab PB Liu Y Hunsaker TL Johnson JE 2003 Zic 1 represses Math expression via interactions with the Math enhancer and modulation of Math autoregula tion Development 130 1949 1959 Ethell IM Irie F Kalo MS Couchman JR Pasquale EB Yamaguchi Y 2001 EohB syndecan 2 signaling in dendritic spine morphogenesis Neuron 31 1001 1013 Gerlach Bank L Cleveland AR Barald KF 2004 DAN directs endolymphatic sac and duct outgrowth in the avian inner ear Dev Dyn 229 219 230 Granata A Quaderi NA 2003 The Opitz syndrome gene MID1 is essential for es tablishing asymmetric gene expression in Hensen s node Dev Biol 258 397 405 Grapin Botton A Majithia AR Melton DA 2001 Key events of pancreas forma tion are triggered in gut endoderm by ectopic expression of pancreatic reg ulatory genes Genes Dev 15 444 454 Haas K Sin W C Javaherian A Cline HT 2001 Single cell electroporation for in vivo neuronal gene expression Hamburger V Hamilton HL 1951 A series of normal stages in the development of the chick embryo J Morphol 88 49 92 Howard EW Newman LA Oleksyn DW Angerer RC Angerer LM 2001 SpKrl a direct target of beta catenin regula tion required for endoderm differentia tion in sea urchin embryos Develop ment 128 365 375 Inoue T Krumlauf R 2001 An impulse to the brain using in vivo electropora
14. in of function analyses paired with the powerful genetics approach in mouse for loss of function studies In the future RNA interference and the application by means of in ovo electroporation of other reagents i e moroholinos that knockdown protein specifically promises to enhance studies of gene function in chickens Predict ably loss of function studies should become more prevalent Another direction for the future in cludes the linking of in ovo electro poration with time lapse imaging of cell behavior Together these ap proaches will allow investigators to exploit the advantages of the chicken embryo which include easy access to develoomental events Novel DNA constructs that allow in vestigators to tightly control the tem poral expression of their gene of in terest should provide enormous power to this approach Moreover in ovo electroporation offers the unique opportunity to identify and analyze enhancer elements quickly Timmer et al 2001 Uchikawa et al 2003 Ebert et al 2003 Clearly the sequencing of the chicken genome will provide enormous opportunities for using in ovo electroporation and the classic strengths of this model system to dissect gene function in the future ACKNOWLEDGMENTS Thanks go to members of the Krull lab for critical discussions REFERENCES Atkins RL Wang D Burke RD 2000 Local ized electroporation a method for tar geting expression of genes in avian em bryos
15. ire Fig 1 4 Lengths of the red and black stranded wire should allow holding of the electrodes by hand or placement in a plastic electrode holder without restriction Red and black stranded wires are then each attached to a double banana BNC connector Fig 1 2 amp 3 with a male end This double banana BNC con nector is then connected to the electroporation system output wiring Fig 1 1 IN OVO ELECTROPORATION 101 TARGETING NEURAL TUBE CELLS Over the past few years in ovo elec troporation into the neural tube has become a routine and relatively easy approach to examine cell specification and axon guidance during chicken embryogenesis If you are a novice using in ovo elec troporation start with neural tube electroporations Once you be come an expert in transfecting the Electroporator Fig 1 and Instructions sections for parts numbers and connections 1 8 neural tube in this manner electro poration of other tissues will be more straightforward Electroporation of the chicken neural tube Fig 2A B can be sub divided into three steps 1 prepara tion of the embryo and DNA for electroporation 2 microinjection of DNA into the lumen of the neural tube and 3 electrode placement and current application Preparation of Embryo and DNA for Electroporation swab eggshell with 70 ethanol re move approximately 2 3 ml of albu men with an 18 g needle attached to a 3 cc syringe and open a smal
16. itive inhibitors 2 RNAi and 3 morpho linos Gene Tools These types of ap proaches allow investigators to dis rupt protein signaling or knockdown protein levels providing important tools for the analysis of gene func tion Each of these techniques is rel atively new and only a few investi gators have published studies using them successfully thus far Expressing Proteins That Act as Competitive Inhibitors We have expressed kinase inactive forms of the EohA4 receptor in cells that express wild type EphA4 to dis rupt EohAd4 signaling Eberhart et al in press kKIEONA4 abolishes the phos phorylation or activation of the WT EphA4 receptor acting as a domi nant negative Ethell et al 2001 Recent studies have used in ovo electroporation to express mutated forms of transcriptional activators which also serve as dominant nega tives Lee and Pfaff 2003 The ex pectation is that this approach will become more widely used by inves tigators as comfort and expertise with in ovo electroporation devel Ops RNAi stoeckli and colleagues recently demonstrated that dsRNA can be introduced into the developing chicken embryo using in ovo elec troporation Pekarik ef al 2003 Rel atively long sequences 200 to 2 000 base pairs in length interfere specif ically with protein translation in chicken embryos as seen in these studies on axon guidance across the midline It is essential to demonstrate that protei
17. ken em bryos in ovo Biochem Biophys Res Commun 230 376 380 Nakamura H Funahashi J 2001 Introduc tion of DNA into chick embryos by in ovo electroporation Methods 24 43 48 Nasevicius A Ekker SC 2000 Effective targeted gene knockdown in ze brafish Nat Genet 26 216 220 Neumann E Schaefer Ridder M Wang Y Hofschneider PH 1982 Gene transfer into mouse lyoma cells by electropora tion in high electric fields EMBO J 1 841 845 Oberg KC Pira CU Revelli JP Ratz B Agui lar Cordova E Eichele G 2002 Efficient ectopic gene expression targeting chick mesoderm Dev Dyn 224 291 302 Osumi N Inoue T 2001 Gene transfer into cultured mammalian embryos by elec troporation Methods 24 35 42 Pekarik V Bourikas D Miglino N Joset P Preiswerk S Stoeckli ET 2003 Screening for gene function in chicken embryo using RNAi and electroporation Nat Biotechnol 21 93 96 Potter H 1988 Electroporation in biology methods applications and instrumen tation Anal Biochem 174 361 373 Pu HF Young AP 1990 Glucocorticoid inducible expression of a glutamine synthetase CAT encoding fusion plas mid after transfection of intact chicken retinal explant cultures Gene 89 259 263 Swartz M Eberhart J Mastick G Krull CE 2001a Sparking new frontiers using in vivo electroporation for genetic ma nipulations Dev Biol 233 13 21 Swartz ME Eberhart J Pasquale EB Krull CE 2001b EohA4 ephrin A5 interac tions in
18. l window 1 2 cm in the eggshell overlying the embryo with fine scis sors Enlarge window so that the em bryo is in clear view Inject a small bolus of a 10 Pelikan Drawing Ink A in Ringer s under the blastoderm to enhance contrast Carefully tear off the vitelline membrane that overlies the neural tube to be electropo rated with a tungsten needle Refer to Methods in Avian Embryology Bronner Fraser 1996 for additional details Typically we have used two DNA constructs for in ovo electropora tion 1 PpCAX which contains a chick beta actin promoter CMV IE enhancer to drive expression of EGFP control Osumi and Inoue 2001 and 2 PMES which contains the same promoter ennancer and an IRES EGFP generating a bicis tronic message Swartz et al 2001a b Eberhart et al 2002 Con structs can be built to include other variants of GFP including RFP One strength of this approach is that there are no apparent limitations to the insert size that can be expressed from these plasmid constructs in contrast to several retroviral vectors Two or theoretically more con structs can be co electroporated at the same time Lee and Pffaff 2003 It is important to sequence your con struct to confirm expression of your DNA insert and EGFP or other marker in cell lines or primary cell cultures Prepare DNA for electroporation by using a Qiagen Plasmid kit to ensure high quality DNA and elute the DNA into water not elutio
19. muscle precursor cell migration inthe avian forelimb Development 128 4669 4680 Timmer J Johnson J Niswander L 2001 The use of in ovo electroporation for the rapid analysis of neural specific murine enhancers Genesis 29 123 132 Uchikawa M Ishida Y Takemoto T Ka machi Y Kondoh H 2003 Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mam mals Dev Cell 4 509 519 Wiliam CM Tanabe Y Jessell TM 2003 Regulation of motor neuron subtype identify by repressor activity of Mnx class homeodomain proteins Devel opment 130 1523 1536 Yasuda K Momose T Takahashi Y 2000 Applications of microelectroporation for studies of chick embryogenesis Dev Growth Differ 42 203 206
20. n Grant number MH059894 MDA Correspondence to Catherine E Krull Biological Sciences 108 Lefevre University of Missouri Columbia Columbia MO 65211 E mail krullc missouri edu DOI 10 1002 dvdy 10473 tissues including the neural tube limb mesoderm and somitic meso derm Swartz et al 2001a b Eber hart et al 2002 We build our own electrodes and have modified sev eral features of our electroporation system Our colleagues in other insti tutions have made additional mod ifications to enhance electropora tion success Below describe the hardware requirements for success ful in ovo electroporation defining both advantages and disadvan tages of four in ovo electroporation systems Each system delivers square wave pulses Four in ovo electroporation sys tems are currently available com mercially 1 BTX electroporator model 8300 www btxonline com 2 CUY 2 electroporator from Pro 2004 Wiley Liss Inc 434 KRULL ELECTROPORATOR ADVANTAGES BIX suspension Electroporation of various tissues highly efficient after 4 investigator made modifications DISADVANTAGES High cost Connectors from the electroporator are flimsy and require modification by the investigator Voltages can be selected easily Low or high voltage modes available for electroporating embryos or cells in TABLE 1 Comparing Electroporators CUY 21 1 Consistent current delivery
21. n buffer Use a soectrometer to measure DNA qual ity if the OD 260 280 1 8 then the DNA is pure with little protein con taminants If the OD is less than 1 6 re prepare the DNA Concentrate the DNA at 2 5 wg pl in either PBS 1x final concentration or water al iquot at 2 4 wl and store at 20 C to prevent degradation Microinjection of DNA Into the Lumen of the Neural Tube If you have used vital dyes by means of a micropipette to mark small numbers of cells this approach will be very similar Add either a few crystals of Fast Green or 0 1 final concentration of phenol red to DNA to verify microinjection Backfill a mi cropipette with 3 ul of your DNA solution by using a Hamilton syringe Place the micropipette into a nee dle holder connected to a micro manipulator which is linked to a pi cospritzer Alternatively DNA can be microinjected by using a mouth pi pette or hand held syringe Align the DNA loaded micropipette with the region of the neural tube to be mi croinjected with DNA and move the micropipette to this area by using the micromanipulator Pierce the IN OVO ELECTROPORATION IN CHICK 435 Constructing electrodes Schematic diagram showing how to build electrodes for in ovo electroporation Refer to the Electrodes dorsal surface of the neural tube where it opposes at midline Expel DNA into the lumen you will visualize your success immediately by the Fast Green or phenol red label If succ
22. n knockdown is specific to one s protein of interest Typically this task is accomplished by means of Western blot analysis using an an tibody that binds the protein of inter est specifically This approach pro vides a powerful method for knockdown studies and should prove extremely valuable for future functional genomics analyses 438 KRULL Morpholinos Morpholinos have served as fantas tic tools to interfere with protein translation for investigators who study develooment in zebrafish Xe nopus and sea urchins Nasevicius and Ekker 2000 Audic ef al 2001 Howard et al 2001 However inves tigators using chickens as model sys tems have encountered some diffi culties introducing these reagents by means of in ovo electroporation most likely due to the reduced charge associated with these mole cules making it problematic to tar get them to some cell types Erickson and colleagues were the first to demonstrate that morpholinos could be applied in chickens with in ovo electroporation Kos et al 2001 see Kos ef al 2003 for methods Re cently a few investigators have used this approach to specifically knockdown proteins during chicken embryonic develooment Gerlach Bank et al 2004 Granata and Qua deri 2003 As with RNAi it is impera tive that the investigator demonstrate that specific protein knockdown has been achieved ANALYSES AFTER IN OVO ELECTROPORATION 1 After embryos develop to the
23. r con structing tissue explants and per forming electroporation in culture dishes 3 Use the enclosed schematic illus trations and electroporation parame ters and other published reports as guidelines Each electroporation sys tem is different and must be cali brated for each tissue to be trans fected 4 After identifying the tissue you wish to transfect consider the elec tric field that you must establish to electroporate that tissue Remem ber that DNA which is negatively charged will move into cells thot lie adjacent to the positive electrode 5 The distance between your electrodes dramatically affects your electroporation success Increased distances between electrodes gen erally require increased voltages to successfully transfect tissues that lie in the electric field generated across the electrodes when passing cur rent Very small distances between electrodes narrow the electric field and can damage cells Therefore voltages must typically be lower As the distance between electrodes is widened and electric field is en larged greater numbers of cells are transfected with in ovo electropora tion As the distance between elec trodes is narrowed and the electric field is reduced a smaller number of cells are electroporated offen re sulting in focal transfections 6 The resistance created by a tis sue must impact the electric field that is generated and thereby influ ence electroporation suc
24. ration has leff many electroporation systems gathering dust in the lab Figure 2C F provides schematic diagrams to illustrate in ovo electroporation of limb mesoderm and the somitic der momyotome and images of these transfected tissues It is imperative that investigators understand that transfecting mesenchyme with in ovo electroporation is a challenging task due to several factors First as a tissue mesenchyme typically con tains cells that are loosely associ ated with each other Thus the ex tracellular space or matrix in mesenchyme provides a medium in which DNA can easily diffuse after microinjection Second luminal spaces in which to deposit DNA may not be obvious in mesenchyme Third it is important to remember that cells in different tissues have dif ferent resistances based on tissue geometry and condensation Tissue resistance affects the electric field generated during electroporation and thus electroporation success This means that investigators must systematically vary electroporation parameters including voltage elec trode placement and pulse num bers when attempting to transfect different tissues A few strategies are available to deal with transfecting loosely packed mesenchyme using in Ovo electroporation One approach is to apply your DNA in a more viscous medium such as Pluronic gel which serves to reduce diffusion Becker et al 1999 A caveat of this approach is that the viscous medium ma
25. s high but well worth it gauge wire Newark Electronics catalog no 92n5737 red stranded 22 gauge wire Newark Electronics catalog no 92n5837 gold plated jack socket Newark Electronics catalog no 40f6130 pin stamped brass Digikey catalog no 82p nd wire insulation Newark Electronics catalog no 03F3 12 and for plati num electrodes platinum rod A M systems catalog no 711000 0 01 inch diameter Instructions Warm the soldering iron Cut plati num wire into 6 to 7 cm lengths yielding three sets of electrodes or purchase precut lengths Solder the pin stamped brass Fig 1 6 onto the platinum wire Fig 1 8 Cut six pieces of wire insulation heat shrink tubing into 5 cm lengths Place the tubing over the platinum wire Fig 1 7 adjacent to the pin stamped brass and apply heat from hair dryer or heat shrink gun to seal the wire insulation to the wire Solder the gold plated jack socket Fig 1 5 1 Low cost 2 Allows measurements of resistance while electrodes are in Grass stimulator 1 Low cost and easy to use 2 Typically available in most departments check with local physiologists Useful for electroporating single or multiple cells Range of voltages available are limited A very workable system Accessibility and low that is low cost cost are strengths Very solid performer see Haas et al 2001 for setup details onto red and black stranded w
26. y in deed have deleterious effects on cells A second approach is to follow the DNA microinjection with a small bolus of mineral oil to localize DNA Oberg et al 2002 Lastly others have reduced the time interval be tween DNA microinjection and elec troporation by microinjecting and apply current by means of a single micropipette see Haas et al 2001 for details Having a luminal surface in which to microinject DNA is a great advantage for in ovo electroporation A lumen serves as a DNA reservor r limiting DNA diffusion and localizing DNA to the tis sue to be transfected To determine whether the tissue you wish To trans fect has a luminal surface go to chick atlases or staging criteria Hamburger and Hamilton 1951 Bellairs and Os mond 1998 Offen you will find that a luminal surface does exist at early stages of development see Swarz et al 20016 Important Tips to Consider 1 Use embryos at stages 10 20 Hamburger and Hamilton 1951 for whole embryo electroporation Younger embryos tolerate less volt age than older embryos Embryos electroporated at stages younger than stage 10 exhibit increased mor tality rates and morphologic de fects due to the delicate tissue or ganization at early stages Embryos older than stage 20 have more com oact tissues and increased Tissue lay ers making microinjection and elec troporation difficult 2 If you wish to transfect tissue from older embryos conside
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