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Electroporation of Embryonic Chick Eyes
鸡胚胎视网膜DNA电转移   

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BMC Biology
May 2014

Abstract

The chick embryo has prevailed as one of the major models to study developmental biology, cell biology and regeneration. From all the anatomical features of the chick embryo, the eye is one of the most studied. In the chick embryo, the eye develops between 26 and 33 h after incubation (Stages 8-9, Hamburger and Hamilton, 1951). It originates from the posterior region of the forebrain, called the diencephalon. However, the vertebrate eye includes tissues from different origins including surface ectoderm (lens and cornea), anterior neural plate (retina, iris, ciliary body and retinal pigmented epithelium) and neural crest/head mesoderm (stroma of the iris and of the ciliary body as well as choroid, sclera and part of the cornea). After gastrulation, a single eye field originates from the anterior neural plate and is characterized by the expression of eye field transcriptional factors (EFTFs) that orchestrate the program for eye development. Later in development, the eye field separates in two and the optic vesicles form. After several inductive interactions with the lens placode, the optic cup forms. At Stages 14-15, the outer layer of the optic cup becomes the retinal pigmented epithelium (RPE) while the inner layer forms the neuroepithelium that eventually differentiates into the retina. One main advantage of the chick embryo, is the possibility to perform experiments to over-express or to down-regulate gene expression in a place and time specific manner to explore gene function and regulation. The aim of this protocol is to describe the electroporation techniques at Stages 8-12 (anterior neural fold and optic vesicle stages) and Stages 19-26 (eye cup, RPE and neuroepithelium). We provide a full description of the equipment, materials and electrode set up as well as a detailed description of the highly reproducible protocol including some representative results. This protocol has been adapted from our previous publications Luz-Madrigal et al. (2014) and Zhu et al. (2014).

Keywords: Electroporation (电穿孔), Chick (小鸡), Eyes (眼睛), Stages 19-26 (eye cup stages) (阶段19-26(眼杯阶段)), Stages 8-12 (anterior neural fold and optic vesicle stages) (阶段8-12(前神经褶和视泡阶段))

Materials and Reagents

  1. Fertilized specific pathogen free (SPF) (Charles River Laboratories) or white Leghorn chicken eggs (Note 1)
  2. 10x Hank's balanced salt solution (HBSS) (Thermo Fisher Scientific, catalog number: 14185-052 )
  3. Fast green FCF (Sigma-Aldrich, catalog number: F7258 )
  4. Indian Ink Type A (Pelikan)
  5. Tris (hydroxymethyl) aminomethane (suitable for cell culture) (Sigma-Aldrich, catalog number: 252859 )
  6. EDTA (Ethylenediaminetetraacetic acid), suitable for cell culture (Sigma-Aldrich, catalog number: E6758 )
  7. pCAG- GFP (Addgene plasmid, catalog number: 11150 ) or pEGFP-N1 (Takara Bio Company, Clontech)
  8. Plasmid and RCAS-DNA [the name RCAS stands for Replication-Competent ASLV long terminal repeat (LTR) with a Splice acceptor] (Note 6)
  9. Morpholinos (Note 7)
  10. NaCl (Fisher Scientific, catalog number: BP358-1 ) (MW 58.44 g/mol)
  11. CaCl2 anhydrous (Acros Organics, catalog number: AC34961-5000 ) (MW 110.98 g/mol)
  12. KCl (Fisher Scientific, catalog number: P217-500 ) (MW 74.55 g/mol)
  13. Na2HPO4 Dibasic anhydrous (Fisher Scientific, catalog number: S374-500 ) (MW 141.96 g/mol)
  14. KH2PO4 Dibasic Anhydrous (Fisher Scientific, catalog number: P290-500 ) (MW 174.18 g/mol)
  15. Ringer’s solution (see Recipes)
  16. 10x Hank's balanced salt solution (HBSS) (see Recipes)
  17. Fast green FCF (see Recipes)
  18. 1 M Tris (see Recipes)
  19. 0.5 M EDTA (see Recipes)
  20. TE buffer for plasmid solutions (see Recipes)

Equipment

  1. Beveled-edge watch glass (to hold the egg during the electroporation) (Thermo Fisher Scientific, catalog number: 15-355 )
  2. 200 μl tips sterile (Corning Incorporated)
  3. Capillary tubing borosilicate for microinjection (1.0 mm OD, 0.5 mm ID/fiber) [Frederick Haer & Co (FHC), catalog number: 30-30-1 ]
  4. 1 ml syringe (Thermo Fisher Scientific)
  5. Pre-pulled beveled glass needles 50 mm long, 20 µm tip diameter and sharpened 10 to 12° (these glass needles are made following the instructions of the Micropipette Puller and Micropipette Beveler- Figure 1d-e)
  6. 35 mm plastic tissue culture plates (Corning Incorporated)
  7. 3/4 inch wide clear tape (Scotch, 3 M)
  8. Micro dissecting tweezers #55 and #5 (Roboz, catalog numbers: RS-4984 and RS-4978 )
  9. ECM 830 High Throughput Electroporation System (Figure 1a), a Square Wave Pulse generator for in vitro and in vivo electroporation with remote operation Footswitch (BTX, Harvard Apparatus, SKU: ECM_830 _for_In_Vivo_Applications)
  10. Microinjector (Figure 1b), MicroJect 1000A (BTX, Harvard Apparatus, SKU: 45- 0750 ) with foot Switches to inject and fill and a stainless steel pipette holder (Figure 1c)
  11. Micropipette beveler (Sutter Instrument Company, model: BV-10 ) (Figure 1d)
  12. Vertical micropipette puller (Sutter Instrument Company, model: P-30 ) (Figure 1e)
  13. Nitrogen tank Compressed 2.2 UN1066 NI NI230PP 230CF PP (CAGA580) (Weiler Welding)
  14. Stereo zoom microscope (Motic, model: SMZ-168 , catalog number: 1100200500322) or equivalent
  15. Tungsten halogen light source (series equipped with Fiber Optic, model: 8375 ) (Fostec ACE)
  16. Rotating incubator (45 of angle rotation every hour) calibrated at 37-39 °C (99 to 103 °F) and relative humidity of 50-55% (83-87 °F or 28-31 °C, on wet bulb thermometer) (e.g. Breeding Technology, 1202E Classic Sportsman, https://www.gqfmfg.com/store/front.asp)
  17. Incubator Thermal Air Hova-Bator (https://www.gqfmfg.com/store/front.asp)
  18. 200 μl micropipette
  19. Blue-light filter (12.5 mm diameter, ~100% transmittance up to 500 nm) (Edmond Optics, catalog number: 52-530 )
  20. Electrodes (Note 8)
    1. Stage-8-12 electrodes (for set up see Figure 2)
      1. Platinum/Iridium (Pt/Ir) Microelectrodes unit of 3 (Frederick Haer&Co, catalog number: UEPMEEVENNND ), use the following Metal Microelectrode Spec Sheet (http://www.fh-co.com/uploads/files/ue-spec-2013.pdf) for ordering
      2. Extreme-Temperature Polyimide Tubing (0.007" ID, 1/16", OD, 0.08" Wall, 1'L,Clear Amber, McMasterCarr, catalog number: 5707K12 )
      3. Plastic holder (made utilizing a 20 µl pipet tip ) with ~2 mm diameter and ~3 mm length (see Figure 2 c1-2)
      4. Bend-and-Stay 302/304 Stainless Steel Wire (0.032" diameter, 1' Long, McMASTER- CARR, catalog number: 6517K66 )
      5. Precision Miniature Stainless Steel Tubing, 304 Stainless Steel, 15 Gauge, 0.072" OD, 0.05" ID, .011" Wall (McMASTER-CARR, catalog number: 8988K31 )
      6. White Delrin® Acetal Resin Rod, 3/8" Diameter (McMASTER-CARR, catalog number: 8572K53 ). This Resin Rod is modified with a press fit to incorporate internally the Stainless Steel Tubing (material #v) and to fit in the polycarbonate tube that functions as a hand holder (material #vii) (Figure 1d).
      7. Impact-Resistant Polycarbonate Round Tube (McMASTER-CARR, catalog number: 8585K11 )
      8. 1 m of 26 gauge (stranded, aluminum wire)
      9. Connector; BNC; Nickel Plated Brass; 20; Gold Plated Beryllium Copper; PVC (Pomona, catalog number: 4969 )
      10. Flow Mix 60 sec (Epoxy, 1250 psi Strength, part number: 21445 ) (Devcon)
    2. ectrodes (for set up see Figure 3)
      1. One genetrode kit (5 mm, Gold plated thick electrode - L-shaped, in ovo gene) (Harvard Apparatus, model: 512, catalog number: 45-0115 )
      2. One platinum/iridium microelectrode unit of 3 (Frederick Haer & Co, catalog number: UEPMEEVENNND), see part “i” from Stage-8-12 electrodes for ordering instructions.
      3. Tygon® microbore tubing ( 0.010" ID x 0.030"OD, 100 ft/roll) (Cole-Parmer, catalog number: EW-06419-00 ) (Characteristics: Extremely flexible, non-toxic; nonpyrogenic; biocompatible) (Formulation Tygon, catalog number: ND-100-80 )
      4. Extreme-Temperature Polyimide Tubing .0089" ID, .0104", OD, .0008" Wall (1 L, Clear Amber) (McMaster-Carr, catalog number: 51085K42 )
      5. One Capillary tubing borosilicate for Microinjection, 1.0 mm OD, 0.5 mm ID/fiber (Frederick Haer&Co, catalog number: 30-30-1)
      6. One 1 ml pipet tip (Corning Incorporated)
      7. 50 cm aluminum wire (stranded), 26 gauge
      8. Connector; BNC; Nickel Plated Brass; 20; Gold Plated Beryllium Copper; PVC (Pomona, catalog number: 4969)
      9. Flow mix 60 sec (Epoxy, 1250 psi Strength, part number: 21445) (Devcon)


        Figure 1. Electroporation equipment. The electroporation equipment consist of the ECM 830 electroporator (a), Microinjector MicroJect 1000A and stainless steel pipette holder (b, c), Micropipette Beveler and Puller necessary to make glass needles (d, e).

Procedure

  1. Chicken egg manipulation and incubation
    1. Prior to incubation, fertilized specific pathogen free (SPF) (Charles River Laboratories, Wilmington) (Note 1) or White Leghorn chicken eggs (Michigan State University, East Lansing, MI) are stored at room temperature up to 5 days without significant decrease in viability (80-85% viability) or defects in development (Note 2).
    2. Before incubating the eggs, check the conditions of the incubator (Note 3).
    3. For electroporations in the eye, we incubate the eggs approximately 35 h for Stages 8-9 (seven somites, the anterior neural folds closes to form the neural tube), 38 h for Stage 10 (ten somites, optic vesicles not constricted at bases), 48 h for Stage 12 (sixteen somites, optic vesicle and optic stalk established), 72 h for Stage 14 (optic vesicle evaginated and lens-placode present) or 4.5 days for Stage 22 [eye pigmented, retinal pigmented epithelium (RPE) and neuroepithelium well established] (Note 4).


      Figure 2. Stage 8-12 electrodes set up. Two isolated Pt/Ir electrodes with Polyimide Tubing (amber tube) having 1 mm of free tip (a) are bent as is indicated (b). Thereafter, the bent electrodes are inserted into a small plastic holder (made from a 20 µl pipet tip) with ~2 mm diameter (c1) and ~3 mm length (c2) keeping at the tip 1.5 mm in between (c). The electrodes are inserted into the holder made of Stainless Steel Tube (SST) and Resin Rod (RR) that has been modified with a press fit (d). Carefully, electrodes are permanently attached to the holder using epoxy. It is critical to keep the shape and 1.5 mm distance between the electrodes during this procedure (e). Finally, the Pt/Ir electrodes are connected to the cable that has been previously connected to the BNC adaptor of the electroporator.


      Figure 3. Stage 19-26 electrodes set up. One Gold plated thick electrode (anode +) is isolated with tygon microbore tubing (a). One Pt/Ir microelectrode (cathode -) is isolated using Polyimide Tubing (amber tube) having 1 mm of free tip (see Figure 2a) and inserted into a glass capillary tube and sealed with epoxy (b), that can be alternatively protected with a 1 ml pipet tip. The assembled electrodes are then connected to the cable that has been previously connected to the BNC adaptor of the electroporator (c).

  2. Electroporations at Stages 8-12
    1. Preparing the embryos for electroporation
      1. Fertilized eggs are incubated horizontally on their sides so the embryo can be properly positioned for electroporation (Figure 4a). If electroporation is planned for more than one dozen, we strongly suggest to space out the incubation times for each dozen in order to have enough time to electroporate the next dozen at the same developmental stage. This is particularly critical for Stages 8 to 12. Once the embryos reach the desired Stage (approximately 38 h for Stage 10, see Chicken egg manipulation and incubation), proceed to open the eggs according with the instructions provided in the Video 1 (steps B1e-h below). It takes about 2 min to open each egg. The electroporation can be made immediately after opening the egg, however if you have enough experience opening the eggs you can open one dozen in about 25 min and then proceed with the electroporation immediately.
      2. Once the eggs reach the desired stage, place them in a carton and at room temperature for about 10 min. This incubation is necessary to facilitate the contraction of the inner shell membrane and promotes the separation from the outer shell membrane to generate the air sack.
      3. Keeping their horizontal position, place the eggs in a small incubator (e,g., HOVA-BATOR) previously calibrated at 37-39 °C (99 to 103 °F) and relative humidity of 50-55%.
      4. With a bright light source, candle the egg and mark the area where the embryo is located, usually the embryo is located in the top part of the yolk (on the top of the horizontal section of the shell).
      5. Mark the air sack that is located between the outer and inner membranes of the egg.
      6. Pierce the shell at the top of the air sack using blunt ended forceps or a syringe with a large bore needle.
      7. Carefully, introduce a fine probe to move the air sack to the top part of the egg, this is possible by gently touching down to lower the inner shell membrane (Figure 4b-c) (Note 5).
      8. Using sharp curved scissors or blunt ended forceps, open a round window on the top of the embryo (around 2 cm of diameter) (Figure 4d) (Video 1 provides visual information of the steps B1e-h) and add 200 μl of HBSS. Close the window using transparent tape and return the egg to the incubator. Discard the embryos older than the desired stage. If the embryos have not reached the desired stage, they can be incubated for an additional time.
        Variations on the embryonic stage in which the electroporation is performed will produce inconsistency on the results and interpretation.
      9. Optionally, to enhance the contrast and visualize the embryo at Stages 8-12, inject 100 µl of Indian ink solution diluted in HBSS using a 1 ml syringe and a 25-26 gauge needle underneath the embryo.
      10. Repeat steps B1e-h for the rest of the eggs until the dozen is completed.


        Figure 4. Preparing eggs for electroporation. Fertilized eggs are incubated in horizontal position (a). After localizing the air sack and the chick embryo, a fine probe is introduced to move the air sack to the top part of the egg. This is possible by gently touching and lowering the inner shell membrane (b, c). Finally, using sharp curved scissors or blunt ended forceps, open a round window on the top of the embryo (around 2 cm of diameter) (d).

        Video 1. Preparing the eggs for electroporation

    2. Embryo electroporation
      1. Adjust the nitrogen tank to 80 psi.
      2. Attach the beveled glass capillary needle to the stainless steel pipette holder of the microinjector (Figure 1c).
      3. Set the microinjector at 18-20 psi and perform a test loading HBSS using the “fill” button or the footswitch and release the solution pushing the button “empty” from the microinjector. Test the microinjection system using the Footswitch.
      4. Attach the blue dichroic filter to the optical path of the Tungsten Halogen Light Source. Configure the electroporator with the following settings: Mode, LV; voltage, 18 V; pulse length, 50 ms; number of pulses, 5; pulse interval, 950 ms; polarity, unipolar.
      5. Connect the Stage-8-12 electrodes (see the equipment section) to the electroporator.
      6. Using a 35mm tissue culture plate, containing 1 ml of HBSS, check the electroporation system placing the electrodes in the solution (Note 9).
      7. Using the footswitch from the microinjector, load the glass capillary needle with the experimental solution (Plasmid, RCAS constructs or MOs in the solutions section).
      8. Add 100 µl of HBSS to the embryo to decrease the resistance between the electrodes.
      9. For electroporations into the eye, inject 0.25 µl (20 microinjection pulses using a 20 µm of tip diameter glass capillary needle) of the solution into the anterior neural fold (anterior neuropore at Stage 8-9, Figure 5a) or into the optic vesicle (Stages 10-11, Figure 5b).
      10. Place the Stage 8-12 electrodes over the vitelline membrane (a clear membrane that is surrounding the egg yolk) in both sides of the embryo (parallel to the neural tube) (Figures 5a, b) and perform the electroporation using the electroporator footswitch.
      11. Carefully, remove the electrodes and add an additional 100 µl of HBSS.
      12. Seal the window with plastic tape and transfer the eggs to the incubator until they reach the desired stage. A representative image of an electroporated embryo at Stage 11 using a control MO (Figure 5c, d), and one collected 3 days after is shown in Figure 5e.


        Figure 5. Electroporations at Stages 8-12. For electroporations in the eye, inject 0.25 µl of the experimental solution into the anterior neural fold (anterior neuropore at Stage 8-9) (a) or into the optic vesicle (Stages 10-11) (b). Representative picture on bright field with india ink for contrast (c) and fluorescence (d) of an embryo electroporated at Stage 11 using control MO. Confocal image of an electroporated eye at Stage 12 using a control MO and collected 24 h after electroporation (e). L: lens, scale bar in (e)= 100 μm.

  3. Electroporations in the optic cup at Stages 19-26
    1. Fertilized eggs are incubated vertically until they reach the desired stage.
    2. Once the eggs reach the desired stage for electroporation, follow the steps a-c as in the section B1 Preparing the embryos for electroporation (keep the eggs in horizontal position starting at step B1c).
    3. Follow steps a to c as in section B2 Embryo electroporation.
    4. Connect the Stage-19-26 electrodes (see the equipment section) of the electroporator.
    5. Configure the electroporator with the following settings: Mode, LV; voltage, 18 V; pulse length, 50 ms; number of pulses, 3; pulse interval, 950 ms; polarity, unipolar.
    6. Check the electroporation system placing the electrodes (1 cm in between) in a 35 mm tissue culture plate containing 1 ml of HBSS (Note 9).
    7. Using the microinjector footswitch, load the glass capillary needle with the experimental solution (RCAS-DNA or morpholinos).
    8. For electroporations in the eye, inject 0.5 µl (40 microinjection pulses) into the eye cup at Stage 18-19 or 0.75 µl (60 microinjection pulses) into the eye at Stage 24-25 using a 20 µm diameter tip glass capillary needle.
    9. Add 200 µl of HBSS to the embryo to decrease the resistance between the electrodes.
    10. To perform the electroporation, the gold plated electrode connected to the anode (+) is placed close to the ventro-temporal section of the eye and the cathode (-) electrode is inserted perpendicular to the head of the embryo (Video 2 essentially describes steps C8-10).

      Video 2 . Electroporation in the chick optic cup at Stages 19-26

    11. Remove the electrodes carefully and add 200 µl of HBSS.
    12. Seal the window with plastic tape and transfer the eggs to the incubator until they reach the desired stage. A representative embryo electroporated at Stage 25 is shown in Figure 6.


      Figure 6. Electroporated eye at Stage 25. Image of electroporated eye at Stage 25 and collected 24 h later using a control morpholino (a). Amplification of the electroporated eye in a (b). Confocal image showing the electroporated neuroepithelium (NE) of the eye (c). RPE: Retinal pigmented epithelium, L: lens. Scale bar = 0.5 mm in (a), 0.1 mm in (b) and 100 μm in (c).

Notes

  1. For studies in which the RCAS [replication-competent ASLV long terminal repeat (LTR) with a Splice acceptor] virus system will be used, we strongly recommend to use Specific Pathogen-Free (SPF) eggs. These eggs have been certified to be pathogen free of avian sarcoma-leukosis virus (ASLV). For additional information see http://www.criver.com/, and a detailed study of endogenous expression of ASLV viral proteins has been published by McNally et al. (2010).
  2. Temperatures below 4 °C are detrimental so special care is advised during shipping in winter season.
  3. Eggs are incubated in a rotating incubator (45 angle rotation every hour) at 37-39 °C (99 to 103 °F) and relative humidity of 50-55%. The incubation times and embryo stages could vary depending on the type of incubator, temperature and humidity, therefore the incubation times for specific embryonic stages should be determined empirically in each laboratory (Hamburger and Hamilton, 1951).
  4. Before performing the electroporation, make sure the embryo is at the desired stage, be aware of some characteristics such as number of somites, optic vesicle, lens-vesicle and eye pigmentation. Small differences in the stage of electroporation can result in tremendous differences in phenotypes. In some cases, an injection of Indian ink could be necessary in order to visualize the embryo and to familiarize with the structures (see step B1i of electroporations at Stages 8-12).
  5. Be gentle when introducing the probe and avoid damage of the inner membranes of the egg. If you notice leaking of albumin is very probable that the embryo will die.
  6. Ensure that the plasmid or RCAS constructs are of high quality. DNA constructs are amplified using commercial kits e.g., PureYieldTM Plasmid Maxiprep System or Qiagen Plasmid Maxi PrepTM and dissolved in TE buffer (see TE buffer in the solutions section). We recommend dissolving the DNA at concentrations between 3.0-5.0 μg/μl for plasmids and 200 ng/μl for RCAS constructs and store at -20 °C for short-term use or at -80 °C for long-term storage. Avoid using samples of DNA containing traces of Phenol or ethanol and high concentration of salts, since they could be detrimental for the viability of the embryo. Determine the concentration of DNA using a spectrophotometer or a Nanodrop. Alternatively, the concentration could be determined using SYBR Green I dsDNA assay. The ratio of absorbance 260/280 will give you an idea about the purity of DNA vs protein or other contaminants and secondary measurement could be 260/230 (phenol carbohydrates and have absorbance close to 230nm) (see technical support bulletin at http://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf). A ratio of ~1.8 or above for 260/280 ratio or 2.0-2.2 for 260/230 are considered good quality DNA. Always verify the integrity of the DNA by electrophoresis. Quantification is not enough to ensure that your DNA is of good quality. For first time users, it is advisable to use plasmids containing a reporter gene such as GFP to evaluate the efficiency of electroporation, for example pCAG-GFP or pEGFP-N1. Additionally, in order to track the expression of your gene of interest it is recommendable to design your constructs to include an IRES-GFP sequence or an alternative way to identify the electroporated areas. To prepare the electroporation solution, mix 9.0 μl of plasmid (3.0-5.0 μg/μl) or RCAS-contruct (200 ng/μl) with 1 μl of 0.05% Fast Green dye.
  7. Morpholino (MO) antisense oligonucleotides are designed to down regulate gene expression. They can be designed and ordered from GeneTools LLC (http://www.gene-tools.com/). In order to trace the electroporated cells, the MOs must be tagged at the 3’-end (e.g., carboxyfluorescein). MO have minimal off-target effects but it is recommendable to use at least two different MOs (e.g., to block translation and splice junctions) in order to observe the same phenotypic effects. Additionally, it is necessary to use a control MO and/or scrambled sequence of the target mRNA. The main disadvantages of using MOs is that they are very expensive and sometimes it is necessary to test more than two sequences in order to find the right MO to efficiently down regulate gene expression. Moreover, during cell division, MOs are diluted so they are inefficient for long term (more than 4 days) and in some cases they are practically undetectable after 72 h of electroporation and in such cases the fluorescence signal needs to be detected using an antibody against fluorescein. MOs are dissolved at 1 mM concentration using sterile Ringer’s solution and stored at room temperature in the original container and kept in the dark. According with Gene Tools, MOs can precipitate at low temperatures and lose their efficiency, so they need to be resuspended again before use. During electroporation, MOs can migrate slightly towards the anode (+) possibly because of the fluorescein that is negatively charged. In order to increase the efficiency of MO electroporation, it is advisable to use 0.5 μg of DNA (e.g., non-biologically active plasmid). To prepare the electroporation solution, mix and heat an aliquot of 10 μl at 65 °C for 10 min to re-dissolve the precipitates. It is not recommendable to use fast green since it can inhibit the electroporation (Kos et al., 2013). The injection of the carboxyfluorescein-tagged morpholino can be visualized using a blue dichroic filter attached to a regular fiber optic lamp.
  8. We provided the list of parts necessary to set up Stage-8-12 and Stage-19-26 electrodes, notice that some of the parts listed could be the same.
  9. You will see gentle bubbling on the electrodes after pushing the electroporator footswitch, (the bubbling is an indication that the system is working properly).

Recipes

  1. Ringer’s solution
    1. Dissolve 7.2 g NaCl, 0.17 g CaCl2, 0.37 g KCl, 0.115 g Na2HPO4, and 0.02 g KH2PO4 in 900 ml of deionized water
    2. Adjust pH to 7.2 using HCl and bring to 1 L with deionized water
      Note: Final concentration: NaCl 123 mM, CaCl2 1.53 mM, KCl 5 mM, Na2HPO4 0.8 mM, KH2PO4 0.1 mM.
    3. Sterilized by filtration using a 0.2 μm Corning disposable plastic vacuum filter
    4. Freeze aliquots of 10 ml at -20 °C
  2. 10x Hank's balanced salt solution (HBSS)
    1. Prepare 250 ml of 1x HBSS using deionized water and adjust pH to 7.2
    2. Sterilize by filtration using a 0.2 μm Corning disposable plastic vacuum filters
    3. Freeze aliquots of 10 ml at -20 °C
  3. Fast green FCF
    1. Prepare a stock solution of 0.05% (wt/vol) using deionized water
    2. Sterilize by filtration using a 0.2 μm Corning syringe disc-type filters
  4. 1 M Tris
    Dissolve 6.057g Tris in 30.0 ml of deionized water andadjust the pH to 8.0 using 1 N HCl and bring to 50.0 ml with deionized water.
  5. 0.5 M EDTA
    Dissolve 18.6 g in 80.0 ml deionized water and adjust the pH to 8.0 using 10 N NaOH (EDTA is soluble until pH reaches 8.0), bring to 100.0 with deionized water.
  6. TE buffer for plasmid solutions (10 mM Tris, 1 mM EDTA, pH=8)
    To prepare 50 ml of TE buffer combine 0.5 ml of 1 M Tris (pH=8) with 0.1 ml of 0.5 M EDTA (pH=8) and adjust to 50 ml with deionized water
    Sterilize by autoclave and freeze aliquots of 2 ml at -20 °C

Acknowledgments

Michael Weeks, Jayson Alexander and Bill Lack, Instrumentation Laboratory at Miami University for their help in the electrodes set up, Leah Stetzel her help on the video recording. This work was supported by EY17319 to KDRT, and CONACYT 162930 and 142523 to AL-M. This protocol has been adapted from our previous publications Luz-Madrigal et al. (2014) and Zhu et al. (2014).

References

  1. Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J Morphol 88(1): 49-92.
  2. Kos, R., Tucker, R. P., Hall, R., Duong, T. D. and Erickson, C. A. (2003). Methods for introducing morpholinos into the chicken embryo. Dev Dyn 226(3): 470-477.
  3. Luz-Madrigal, A., Grajales-Esquivel, E., McCorkle, A., DiLorenzo, A. M., Barbosa-Sabanero, K., Tsonis, P. A. and Del Rio-Tsonis, K. (2014). Reprogramming of the chick retinal pigmented epithelium after retinal injury. BMC Biol 12: 28.
  4. McNally, M. M., Wahlin, K. J. and Canto-Soler, M. V. (2010). Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: relevance for the developmental biology research field. BMC Dev Biol 10: 106.
  5. Zhu, J., Luz-Madrigal, A., Haynes, T., Zavada, J., Burke, A. K. and Del Rio-Tsonis, K. (2014). beta-Catenin inactivation is a pre-requisite for chick retina regeneration. PLoS One 9(7): e101748.

简介

鸡胚已成为研究发育生物学,细胞生物学和再生的主要模型之一。从鸡胚的所有解剖特征,眼睛是研究最多的之一。在鸡胚中,眼睛在孵育后26和33小时之间发育(阶段8-9,Hamburger和Hamilton,1951)。它起源于前脑的后部区域,称为间脑。然而,脊椎动物眼睛包括来自不同起源的组织,包括表面外胚层(晶状体和角膜),前神经板(视网膜,虹膜,睫状体和视网膜色素上皮)和神经嵴/头中胚层(虹膜和睫状体的基质以及脉络膜,巩膜和角膜的一部分)。在预处理后,单个眼视野源自前神经板,并且其特征在于协调眼部发育程序的眼场转录因子(EFTF)的表达。在后来的发展中,眼球分成两部分和视囊泡形式。在与透镜阵列几次感应相互作用之后,形成视杯。在阶段14-15,视杯的外层变成视网膜色素上皮(RPE),而内层形成最终分化成视网膜的神经上皮。鸡胚的一个主要优点是进行实验以过表达或下调基因表达在一个地方和时间特异性方式来探索基因功能和调节的可能性。这个协议的目的是描述电穿孔技术在阶段8-12(前神经折叠和光囊泡阶段)和阶段19-26(眼杯,RPE和神经上皮)。我们提供设备,材料和电极设置的完整描述,以及高度可重现的协议的详细描述,包括一些有代表性的结果。该协议已经从我们以前的出版物Luz-Madrigal等人(2014)和Zhu等人(2014)中改编而来。

关键字:电穿孔, 小鸡, 眼睛, 阶段19-26(眼杯阶段), 阶段8-12(前神经褶和视泡阶段)

  • Manders,E.M.,Stap,J.,Brakenhoff,G.J.,van Driel,R。和Aten,J.A。(1992)。 S期三维复制模式的动力学,通过双重标记DNA和共聚焦 显微镜。细胞科学 103(Pt 3):857-862。
  • Tsuda,K.,Qi,Y.,Nguyen le,V.,Bethke,G.,Tsuda,Y.,Glazebrook,J.and Katagiri,F。(2012)。 有效的土壤杆菌 - 介导的拟南芥的瞬时转化 69(4):713-719。
  • Voinnet,O.,Rivas,S.,Mestre,P。和Baulcombe,D。(2003)。 基于通过番茄丛生特技的p19蛋白对基因沉默的抑制而在植物中增强的瞬时表达系统 病毒。植物J 33(5):949-956。
  • ...
  • EDTA (Ethylenediaminetetraacetic acid), suitable for cell culture (Sigma-Aldrich, catalog number: E6758)
  • pCAG- GFP (Addgene plasmid, catalog number: 11150) or pEGFP-N1 (Takara Bio Company, Clontech)
  • Plasmid and RCAS-DNA [the name RCAS stands for Replication-Competent ASLV long terminal repeat (LTR) with a Splice acceptor] (Note 6)
  • Morpholinos (Note 7)
  • NaCl (Fisher Scientific, catalog number: BP358-1) (MW 58.44 g/mol)
  • CaCl2 anhydrous (Acros Organics, catalog number: AC34961-5000) (MW 110.98 g/mol)
  • KCl(Fisher Scientific,目录号:P217-500)(MW 74.55g/mol)
  • Na 2 HPO 4无水二碱(Fisher Scientific,目录号:S374-500)(MW 141.96g/mol)
  • KH 2 PO 4无水二碱(Fisher Scientific,目录号:P290-500)(MW 174.18g/mol)
  • 林格的解决方案(参见配方)
  • 10x Hank's平衡盐溶液(HBSS)(参见配方)
  • 快速绿色FCF(请参阅配方)
  • 1 M Tris(见配方)
  • 0.5 M EDTA(见配方)
  • 质粒溶液的TE缓冲液(参见配方)
  • 设备

    1. 斜边表玻璃(在电穿孔期间固定鸡蛋)(Thermo Fisher Scientific,目录号:15-355)
    2. 200μl无菌提示(Corning Incorporated)
    3. 用于显微注射的毛细管硼硅酸盐(1.0mm OD,0.5mm ID /纤维)[Frederick Haer& Co(FHC),目录号:30-30-1]
    4. 1ml注射器(Thermo Fisher Scientific)
    5. 预拉玻璃针头50毫米长,尖头直径20毫米,锐化10至12°(这些玻璃针按照Micropipette Puller和Micropipette Beveler的说明制作 - 图1d-e)
    6. 35mm塑料组织培养板(Corning Incorporated)
    7. 3/4英寸宽的透明胶带(Scotch,3M)
    8. 微切割镊子#55和#5(Roboz,目录号:RS-4984和RS-4978)
    9. ECM 830高通量电穿孔系统(图1a),用于体外和体内电穿孔的方波脉冲发生器用远程操作脚踏开关(BTX,Harvard Apparatus,SKU: ECM_830_for_In_Vivo_Applications)
    10. 具有用于注射和填充的脚踏开关的微注射器(图1b),MicroJect 1000A(BTX,Harvard Apparatus,SKU:45-0705)和不锈钢移液管支架(图1c)
    11. Micropipette beveler(Sutter Instrument Company,型号:BV-10)(图1d)
    12. 垂直微量移液器(Sutter仪器公司,型号:P-30)(图1e)
    13. 氮气罐压缩2.2 UN1066 NI NI230PP 230CF PP(CAGA580)(Weiler焊接)
    14. 立体变焦显微镜(Motic,型号:SMZ-168,目录号:1100200500322)或等效物
    15. 钨卤素光源(配备有光纤的系列,型号:8375)(Fostec ACE)
    16. 在37-39℃(99-103°F)和50-55%的相对湿度(83-87°F或28-31℃,在湿球温度计上)上校准的旋转培养箱(每小时角度旋转45°) (例如育种技术,1202E经典运动员, https://www.gqfmfg .com/store/front.asp
    17. 孵化器热空气Hova-Bator(https://www.gqfmfg.com/store/front.asp)
    18. 200μl微量移液器
    19. 蓝光滤光片(直径12.5mm,〜100%透射率高达500nm)(Edmond Optics,目录号:52-530)
    20. 电极(注8)
      1. Stage-8-12电极(设置见图2)
        1. 铂/铱 (Pt/Ir)微电极单元3(Frederick Haer& Co,目录 编号:UEPMEEVENNND),使用以下Metal Microelectrode Spec Sheet   ( http://www.fh-co.com/uploads/files/ue-spec-2013.pdf )订购
        2. 极端温度聚酰亚胺管(0.007"ID,1/16",OD,0.08" Wall,1'L,Clear Amber,McMasterCarr,catalogue number:5707K12)
        3. 使用直径约2 mm和长度约3 mm的塑料支架(使用20μl移液管吸头制成)(见图2c1-2)
        4. 弯曲和停留302/304不锈钢丝(0.032"直径,1'长,McMASTER-CARR,目录号:6517K66)
        5. 精密微型不锈钢管,304不锈钢,15 规格,0.072"OD,0.05"ID,0.011"壁(McMASTER-CARR,目录号: 8988K31)
        6. 白色Delrin 缩醛树脂棒,3/8"直径 (McMASTER-CARR,目录号:8572K53)。 这种树脂棒是改性的 具有压配合以在内部结合不锈钢管 (材料#v)并且装配在用作a的聚碳酸酯管中 手持器(材料#vii)(图1d)
        7. 耐冲击聚碳酸酯圆管(McMASTER-CARR,目录号:8585K11)
        8. 1米的26号(绞合的铝线)
        9. 连接器; BNC; 镀镍黄铜; 20; 镀金铍铜; PVC(Pomona,目录号:4969)
        10. 流动混合60秒(环氧树脂,1250psi强度,部件号:21445)(Devcon)
      2. ectrodes(设置见图3)
        1. 一个genetrode套件(5毫米,镀金厚电极 - L形, ovo基因)(Harvard Apparatus,型号:512,目录号:45-0115)
        2. 一个铂/铱微电极单元3(Frederick Haer& Co,目录号:UEPMEEVENNND),参见来自阶段-8-12的部分"i" 订购指令的电极
        3. Tygon ®微孔管( 0.010"ID×0.030"OD,100ft/roll)(Cole-Parmer,目录号: EW-06419-00)(特性:极柔软,无毒; 无热原 生物相容性)(Formulation Tygon,目录号: ND-100-80)
        4. 极端温度聚酰亚胺管.0089"ID, .0104",OD,.0008"Wall(1L,Clear Amber)(McMaster-Carr, number:51085K42)
        5. 一个毛细管管硼硅酸盐 显微注射,1.0mm OD,0.5mm ID /纤维(Frederick Haer& Co, 目录号:30-30-1)
        6. 一个1ml移液管吸头(Corning Incorporated)
        7. 50厘米铝线(绞合),26规格
        8. 连接器; BNC; 镀镍黄铜; 20; 镀金铍铜; PVC(Pomona,目录号:4969)
        9. 流动混合60秒(环氧树脂,1250psi强度,部件号:21445)(Devcon)


          图1.电穿孔设备。电穿孔设备 由ECM 830电穿孔仪(a),Microinjector MicroJect 1000A组成   和不锈钢移液管支架(b,c),Micropipette Beveler和 制造玻璃针(d,e)所需的拉拔器。

    程序

    1. 鸡蛋操作和孵化
      1. 在孵育之前,受精的无特异性病原体(SPF)(Charles River Laboratories,Wilmington)(注1)或白色来鸡鸡蛋 (密歇根州立大学,East Lansing,MI) 温度高达5天,生存力没有显着降低 (80-85%存活率)或发育缺陷(注2)。
      2. 孵化卵前,检查孵化器的条件(注3)。
      3. 对于眼中的电穿孔,我们大约孵化蛋 35 h为阶段8-9(七个体节,前神经折叠关闭 形成神经管),38 h阶段10(十个体节,视神经囊泡 不收缩在基地),48 h阶段12(十六个体节,光学 囊泡和视杆建立),72小时为阶段14(光学囊泡 evaginated和镜片placode存在)或4.5天为阶段22 [眼睛 色素沉着,视网膜色素上皮(RPE)和神经上皮孔 建立](注4)。


        图2. 8-12电极设置。两个 隔离的Pt/Ir电极与具有1mm的聚酰亚胺管(琥珀色管)  的自由末端(a)如图所示弯曲(b)。此后,弯曲 电极插入小塑料保持器(由20μl 移液管末端),直径约2 mm(c1),长度约3 mm(c2)  尖端1.5mm之间(c)。电极插入保持器中 由不锈钢管(SST)和树脂棒(RR)制成 用压配合(d)修改。仔细地,电极是永久的 使用环氧树脂附接到支架。保持形状和  在该过程期间电极之间的距离为1.5mm(e)。 最后,Pt/Ir电极连接到已经的电缆 以前连接到电穿孔仪的BNC适配器

        图3. 19-26级电极设置。一个镀金厚电极   (阳极+)用Tygon微孔管(a)隔离。 一个Pt/Ir 微电极(阴极 - )使用聚酰亚胺管(琥珀 管),其具有1mm的自由末端(参见图2a)并插入玻璃中 毛细管并用环氧树脂(b)密封,可替代地 用1ml移液管吸头保护。 然后组装电极 连接到先前已连接到BNC的电缆 电穿孔仪(c)的适配器

    2. 第8-12阶段的电穿孔
        最后,Pt/Ir电极连接到已经的电缆 以前连接到电穿孔仪的BNC适配器

        图3. 19-26级电极设置。一个镀金厚电极   (阳极+)用Tygon微孔管(a)隔离。 一个Pt/Ir 微电极(阴极 - )使用聚酰亚胺管(琥珀 管),其具有1mm的自由末端(参见图2a)并插入玻璃中 毛细管并用环氧树脂(b)密封,可替代地 用1ml移液管吸头保护。 然后组装电极 连接到先前已连接到BNC的电缆 电穿孔仪(c)的适配器

    3. 第8-12阶段的电穿孔
        ...
      1. Once the eggs reach the desired stage, place them in a carton and at room temperature for about 10 min. This incubation is necessary to facilitate the contraction of the inner shell membrane and promotes the separation from the outer shell membrane to generate the air sack.
      2. Keeping their horizontal position, place the eggs in a small incubator (e,g., HOVA-BATOR) previously calibrated at 37-39 °C (99 to 103 °F) and relative humidity of 50-55%.
      3. With a bright light source, candle the egg and mark the area where the embryo is located, usually the embryo is located in the top part of the yolk (on the top of the horizontal section of the shell).
      4. Mark the air sack that is located between the outer and inner membranes of the egg.
      5. Pierce the shell at the top of the air sack using blunt ended forceps or a syringe with a large bore needle.
      6. Carefully, introduce a fine probe to move the air sack to the top part of the egg, this is possible by gently touching down to lower the inner shell membrane (Figure 4b-c) (Note 5).
      7. Using sharp curved scissors or blunt ended forceps, open a round window on the top of the embryo (around 2 cm of diameter) (Figure 4d) (Video 1 provides visual information of the steps B1e-h) and add 200 μl of HBSS. Close the window using transparent tape and return the egg to the incubator. Discard the embryos older than the desired stage. If the embryos have not reached the desired stage, they can be incubated for an additional time.
        在其中电穿孔的胚胎阶段的变化 执行会对结果和解释产生不一致
      8. 可选地,增强对比度和可视化胚胎 阶段8-12,注射100微升稀释在HBSS中的印度油墨溶液,使用a  1毫升注射器和25-26号针在胚胎下面
      9. 对其余的鸡蛋重复步骤B1e-h,直到十几个完成。


        图4.准备鸡蛋进行电穿孔。受精卵是 在水平位置(a)温育。当地化了空气袋和 鸡胚,一个细探针被引入将空气袋移动到  蛋的顶部。这可以通过轻轻地触摸和降低 内壳膜(b,c)。最后,使用锋利的弯曲剪刀  钝端镊子,在胚胎的顶部打开一个圆窗 (直径约2cm)(d)。

        视频1. 准备用于电穿孔的鸡蛋
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    4. 胚胎电穿孔
      1. 将氮气罐调整到80 psi
      2. 将斜角玻璃毛细管针连接到微量注射器的不锈钢移液管支架(图1c)。
      3. 将微注射器设置在18-20 psi,并执行测试加载HBSS 使用"填充"按钮或脚踏开关并释放解决方案 从微注射器推动按钮"空"。 测试 使用脚踏开关的微注射系统
      4. 附上蓝色 二向色滤光片到钨卤素灯的光路 资源。 使用以下设置配置电穿孔仪:模式, LV; 电压,18V; 脉冲长度,50 ms; 脉冲数,5; 脉冲 间隔,950ms; 极性,单极性
      5. 将Stage-8-12电极(见设备部分)连接到电穿孔仪
      6. 使用含有1ml HBSS的35mm组织培养板,检查 电穿孔系统将电极放置在溶液中(注意 9)。
      7. 使用微型注射器的脚踏开关,装载玻璃 毛细管针与实验溶液(质粒,RCAS 构造或MO在解决方案部分)。
      8. 添加100微升的HBSS胚胎,以减少电极之间的电阻
      9. 对于电穿孔进入眼睛,注射0.25μl(20微注射   脉冲使用20μm的尖端直径的玻璃毛细管针) 溶液进入前神经折叠(前神经细胞在阶段8-9, 图5a)或进入光学囊泡(阶段10-11,图5b)
      10. 将阶段8-12电极放在卵黄膜上(一个清晰的 在蛋黄周围的膜)在胚胎的两侧 (平行于神经管)(图5a,b)并执行 使用电穿孔器脚踏开关进行电穿孔
      11. 小心地,删除电极,并添加额外100微升的HBSS
      12. 用塑料胶带密封窗口并将蛋转移到 孵育器直到它们达到期望的阶段。 代表性的形象 在阶段11使用对照MO(图5c,d)的电穿孔胚胎, 并且3天后收集的数据如图5e所示

        图5。 阶段8-12电穿孔。 对于眼中的电穿孔,注射   0.25μl的实验溶液进入前神经折叠 (在阶段8-9的前神经)(a)或进入视神经囊(阶段 10-11)(b)。 在明亮的领域的代表性图片与印度墨水为 在阶段电穿孔的胚胎的对比度(c)和荧光(d) 11使用控制MO。 第12阶段电穿孔眼的共焦图像   使用对照MO并在电穿孔后(e)收集24小时。 L: 透镜,(e)中的比例尺=100μm。

  • 在阶段19-26的视杯中电穿孔
    1. 将受精卵垂直温育直至达到所需阶段 阶段8-12电穿孔。 对于眼中的电穿孔,注射   0.25μl的实验溶液进入前神经折叠 (在阶段8-9的前神经)(a)或进入视神经囊(阶段 10-11)(b)。 在明亮的领域的代表性图片与印度墨水为 在阶段电穿孔的胚胎的对比度(c)和荧光(d) 11使用控制MO。 第12阶段电穿孔眼的共焦图像   使用对照MO并在电穿孔后(e)收集24小时。 L: 透镜,(e)中的比例尺=100μm。

  • 在阶段19-26的视杯中电穿孔
    1. 将受精卵垂直温育直至达到所需阶段... Check the electroporation system placing the electrodes (1 cm in between) in a 35 mm tissue culture plate containing 1 ml of HBSS (Note 9).
    2. Using the microinjector footswitch, load the glass capillary needle with the experimental solution (RCAS-DNA or morpholinos).
    3. For electroporations in the eye, inject 0.5 µl (40 microinjection pulses) into the eye cup at Stage 18-19 or 0.75 µl (60 microinjection pulses) into the eye at Stage 24-25 using a 20 µm diameter tip glass capillary needle.
    4. Add 200 µl of HBSS to the embryo to decrease the resistance between the electrodes.
    5. To perform the electroporation, the gold plated electrode connected to the anode (+) is placed close to the ventro-temporal section of the eye and the cathode (-) electrode is inserted perpendicular to the head of the embryo (Video 2 essentially describes steps C8-10).

      Video 2 . Electroporation in the chick optic cup at Stages 19-26

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    6. 小心取出电极,加入200μlHBSS
    7. 用塑料胶带密封窗口并将蛋转移到 孵育器直到它们达到期望的阶段。 代表性胚胎 在阶段25电穿孔的如图6所示

      图6。 第25阶段的电穿孔眼。第25阶段电穿孔眼的图像 并使用对照吗啉(a)在24小时后收集。 放大 的电穿孔眼在(b)。 显示的共聚焦图象 电穿孔的神经上皮(NE)(c)。 RPE:视网膜 色素上皮,L:晶状体。 比例尺= 0.5mm(a),0.1mm(b) 在(c)中为100μm。
  • 笔记

    1. 对于将使用RCAS [具有剪接受体]复制能力的ASLV长末端重复(LTR)病毒系统的研究,我们强烈建议使用无特定病原体(SPF)的卵。这些卵已经被证明是没有禽肉瘤白血病病毒(ASLV)的病原体。有关其他信息,请参见http://www.criver.com/,McNally等人(2010)发表了关于ASLV病毒蛋白内源性表达的详细研究。
    2. 低于4°C的温度是有害的,因此在冬季运输期间要特别小心
    3. 将蛋在37-39℃(99-103°F)和50-55%的相对湿度下在旋转培养箱中孵育(每小时45度角旋转)。孵化时间和胚胎阶段可以根据培养箱的类型,温度和湿度而变化,因此特定胚胎阶段的孵育时间应该在每个实验室中经验地确定(Hamburger和Hamilton,1951)。
    4. 在进行电穿孔之前,确保胚胎处于期望的阶段,注意一些特征,例如体节数,视神经囊泡,晶状体囊泡和眼睛色素沉着的特征。电穿孔阶段的小差异可导致表型的巨大差异。在某些情况下,印度墨水的注入可能是必要的,以便可视化胚胎和熟悉结构(参见阶段8-12的电穿孔步骤B1i)。
    5. 在引入探针时要温和,避免损坏蛋的内膜。如果你注意到白蛋白的泄漏很可能是胚胎死亡
    6. 确保质粒或RCAS构建体具有高质量。使用市售试剂盒例如PureYield TM质粒Maxiprep系统或Qiagen Plasmid Maxi Prep TM扩增DNA构建体并溶解于TE缓冲液中(参见TE缓冲液在解决方案部分)。我们建议将DNA溶解在质量为3.0-5.0μg/μl和RCAS构建体的浓度为200 ng /μl,短期使用时保存在-20°C,长期保存在-80°C保存。避免使用含有痕量苯酚或乙醇和高浓度盐的DNA样品,因为它们可能对胚胎的生存能力有害。使用分光光度计或Nanodrop确定DNA的浓度。或者,可以使用SYBR Green I dsDNA测定法测定浓度。吸光度260/280的比例将给你一个关于DNA纯度与蛋白质或其他污染物的想法,次级测量可以是260/230(苯酚碳水化合物,吸光度接近230nm)(参见技术支持公告 http://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf )。对于260/280比率〜1.8或更高的比率或对于260/230的2.0-2.2比率被认为是高质量的DNA。始终通过电泳验证DNA的完整性。定量不足以确保您的DNA具有良好的质量。对于第一次使用者,建议使用含有报道基因如GFP的质粒来评估电穿孔的效率,例如pCAG-GFP或pEGFP-N1。此外,为了跟踪您感兴趣的基因的表达,建议设计您的构建包括IRES-GFP序列或识别电穿孔区域的替代方法。为了制备电穿孔溶液,将9.0μl质粒(3.0-5.0μg/μl)或RCAS构成(200ng /μl)与1μl的0.05%Fast Green染料混合。
    7. 设计吗啉(MO)反义寡核苷酸以下调基因表达。它们可以从GeneTools LLC(http://www.gene-tools.com/)设计和订购。为了追踪电穿孔细胞,MO必须在3'-末端标记(例如羧基荧光素)。 MO具有最小的脱靶效应,但是为了观察相同的表型效应,推荐使用至少两种不同的MO(例如,阻断翻译和剪接连接)。另外,有必要使用靶mRNA的对照MO和/或加扰序列。使用MO的主要缺点是它们非常昂贵,有时需要 测试两个以上的序列,以找到合适的MO有效地下调基因表达。此外,在细胞分裂期间,MO被稀释,因此它们长期(超过4天)是无效的,并且在一些情况下,它们在电穿孔72小时后几乎不可检测,并且在这种情况下,荧光信号需要使用抗体荧光素。 MO使用无菌林格氏溶液以1mM浓度溶解,并在室温下储存在原始容器中并保存在黑暗中。根据Gene Tools,MO可以在低温下沉淀并且失去其效率,因此它们需要在使用前再次重悬。在电穿孔期间,MO可能轻微地朝阳极(+)迁移,可能是因为带负电的荧光素。为了提高MO电穿孔的效率,建议使用0.5μgDNA(例如非生物活性质粒)。为了制备电穿孔溶液,混合并加热10μl的等分试样在65℃下10分钟以重新溶解沉淀物。不推荐使用快速绿色,因为它可以抑制电穿孔(Kos等人,2013)。可以使用附接到常规光纤灯的蓝色二向色滤光器来观察羧基荧光素标记的吗啉代的注射。
    8. 我们提供了设置Stage-8-12和Stage-19-26电极所需的部件列表,请注意,列出的某些部件可以相同。
    9. 按下电穿孔器脚踏开关后,您会看到电极上有轻微的气泡(气泡表示系统正常工作)。

    食谱

    1. 林格的解决方案
      1. 将7.2g NaCl,0.17g CaCl 2,0.37g KCl,0.115g Na 2 HPO 4和0.02g KH 2溶液 PO 4在900ml去离子水中的溶液
      2. 用HCl调节pH至7.2,用去离子水调至1L 注意:最终浓度:NaCl 123mM,CaCl 2 2mM,1.53mM,KCl 5mM,Na 2 SO 4, 2
      3. 使用0.2μmCorning一次性塑料真空过滤器过滤灭菌
      4. 在-20℃下冷冻10ml的等分试样
    2. 10x Hank平衡盐溶液(HBSS)
      1. 使用去离子水制备250ml的1×HBSS,并将pH调节至7.2
      2. 使用0.2μmCorning一次性塑料真空过滤器过滤灭菌
      3. 在-20℃下冷冻10ml的等分试样
    3. 快速绿色FCF
      1. 使用去离子水制备0.05%(wt/vol)的储备溶液
      2. 使用0.2μmCorning注射器圆盘型过滤器过滤灭菌
    4. 1 M Tris
      将6.057g Tris溶解在30.0ml去离子水中,用1N HCl调节pH至8.0,用去离子水调至50.0ml。
    5. 0.5 M EDTA
      将18.6g溶于80.0ml去离子水中,并用10N NaOH(EDTA溶解直到pH达到8.0)将pH调节至8.0,用去离子水调至100.0。
    6. 用于质粒溶液(10mM Tris,1mM EDTA,pH = 8)的TE缓冲液 为了制备50ml TE缓冲液,将0.5ml 1M Tris(pH = 8)与0.1ml 0.5M EDTA(pH = 8)混合,用去离子水调节至50ml。 通过高压灭菌器灭菌并在-20℃下冷冻2ml的等分试样

    致谢

    Michael Weeks,Jayson Alexander和Bill Lack,迈阿密大学仪器实验室在电极设置方面的帮助,Leah Stetzel对视频录制的帮助。这项工作由EY17319到KDRT,CONACYT 162930和142523到AL-M支持。该协议已经从我们以前的出版物Luz-Madrigal等人(2014)和Zhu等人(2014)中改编而来。

    参考文献

    1. Hamburger,V。和Hamilton,H.L。(1951)。 鸡胚发育的一系列正常阶段。 J Morphol 88(1):49-92。
    2. Kos,R.,Tucker,R.P.,Hall,R.,Duong,T.D.and Erickson,C.A。(2003)。 将morpholinos引入鸡胚中的方法 Dev Dyn 226(3):470-477。
    3. Luz-Madrigal,A.,Grajales-Esquivel,E.,McCorkle,A.,DiLorenzo,A.M.,Barbosa-Sabanero,K.,Tsonis,P.A.and Del Rio-Tsonis,K。 视网膜损伤后鸡视网膜色素上皮的重编程。 BMC Biol 12:28.
    4. McNally,M.M.,Wahlin,K.J.and Canto-Soler,M.V。(2010)。 ASLV病毒蛋白在特定病原体游离鸡胚中的内源性表达:与发育生物学研究领域的相关性。 BMC Dev Biol 10:106.
    5. Zhu,J.,Luz-Madrigal,A.,Haynes,T.,Zavada,J.,Burke,A.K.and Del Rio-Tsonis,K。 β-联蛋白失活是雏鸡视网膜再生的先决条件。 PLoS One 9(7):e101748。
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    引用:Luz-Madrigal, A., Grajales-Esquivel, E. and Del Rio-Tsonis, K. (2015). Electroporation of Embryonic Chick Eyes. Bio-protocol 5(12): e1498. DOI: 10.21769/BioProtoc.1498.
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